WO2025255166A2 - Procédés et systèmes de détection de biomarqueurs protéiques de la polyarthrite rhumatoïde dans un échantillon biologique à l'aide de lc-ms/ms - Google Patents

Procédés et systèmes de détection de biomarqueurs protéiques de la polyarthrite rhumatoïde dans un échantillon biologique à l'aide de lc-ms/ms

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Publication number
WO2025255166A2
WO2025255166A2 PCT/US2025/032142 US2025032142W WO2025255166A2 WO 2025255166 A2 WO2025255166 A2 WO 2025255166A2 US 2025032142 W US2025032142 W US 2025032142W WO 2025255166 A2 WO2025255166 A2 WO 2025255166A2
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WIPO (PCT)
Prior art keywords
protein
sample
surrogate
peptides
ions
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English (en)
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WO2025255166A3 (fr
WO2025255166A9 (fr
Inventor
Christopher Michael SHUFORD
Russell Philip Grant
Lyelle Nathanial DAVIS
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Labcorp Holdings Inc
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Laboratory Corp of America Holdings
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Publication of WO2025255166A2 publication Critical patent/WO2025255166A2/fr
Publication of WO2025255166A3 publication Critical patent/WO2025255166A3/fr
Publication of WO2025255166A9 publication Critical patent/WO2025255166A9/fr
Pending legal-status Critical Current
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present disclosure generally relates to improved methods and systems for detecting and/or quantifying one or more 14-3-3 proteins (e.g, 14-3-3q protein), or surrogate peptide thereof, in a sample using mass spectrometry (e.g, LC-MS/MS).
  • 14-3-3 proteins e.g, 14-3-3q protein
  • surrogate peptide thereof e.g, LC-MS/MS
  • Rheumatoid arthritis is one of the most common systemic autoimmune diseases. There are multiple pathophysiological factors that contribute to the onset of Rheumatoid arthritis and its symptoms, and there is high heterogeneity among patients throughout the disease. If untreated, Rheumatoid arthritis results in severe joint destruction, leading to impaired physical activity and disability. The disease outcome and patient prognosis can be significantly improved by early identification of Rheumatoid arthritis and prediction of the disease severity at diagnosis for the implementation of an effective treatment strategy.
  • the present disclosure is related to methods and systems for detecting or quantifying a 14-3-3q protein in a sample using LC-MS/MS.
  • the method may be embodied in a variety of ways.
  • the method for determining the amount of 14-3-3q protein in a sample includes: (a) generating a precursor ion of 14-3-3 q protein; (b) optionally, generating one or more fragment ions of the precursor ion; and (c) detecting the amount of one or more ions in step (a) or (b) or both to determine the amount of 14-3-3q protein in the sample.
  • the method further comprises purifying the sample prior to step (a).
  • the purification step comprises liquid chromatography.
  • step (a) comprises generating one or more precursor ions of the 14-3-3q.
  • the method comprises enriching the sample in the 14-3-3q protein prior to step (a).
  • the sample can be enriched in the 14-3-3q protein using solid-phase extraction, precipitation, affinity enrichment, immunoaffinity enrichment, or combinations thereof to produce an enriched sample.
  • the antibodies used for immunoaffinity enrichment can be specific to 14-3-3q protein or fragments thereof.
  • the method for determining the amount of 14-3-3q protein in a sample includes: (a) generating a precursor ion of a surrogate peptide of 14-3-3q protein; (b) generating one or more fragment ions of the precursor ion; and (c) detecting the amount of one or more ions in step (a) or (b) or both to determine the amount of 14-3-3q protein in the sample.
  • the method further comprises purify ing the sample prior to step (a).
  • the purification step comprises liquid chromatography.
  • step (a) comprises generating one or more precursor ions of the 14-3-3q surrogate peptide having a mass to charge ratio selected from the group consisting of 408.7+0.5, 516.2+0.5, 739.9+0.5, 454.3+0.5, 308.2+0.5, 452.3+0.5, 433.2.7+0.5, 874.4+0.5, 652.8+0.5, 533.8+0.5, 634.3+0.5, 412.2+0.5, 412.9+0.5, 991.5+0.5, 720.0+0.5, 595.3+0.5, and 708.0+0.5.
  • the method comprises enriching the sample in the 14- 3-3q protein prior to step (a).
  • the sample can be enriched in the 14-3- 3q protein using solid-phase extraction, precipitation, affinity enrichment, immunoaffinity enrichment, or combinations thereof to produce an enriched sample.
  • the antibodies used for immunoaffinity enrichment can be specific to 14-3-31 ⁇ protein or fragments thereof.
  • the antibody used for immunoaffinity enrichment can be specific to 14-3-31 ⁇ protein and at least one other family member of 14-3-3 protein (e.g., 14-3-3fy 14-3-3y, 14-3- 3a, etc.) or fragments thereof.
  • the antibody used for immunoaffinity enrichment is a pan- 14-3-3 protein antibody.
  • affinity enrichment may be performed with a binding partner of 14-3-31 ⁇ or 14-3-3 protein family.
  • the method includes digesting the enriched sample to produce one or more surrogate peptides of 14-3-31 ⁇ protein or 14-3-3 protein family.
  • a method may include providing a biological sample comprising a plurality of proteins including 14-3-31 protein; adding a binding agent that specifically binds to 14-3-3i ⁇ protein to the biological sample; separating 14-3-3i ⁇ protein bound to the binding agent from unbound proteins in the biological sample to produce an enriched sample; contacting the enriched sample with a proteolytic enzyme to produce a proteolytic digest comprising peptides, wherein at least one of the peptides comprises a surrogate peptide of 14-3-3i ⁇ protein; performing liquid chromatography on the proteolytic digest to purify the sample; and measuring the amount of 14-3-3q protein in the biological sample by detection of the surrogate peptide of 14-3-31 ⁇ protein using mass spectrometry.
  • the binding agent that specifically binds to 14-3-31 ⁇ protein comprises an antibody or an aptamer or a binding partner.
  • the proteolytic enzyme may be trypsin or an isozyme thereof.
  • the biological sample may be serum or plasma.
  • the method may include providing a biological sample comprising a plurality of proteins including at least one 14-3-3 protein family member; adding a binding agent that specifically binds to one or more 14-3-3 protein family members to the biological sample; separating 14-3-3 protein family members bound to the binding agent from unbound proteins in the biological sample to produce an enriched sample; contacting the enriched sample with a proteolytic enzyme to produce a proteolytic digest comprising peptides, wherein at least one of the peptides comprises a surrogate peptide of one or more 14-3-3 protein family members; performing liquid chromatography on the proteolytic digest to purify the sample; and measuring the total amount of 14-3-3 protein family members in the biological sample by detection of the surrogate peptide of the one or more 14-3-3 protein family members using mass spectrometry.
  • the binding agent that specifically binds to 14-3-3 protein family members comprises an antibody or an aptamer or a binding partner.
  • the proteolytic enzyme may be trypsin.
  • the biological sample may be serum or plasma.
  • the total amount of 14-3-3 protein family members measured includes 14-3-3q protein.
  • the 14-3-3q protein or 14-3-3 protein family members bound to the binding agent prior to liquid chromatography are separated by first coupling the binding agent to a solid phase substrate.
  • the solid phase substrate can be washed to remove unbound proteins from the solid phase substrate and eluting the 14-3-3q protein or 14-3-3 protein family members bound to the binding agent from the solid phase substrate.
  • measuring the amount of 14-3-3q protein in the sample using mass spectrometry may comprise the steps of: (i) generating at least one precursor ion of 14-3 -3 q surrogate peptide; (ii) generating one or more fragment ions of the precursor ion; and (iii) detecting the presence or amount of the precursor ion generated in step (i) and/or the at least one or more fragment ions generated in step (ii), or both, and relating the detected ions to the presence or amount of the 14-3-3p protein in the biological sample.
  • the at least one precursor ion is formed by electrospray ionization. The electrospray ionization may be performed in positive or negative ion mode.
  • the mass spectrometry detection of the 14-3-3q protein surrogate peptide or 14-3-3 protein family surrogate peptide may be performed in selected reaction monitoring mode (SRM). In some embodiments, more than one surrogate peptide may be detected by mass spectrometry.
  • the method may further comprise adding an internal standard to the biological sample, wherein the internal standard is a stable isotope of 14-3-3q protein. In some embodiments, the method may comprise adding an internal standard to the enriched sample, wherein the internal standard includes a stable isotope of the surrogate peptide and can be proteolyzed to form a stable isotope surrogate peptide.
  • the method may comprise adding an internal standard to the enriched or proteolyzed sample, wherein the internal standard is a stable isotope of the surrogate peptide.
  • the detection of the 14-3-3q protein may be over a range of from 0.1 ng/mL to 100 ng/mL.
  • the mass spectrometry detection of the stable isotope of 14-3-3q protein may be performed in selected reaction monitoring (SRM) mode.
  • the at least one precursor ion for a surrogate peptide 14-3-3q protein may have a mass/charge ratio (m/z) of about 516.2 ⁇ 0.5 and the one or more fragment ions comprise a fragment ion that may have an m/z of about 868.4 ⁇ 0.5, 753.3 ⁇ 0.5, 638.3 ⁇ 0.5, 507.3 ⁇ 0.5, 436.2 ⁇ 0.5, 279. H0.5, 394.H0.5, 136.1 ⁇ 0.5, or 251.1 ⁇ 0.5.
  • the liquid chromatography may include high performance liquid chromatography (HPLC). The liquid chromatography may be performed in reverse phase separation or hydrophilic interaction liquid chromatography.
  • the disclosure provides a method for determining the presence or amount of at least one biomarker of interest in a biological sample, the method comprising: providing a biological sample comprising a plurality of proteins including 14-3- 3q protein; contacting a proteolytic enzy me with the biological sample to produce a proteolytic digest including peptides, wherein at least one of the peptides comprises a surrogate peptide of 14-3-3 q protein; adding one or more binding agent to the proteolytic digest of the biological sample, wherein the binding agent specifically binds to the surrogate peptide of 14-3-3 r] protein; separating the peptides bound to the binding agent from unbound peptides in the proteolytic digest to produce an enriched sample; purifying the sample enriched with the surrogate peptide of 14-3-3q protein using liquid chromatography; and measuring the amount of 14-3-3 i protein in the biological sample by detection of the surrogate peptide of 14-3-3iq protein using mass
  • the disclosure provides a method for determining the presence or amount of at least one biomarker of interest in a biological sample, the method comprising: providing a biological sample comprising a plurality 7 of proteins including at least one 14-3-3 protein family member; contacting a proteolytic enzyme with the biological sample to produce a proteolytic digest comprising surrogate peptides of the plurality of proteins, wherein at least one of the surrogate peptides comprises a surrogate peptide of one or more 14-3-3 protein family members; adding one or more binding agents to the proteolytic digest of the biological sample, wherein the binding agent specifically binds to the surrogate peptides of one or more 14-3-3 protein family members; separating the surrogate peptides bound to the binding agent from unbound peptides in the proteolytic digest to produce an enriched sample; purifying the sample enriched with surrogate peptide of the one or more 14- 3-3 protein family members using liquid chromatography; and detecting the surrog
  • the total amount of 14-3-3 protein family members measured using surrogate peptides of one or more 14-3-3 protein family members is predominantly from 14-3-3q protein.
  • the binding agent that specifically binds to the surrogate peptides of the one or more 14-3-3 protein family members comprises an antibody or an aptamer.
  • the proteolytic enzyme may include trypsin, and the biological sample may be serum or plasma.
  • the method may include measuring the amount of 14-3-3q protein or 14-3-3 protein family members in the biological sample using mass spectrometry.
  • the method may include the steps of: (i) generating a precursor ion of a 14-3-3r
  • the precursor ions are formed by electrospray ionization wherein the electrospray ionization is performed in positive or negative ion mode.
  • the method may further include adding an internal standard to the biological sample, wherein the internal standard is a stable isotope of l4-3-3q protein or fragment thereof.
  • the mass spectrometry detection of surrogate peptides derived from 14-3-3r] protein or 14-3-3 protein family members is performed in selected reaction monitoring (SRM) mode.
  • SRM selected reaction monitoring
  • the detection of the 14-3-31] protein or 14-3-3 protein family members may be over a range of from 0. 1 ng/mL to 100 ng/mL.
  • the precursor ion for the surrogate peptides derived from 14-3-3i] protein has a mass/charge ratio (m/z) of about 412.2 ⁇ 0.5 and the one or more fragment ions comprise a fragment ion with a m/z of about 623.3 ⁇ 0.5, 476.3 ⁇ 0.5, 347.2 ⁇ 0.5, 234. l ⁇ 0.5, or combinations thereof.
  • the method may include performing liquid chromatography wherein the liquid chromatography includes high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the liquid chromatography technique may be performed in reverse phase separation or hydrophilic interaction liquid chromatography.
  • the present disclosure provides systems for performing the methods of the invention.
  • a system for determining the presence and/or amount of a biomarker of interest in a biological sample comprising: a station for providing a biological sample comprising 14-3-3q protein; a station for partially- purifying 14-3-3q protein from other components in the sample; a station for chromatographically separating 14-3-3q protein from other components in the sample; and a station for analyzing the chromatographically separated 14-3-3q protein by mass spectrometry to determine the presence or amount of 14-3-3q protein in the biological sample.
  • the present disclosure provides a computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to cause one or more computers to perform actions to measure a presence or amount of 14-3-3q protein in a biological sample comprising at least one of the following steps: (a) obtaining a biological sample from a subject; (b) optionally adding a stable isotope-labeled 14-3-3q protein to the sample as an internal standard; (c) performing liquid chromatography; and (d) measuring 14-3-31] protein by tandem mass spectrometry.
  • the biomarker of interest is a protein
  • the biological sample may be subjected to proteolytic digestion to generate a peptide from the protein.
  • the station for mass spectrometry may comprise a tandem mass spectrometer.
  • the system may comprise a station for chromatographic purification of the biomarker of interest prior to mass spectrometry, as for example by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the mass spectrometry may comprise liquid chromatography tandem mass spectrometry (LC-MS/MS), or 2 dimensional LC-MS/MS.
  • the system may comprise a station for subjecting the sample to protease digestion.
  • the system may comprise a computer such that at least one of the stations is controlled by the computer.
  • the present disclosure provides a method for determining a presence or amount of a l4-3-3q protein in a sample including: (a) providing one or more surrogate peptides of the 14-3-3q protein; (b) ionizing the one or more surrogate peptides of the 14-3-3q protein to generate one or more ions of the surrogate peptides of 14-3-3q protein detectable by mass spectrometry; (c) determining the presence or amount of the one or more ions of the surrogate peptides of the 14-3-3 i protein by mass spectrometry; and (d) determining the presence or amount of the 14-3 -3iq protein in the sample according to the presence or amount of the one or more ions determined in (c).
  • the sample further comprises one or more surrogate peptides derived from other 14-3-3 protein family members.
  • providing the one or more peptides of (a) comprises contacting a biological sample with a proteolytic enzyme to produce a proteolytic digest comprising the one or more surrogate peptides of the 14-3-3q protein.
  • the method includes, prior to (a), and/or prior to producing the proteolytic digest, enriching the biological sample for one or more 14-3-3 protein family members.
  • the one or more 14-3-3 protein family members comprises the 14-3-3q protein.
  • the enriching of the biological sample comprises use of one or more binding agents that bind to one or more of the 14-3-3 protein family members or one or more binding agents that bind specifically to the 14-3-3q protein.
  • the enriching of the biological sample comprises an immunoprecipitation method.
  • the one or more binding agents comprise one or more monoclonal antibodies.
  • the proteolytic enzyme comprises trypsin or an isozyme thereof.
  • providing the one or more peptides of (a) comprises enriching the proteolytic digest for the one or more surrogate peptides of 14-3-3q.
  • the enriching of the proteolytic digest comprises contacting the proteolytic digest with one or more binding agents that bind to the one or more surrogate peptides of the 14-3-3q protein.
  • binding agents comprise one or more antibodies.
  • the one or more antibodies comprise one or more monoclonal antibodies.
  • the enriching of the proteolytic digest comprises an immunoprecipitation method.
  • the one or more binding agents comprise a binding agent that specifically binds to a peptide sequence selected from the peptide sequences of SEQ ID NOs. 2, 3, 7-15 and 17.
  • the one or more binding agents comprise a binding agent that specifically binds to a peptide sequence selected from the peptide sequences of SEQ ID NOs. 2. 7 and 12.
  • the proteolytic digest is enriched for a surrogate peptide comprising the sequence of one or more of SEQ ID NOs. 2, 7 and 12.
  • the one or more surrogate peptides provided of (a) comprise one or more of SEQ ID NOs. 2, 7 and 12.
  • the one or more surrogate peptides provided in (a) comprise one or more internal standard peptides.
  • one or more of the internal standard peptides comprise a stable isotope label.
  • enriching the biological sample or enriching the proteolytic digest comprises solid-phase extraction, precipitation, affinity enrichment, immunoaffimly enrichment, or combinations thereof.
  • ionizing the one or more surrogate peptides of 14-3-3q protein comprises: (i) generating at least one precursor ion of the one or more surrogate peptides of 14-3-3q protein; (ii) generating one or more product ions of the precursor ion; and detecting the presence or amount of the at least one precursor ion generated in step (i) and/or the one or more product ions generated in step (ii), or both, to determine the presence or amount of the 14-3-3i] protein in the biological sample.
  • the at least one precursor ion has a mass/charge ratio of 408.7 ⁇ 0.5.
  • the one or more product ions of the precursor ion has a mass/charge ratio of one or more of 703.3 ⁇ 0.5, 632.3 ⁇ 0.5, 503.3 ⁇ 0.5, 375.2 ⁇ 0.5, 304.2 ⁇ 0.5, 175.1 ⁇ 0.5, 185.1 ⁇ 0.5, 314.2+0.5, and 442.2+0.5.
  • protein comprise a mass/charge ratio selected from the group consisting of 703.3 ⁇ 0.5, 632.3 ⁇ 0.5, 503.3+0.5, and 185.1 ⁇ 0.5.
  • the one or more ions of the surrogate peptides of the 14-3-3i] protein comprise a mass/charge ratio selected from the group consisting of 868.4+0.5, 753.3+0.5, 507.3+0.5, and 279.1+0.5. In some embodiments, the one or more ions of the surrogate peptides of the 14-3-3p protein comprises a mass/charge ratio selected from the group consisting of 804.4 ⁇ 0.5. 691.4+0.5, 213.2+0.5, and 185.2+0.5.
  • the one or more ions of the surrogate peptides of the 14-3-3p protein comprises a mass/charge ratio selected from the group consisting of 1066.5+0.5, 967.5+0.5, 868.4+0.5, and 301.2+0.5.
  • the one or more ions of the surrogate peptides of the 14-3-3q protein comprises a mass/charge ratio selected from the group consisting of 623.3 ⁇ 0.5. 476.3 ⁇ 0.5, 347.2 ⁇ 0.5, and 234. l ⁇ 0.5.
  • the mass spectrometry comprises tandem mass spectrometry.
  • the mass spectrometry comprises LC-MS, LC-MS/MS, or 2D-LC-MS/MS.
  • the biological sample comprises serum or plasma.
  • the biological sample is derived from a subject.
  • the subject is a mammal or wherein the subject is a human.
  • the subject has or is suspected of having rheumatoid arthritis.
  • the presence or amount of 14-3-3i] determined in (d) indicates the subject has rheumatoid arthritis.
  • the amount of 14-3-3i] determined in (d) is detected in a biological sample with a lower limit of detection of about 1 ng/ml. 0. 1 ng/ml or about 0. 1 ng/ml.
  • the amount of the one or more ions of the surrogate peptides of (c) are detected with a lower limit of detection of about 1 ng/ml, 0.1 ng/ml or about 0.1 ng/ml.
  • a method for determining a presence or amount of 14-3-3i] protein in a biological sample comprises: providing a biological sample comprising a plurality of proteins including one or more 14-3-3 protein family members; contacting the biological sample with a proteolytic enzyme to produce a proteolytic digest comprising peptides, wherein at least one of the peptides comprises a surrogate peptide derived from a 14-3 -3q protein; contacting the proteolytic digest with a binding agent, wherein the binding agent specifically binds the surrogate peptide derived from the 14-3-3q protein; separating unbound peptides from the surrogate peptide to produce a sample enriched for the surrogate peptide; purifying the surrogate peptide from the enriched sample using liquid chromatography; generating at least one precursor ion of the surrogate peptide; generating one or more product ions of the precursor ion; and detecting the presence or amount of the at least one precursor i
  • the proteolytic enzyme comprises trypsin or an isozyme thereof.
  • the at least one precursor ion is formed by electrospray ionization.
  • the electrospray ionization is performed in positive ion mode.
  • the detecting the presence or amount of the at least one precursor ion, the one or more product ions of the precursor ion, or both, is performed by a process comprising mass spectrometry’.
  • the mass spectrometry detection of the surrogate peptide is performed in selected reaction monitoring mode (SRM).
  • the at least one precursor ion of the surrogate peptide has a mass/charge ratio (m/z) of about 516.2 ⁇ 0.5.
  • the one or more product ions comprise an ion with a m/z of about 868.4 ⁇ 0.5, 753.3 ⁇ 0.5, 638.3 ⁇ 0.5, 507.3 ⁇ 0.5, 436.2 ⁇ 0.5, 279.1 ⁇ 0.5, 394.1 ⁇ 0.5, 136.1 ⁇ 0.5, or 251.1 ⁇ 0.5.
  • the mass spectrometry’ comprises LC-MS or LC-MS/MS.
  • the liquid chromatography comprises high performance liquid chromatography (HPLC).
  • the liquid chromatography is performed in reverse phase liquid chromatography or by hydrophilic interaction liquid chromatography.
  • a method for determining a presence or amount of 14-3-3q protein in a biological sample comprises: subjecting a surrogate peptide of 14-3-3q protein from a biological sample to an ionization source under conditions suitable to generate one or more precursor ions with a mass to charge ratio (m/z) of 452.3 ⁇ 0.5; fragmenting at least one of the precursor ions to generate one or more fragment ions detectable by mass spectrometry, wherein the one or more fragment ions comprise one or more ions selected from the group consisting of ions with m/z of 804.4 ⁇ 0.5, 691.4 ⁇ 0.5, 213.2 ⁇ 0.5, and 185.2 ⁇ 0.5; and determining the amount of one or more of the precursor ions and/or fragment ions by mass spectrometry to determine the amount of 14-3-3q protein in the sample.
  • the surrogate peptide of 14-3-3q protein is at least 80 % identical to any one of SEQ ID NOs. : 1 -17.
  • a method for determining a presence or amount of 14-3-3q protein in a biological sample comprises: providing a biological sample comprising one or more 14- 3-3 protein family members or fragments thereof; enriching the one or more 14-3-3 protein family members in the biological sample to produce an enriched sample; contacting a proteolytic enzyme with the enriched sample to produce a proteolytic digest comprising peptides, wherein the peptides comprise a surrogate peptide derived from a 14-3-3q protein; generating at least one precursor ion of the surrogate peptide; generating one or more product ions of the precursor ion; and detecting the presence or amount of the at least one precursor ion and/or the one or more fragment ions, or both, and determining a presence or amount of the 14-3-31] protein in the biological sample according to the detecting.
  • the detecting comprises use of mass spectrometry.
  • the proteolytic enzyme comprises trypsin or an isozyme thereof.
  • the biological sample comprises serum or plasma.
  • the one or more precursor ions of the surrogate peptide has a mass to charge (m/z) ratio selected from the group consisting of 408.7 ⁇ 0.5, 516.2 ⁇ 0.5, 739.9 ⁇ 0.5, 454.3 ⁇ 0.5, 308.2 ⁇ 0.5, 452.3 ⁇ 0.5, 433.2.7 ⁇ 0.5, 874.4 ⁇ 0.5, 652.8 ⁇ 0.5, 533.8 ⁇ 0.5, 634.3 ⁇ 0.5. 412.2 ⁇ 0.5. 412.9 ⁇ 0.5, 991.5 ⁇ 0.5, 720.0 ⁇ 0.5, 595.3 ⁇ 0.5, and 708.0 ⁇ 0.5.
  • the method further comprises purifying the enriched sample using liquid chromatography, wherein the liquid chromatography is performed in reverse phase separation or hydrophilic interaction liquid chromatography.
  • the present disclosure provides a system for conducting the method of any one of the embodiments described herein, wherein the system comprises: a station for providing a biological sample comprising a 14-3-3q protein; a station for partially purifying the 14-3-3q protein from other components in the sample; a station for chromatographically separating 14-3-3q protein from other components in the sample; and a station for analyzing the chromatographically separated 14-3-3q protein by mass spectrometry to determine the presence or amount of the 14-3-3q protein in the biological sample.
  • the sy stem further comprises a station for contacting the biological sample with a proteolytic enzyme, and optionally a station for partially purifying a surrogate peptide of the 14-3-3q protein from a proteolytic digest.
  • a computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to cause one or more computers and/or devices to perform the methods described herein.
  • FIG. 1 shows a flowchart for a method for detecting or quantifying 14-3-3q protein in a biological sample using LC-MS/MS.
  • FIG. 2 shows a flowchart for another method for detecting or quantifying 14-3-3q protein in a biological sample using LC-MS/MS.
  • FIG. 3 shows a system in accordance with an embodiment of the disclosure.
  • FIG. 4 shows an exemplary sequence of full-length human 14-3-3p protein (SEQ ID NO. 19).
  • the bold sequences correspond to surrogate peptides formed from trypsin digestion of 14-3-3p (i.e., YDDMASAMK (SEQ ID NO. 2), VISSIEQK (SEQ ID NO. 6), TMADGNEK (SEQ ID NO. 7), and EAFEISK (SEQ ID NO 12).
  • SEQ ID NOs. 2, 7 and 12 correspond to surrogate peptides produced from 14-3-3p which are not found in other 14-3-3 protein family members.
  • SEQ ID NO. 19 The bold sequences correspond to surrogate peptides formed from trypsin digestion of 14-3-3p (i.e., YDDMASAMK (SEQ ID NO. 2), VISSIEQK (SEQ ID NO. 6), TMADGNEK (SEQ ID NO. 7), and EAFEISK (SEQ ID NO 12).
  • SYKDSTLIMQLLRDNLTLWT SEQ ID NO. 18
  • SEQ ID NO. 18 The underlined sequence SYKDSTLIMQLLRDNLTLWT is conserved across all 14-3-3 family members and contains an epitope for the anti-14-3-3 (pan) antibody used for 14-3-3 protein enrichment workflows (e.g., workflow' 2 of Example 2).
  • FIGs. 5A-5F show 7 exemplary chromatograms for a blank/zero calibrator (FIGs. 5 A and 5B), a 20 ng/mL calibrator (FIGs. 5C and 5D), and native serum sample (FIGs. 5E and 5F) for the 14-3-3_VIS surrogate peptide (SEQ ID NO. 6) shown in top panels FIGs. 5 A, 5C and 5E) and its stable isotope internal standard (shown in bottom panels FIGs. 5B, 5D and 5F) produced from the workflow of Example 1. All native serum samples measured > 70 ng/mL for 14-3-3_VIS using the workflow of Example 1.
  • FIGs. 6A-6F show exemplary chromatograms for a blank/zero calibrator (FIGs. 6A and 6B), a 1 ng/mL calibrator (FIGs. 6C and 6D), and native serum sample (FIGs. 6E and 6F) for the 14-3-3_TMA surrogate peptide (SEQ ID NO. 7) shown in top panels FIGs. 6A, 6C and 6E) and its stable isotope internal standard (shown in bottom panels FIGs. 6B, 6D and 6F) produced from the workflow of Example 1. All native serum samples measured > 9 ng/mL for 14-3-3_TMA using the workflow of Example 1.
  • FIGs. 7A-7F show exemplary chromatograms for a blank/zero calibrator (FIGs. 7A and 7B), a 1 ng/mL calibrator (FIGs. 7C and 7D), and native serum sample (FIGs. 7E and 7F) for the 14-3-3 YDD surrogate peptide (SEQ ID NO. 2) shown in top panels FIGs. 7A. 7C and 7E) and its stable isotope internal standard (shown in bottom panels FIGs. 7B, 7D and 7F) produced from the workflow of Example 1. All native serum samples measured > 7 ng/mL for 14-3-3_YDD using the workflow of Example 1.
  • FIGs. 8A-8F show exemplary chromatograms for a blank/zero calibrator (FIGs. 8A and 8B), a 10 ng/mL calibrator (FIGs. 8C and 8D), and native serum sample (FIGs. 8E and 8F) for the 14-3-3_EAF surrogate peptide (SEQ ID NO. 12) shown in top panels FIGs. 8 A, 8C and 8E) and its stable isotope internal standard (shown in bottom panels FIGs. 8B, 8D and 78F) produced from the workflow of Example 1. All native serum samples measured > 11 ng/mL for 14-3-3_EAF using the workflow of Example 1.
  • FIGs. 9A-9C show Demming regression plots for the measured values of surrogate peptides of 14-3-3 proteins in serum: 14-3-3_VIS, SEQ ID NO. 6 (FIG. 9 A); 14-3-3 YDD, SEQ ID NO. 2 (FIG. 9B); and 14-3-3 EAF (SEQ ID NO. 12)[Y-axis] vs. 14-3-3 TMA, SEQ ID NO. 7 [X-axis] produced from the workflow of Example 1.
  • FIG. 10 shows a mean and standard deviation box plot of the measured values of surrogate peptides of 14-3-3 proteins in serum (14-3-3 VIS, 14-3-3 TMA. 14-3-3 YDD, and 14-3-3_EAF) produced from the workflow of Example 1 and Example 2.
  • FIGs. 11A-11D show example chromatograms for the surrogate peptides (top panel) and corresponding stable isotope labeled peptides (bottom panel) measured according to the workflow of Example 2 (FIGs. HA and 11C) and a hybrid workflow of Example 3 (FIGs. 1 IB and 1 ID).
  • FIGs. 11 A and 1 IB show example chromatograms for the 14-3- 3 TMA peptide
  • FIGs. 11C and 1 ID shows example chromatograms for the 14-3-3 YDD peptide.
  • the target analytes in a sample are 14-3-3 proteins (e.g. 14-3-3p).
  • the quantification or detection of specific 14-3-3 proteins in a sample can be used to aid in the diagnosis, prognosis, and/or monitoring of disease (e.g., rheumatoid arthritis) in a subject.
  • a target analyte for detection via LC-MS/MS is a peptide used as a surrogate for detection (e.g., quantification) of a specific 14-3-3 protein.
  • surrogate peptides may be produced from a target protein by digestion with a protease (e.g, trypsin).
  • one or more surrogate peptides may be used to detect and/or quantify a specific 14-3-3 protein in a sample.
  • the methods and systems described herein improve sensitivity, selectivity, and accuracy for quantifying and/or detecting one or more 14-3-3 proteins in a sample.
  • the methods described herein can reduce auto-antibody interferences caused by antibodies in a sample that interfere with other methods used to detect 14-3-3 proteins in a sample, often leading to false-positive or false-negative results.
  • a sample can be obtained from a suitable subject.
  • a sample can be isolated or obtained directly from a subject or part thereof.
  • a sample is obtained indirectly from an individual or medical professional.
  • a sample is derived from a subject.
  • a sample can be any specimen that is isolated or obtained from a subject or part thereof.
  • a sample comprises a bodily fluid obtained from a subject.
  • a sample comprises an extract or lysate of a tissue or cells.
  • Non-limiting examples of a sample include blood or a blood product (e.g, serum, plasma, platelets, buffy coats, or the like), umbilical cord blood, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., lung, gastric, peritoneal, ductal, ear, arthroscopic), urine, sputum, saliva, nasal mucous, prostate fluid, lavage, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, a liquid biopsy, as well as lysates or extracts obtained from cells (blood cells, lymphocytes, placental cells, stem cells, bone marrow derived cells, embryo or fetal cells), feces, tissues, the like or combinations thereof.
  • a blood product e.g, serum, plasma, platelets, buffy coats, or the like
  • umbilical cord blood e.g., amniotic fluid, cerebrospinal fluid, spinal fluid
  • lavage fluid e.
  • a sample is blood or a blood product, e.g., serum.
  • a sample may be processed prior to, or while performing a method described herein. For example, a sample may be partially purified, enriched, filtered, concentrated, and/or diluted.
  • a subject refers to an animal, typically a mammalian animal.
  • a subject is a mammal.
  • mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig).
  • a subject is a primate.
  • a subject is a human.
  • the methods and systems described herein are configured to detect a presence or absence of, and/or determine an amount of. a 14-3-3 protein in a sample. In some embodiments, the methods and systems described herein are configured to detect a presence, absence, and/or amount of one or more of 14-3-3q, 14-3-3(3, 14-3-3e, 14-3-3y, 14- 3-3T, 14-3-3O, and 14-3-3 ⁇ in a sample.
  • the methods and systems described herein are configured to detect a presence or absence of, and/or determine an amount of, surrogate peptides derived from one or more of 14-3-3p, 14-3-3(3, 14-3-3e, 14-3- 3y, 14-3-31, 14-3-3o, and 14-3-3 .
  • a method described herein has a limit of detection (LOD) of a 14-3-3 protein in a sample in a range of 50 ng/ml to about 0.01 ng/ml, or 20 ng/ml to about 0.001 ng/ml. In some embodiments, a method described herein has a limit of detection for a 14-3-3 protein in a sample of 20 ng/ml or less, 10 ng/ml or less, 5 ng/ml or less, 11 ng/ml or less, 0.1 ng/ml or less, or about 0.01 ng/ml or less. In some embodiments, a method described herein has a limit of detection for a 14-3-3 protein in a sample of about 5 ng/ml, about 1 ng/ml, about 0.1 ng/ml or about 0.01 ng/ml.
  • LOD limit of detection
  • a method described herein has a limit of detection for a surrogate peptide of a 14-3-3 protein (e.g., 14-3-3p) in a range of 50 ng/ml to about 0.001 ng/ml, or 20 ng/ml to about 0.001 ng/ml. In some embodiments, a method described herein has a limit of detection for a surrogate peptide of a 14-3-3 protein of 20 ng/ml or less, 10 ng/ml or less, 5 ng/ml or less, 11 ng/ml or less, 0.1 ng/ml or less, or about 0.01 ng/ml or less.
  • a method described herein has a limit of detection for surrogate peptides of a 14-3-3 protein of about 5 ng/ml, about 1 ng/ml, about 0.1 ng/ml or about 0.01 ng/ml.
  • biomarker or a “biomarker of interest” is any biomolecule that may provide biological information about the physiological state of an organism.
  • the presence or absence of a biomarker may be informative.
  • the level of a biomarker may be informative.
  • the biomarker of interest may comprise a peptide, a hormone, a nucleic acid, a lipid or a protein.
  • biological sample refers to a sample obtained from a biological source.
  • body fluid refers to a liquid sample obtained from a biological source, including, but not limited to. an animal, a cell culture, an organ culture, and the like.
  • the term “preferentially binds” refers to a reagent that predominantly binds to a protein. Preferential binding of a reagent is meant to include at least 90% of the times a reagent will bind to a protein and discriminate between other proteins.
  • the term "specifically binds" refers to a binding agent that binds to a target analyte (e.g.. one or more 14-3-3 protein family members) in preference to binding other molecules or other peptides as determined by. for example, a suitable in vitro assay (e.g., an Elisa. Immunoblot. Flow cytometry, and the like).
  • a specific binding interaction discriminates over non-specific binding interactions by about 2-fold or more, often about 10-fold or more, and sometimes about 100-fold or more, 1000-fold or more, 10,000- fold or more, 100,000-fold or more, or 1,000,000-fold or more.
  • a “surrogate peptide” refers to a peptide produced through proteolysis and which can be derived from a single protein or collection of proteins based on its unique amino acid sequence. As such, detection of a surrogate peptide following proteolysis often provides evidence to the presence of the protein or collection of proteins from which it was derived. If calibration is performed, measurement of a surrogate peptide following proteolysis can be used to determine the amount of protein or the collective amount of protein from which it was derived in the sample prior to proteolysis.
  • the terms “purify” or “separate” or derivations thereof do not necessarily refer to the removal of all materials other than the analyte(s) of interest from a sample matrix. Instead, in some embodiments, the terms “purify” or “separate” refer to a procedure that enriches the amount of one or more analytes of interest relative to one or more other components present in the sample matrix. In some embodiments, a “purification” or “separation” procedure can be used to remove one or more components of a sample that could interfere with the detection of the biomarker of interest, for example, one or more components that could interfere with detection of an analyte by mass spectrometry.
  • within group variability refers to coefficient of variability of a measurement between subjects in a group.
  • a “calibrator” is a sample created with known biomarker concentration in a matrix which is ideally but not necessarily free from a biomarker of interest. Calibrators are often used to generate a dose response curve used to determined concentrations of biomarkers in unknown samples.
  • a “quality control” is a sample with a target concentration range which is used to verify the quality of results from an experiment.
  • chromatography refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.
  • liquid chromatography means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s).
  • Liquid chromatography includes reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), high turbulence liquid chromatography (HTLC), and hydrophilic interaction liquid chromatography (HILIC).
  • RPLC reverse phase liquid chromatography
  • HPLC high performance liquid chromatography
  • HTLC high turbulence liquid chromatography
  • HILIC hydrophilic interaction liquid chromatography
  • the chromatographic column typically includes a medium (i.e.. a packing material) to facilitate separation of chemical moieties (i.e., fractionation).
  • the medium may include minute particles.
  • the particles may include a bonded surface that interacts with the various chemical moieties to facilitate separation of the chemical moieties such as the biomarker analytes quantified in the experiments herein.
  • One suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded surface.
  • Alkyl bonded surfaces may include C-4, C- 8, or C-18 bonded alkyl groups, preferably C-18 bonded groups.
  • the chromatographic column may include an inlet port for receiving a sample and an outlet port for discharging an effluent that includes the fractionated sample.
  • the sample (or pre-purified sample) may be applied to the column at the inlet port, eluted with a solvent or solvent mixture, and discharged at the outlet port.
  • Different solvent modes may be selected for eluting different analytes of interest.
  • liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytyptic (i.e., mixed) mode.
  • HPLC may be performed on a multiplexed analytical HPLC system with a Cl 8 solid phase using isocratic separation with water: methanol as the mobile phase.
  • the term “analytical column” refers to a chromatography column having sufficient chromatographic plates to effect a separation of the components of a test sample matrix.
  • the components eluted from the analytical column are separated in such a way to allow the presence or amount of an analyte(s) of interest to be determined.
  • the analytical column comprises particles having an average diameter of about 5 pm.
  • the analytical column is a functionalized silica or polymer-silica hybrid, or a polymeric particle or monolithic silica stationary phase, such as a phenyl-hexyl functionalized analytical column.
  • Analytical columns can be distinguished from “extraction columns/’ which typically are used to separate or extract retained materials from non-retained materials to obtain a “purified” sample for further purification or analysis.
  • the extraction column is a functionalized silica or polymer-silica hybrid or polymeric particle or monolithic silica stationary phase, such as a Poroshell SBC-18 column.
  • heart-cutting refers to the selection of a region of interest in a chromatogram and subjecting the analytes eluting within that region of interest to a second separation, e.g., a separation in a second dimension.
  • electrospray ionization refers to methods in which an analyte of interest in a gaseous or vapor phase interacts with a flow of electrons. Impact of the electrons with the analyte produces analyte ions, which may then be subjected to a mass spectrometry technique.
  • chemical ionization refers to methods in which a reagent gas (e.g., ammonia) is subjected to electron impact, and analyte ions are formed by the interaction of reagent gas ions and analyte molecules.
  • a reagent gas e.g., ammonia
  • field desorption refers to methods in which anon-volatile test sample is placed on an ionization surface, and an intense electric field is used to generate analyte ions.
  • matrix-assisted laser desorption ionization refers to methods in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo-ionization, protonation, deprotonation, and cluster decay.
  • MALDI matrix-assisted laser desorption ionization
  • the sample is mixed with an energy-absorbing matrix, which facilitates desorption of analyte molecules.
  • SELDI surface enhanced laser desorption ionization
  • the sample is typically bound to a surface that specifically retains one or more analytes of interest.
  • this process may also employ an energy-absorbing material to facilitate ionization.
  • electrospray ionization refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Upon reaching the end of the tube, the solution may be vaporized (nebulized) into ajet or spray of very small droplets of solution in solvent vapor. This mist of droplet can flow through an evaporation chamber which is heated slightly to prevent condensation and to evaporate solvent. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.
  • APCI Bactmospheric Pressure Chemical Ionization, 7 or “APCI,” as used herein refers to mass spectroscopy methods that are similar to ESI, however, APCI produces ions by ion-molecule reactions that occur within a plasma at atmospheric pressure. The plasma is maintained by an electric discharge between the spray capillary and a counter electrode. Then, ions are typically extracted into a mass analyzer by use of a set of differentially pumped skimmer stages. A counterflow of dry and preheated N2 gas may be used to improve removal of solvent. The gas-phase ionization in APCI can be more effective than ESI for analyzing less-polar species.
  • APPI Atmospheric Pressure Photoionization
  • M is photon absorption and electron ejection to form the molecular M+.
  • the molecular ion is less susceptible to dissociation. In many cases it may be possible to analyze samples without the need for chromatography, thus saving significant time and expense. In the presence of water vapor or protic solvents, the molecular ion can extract H to form MH+. This tends to occur if M has a high proton affinity.
  • inductively coupled plasma refers to methods in which a sample is interacted wi th a partially ionized gas at a sufficiently high temperature to atomize and ionize most elements.
  • ionization and “ionizing” as used herein refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Negative ions are those ions having a net negative charge of one or more electron units, while positive ions are those ions having a net positive charge of one or more electron units.
  • the term “desorption” as used herein refers to the removal of an analyte from a surface and/or the entry of an analyte into a gaseous phase.
  • the term “hemolyzed” refers to the rupturing of the red blood cell membrane, which results in the release of hemoglobin and other cellular contents into the plasma or serum and the term “lipemic” refers to an excess of fats or lipids in blood.
  • liquid plasma is plasma that is obtained from drawing blood from a patient and that is separated from the red blood cells but that remains in a liquid state. Liquid plasma is generally obtained from subjects by phlebotomy or venipuncture.
  • Dried plasma is plasma that has been allowed to dry. Dried plasma may be produced following separation from red blood cells by migration of the plasma through pores of a solid-phase substrate which restrict migration of cells as is described in more detail herein.
  • a “protein variant” is a protein that has an amino acid sequence that is different from the most common or wild-type sequence.
  • a “protein family” refers to a collection of different oligopeptides, derived from expression of different genes, but which have homologous or analogous amino acid sequences. In some cases, a “protein family” could also be used to define a collection of protein isoforms derived from a single gene, but which differ in their amino acid sequence through, for example, alternative splicing and/or post-translational processing.
  • the present disclosure is directed to a method for detecting or determining the presence or amount of at least one biomarker of interest in a biological sample using liquid chromatography and tandem mass spectrometry (LC-MS/MS).
  • the methods described herein may be employed for detecting and/or measuring one or more 14-3-3 proteins in a biological sample.
  • 14-3-3 proteins are a family of proteins found in all eukaryotic cells, from plants to humans.
  • 14-3-3 proteins regulate a vast array of cellular functions, and dysregulation of 14-3- 3 proteins or their interactions is implicated in numerous diseases.
  • beta (
  • a method of detecting or determining an amount of one or more 14-3-3 proteins in a sample includes detecting the amount of intact 14-3-3 proteins, surrogate peptides derived from 14-3-3 proteins, or combinations thereof. In some embodiments, a method of detecting or determining an amount of one or more 14-3-3 proteins includes detecting one or more intact 14-3-3 proteins (e.g., proteins that are not subject to proteolysis) using LC-MS/MS.
  • a method of detecting or determining an amount of one or more 14-3-3 proteins does not include directly detecting or measuring an amount of one or more intact 14-3-3 proteins ( ⁇ ?.g., proteins that were not subject to proteolysis) using mass spectrometry (e.g., LC-MS/MS).
  • a method of detecting or determining an amount of one or more 14-3-3 proteins includes detecting one or more surrogate peptides of 14-3-3 proteins using LC-MS/MS.
  • a method of detecting or determining an amount of one or more 14-3-3 proteins in a sample includes purifying or enriching a sample (e.g., a biological sample).
  • a 14-3-3 proteins may be derived, isolated, extracted, purified or partially purified from one or more subjects, one or more samples or one or more sources.
  • purification of the sample may include one or more of liquid chromatography, solid-phase extraction, precipitation (e.g, immunoprecipitation), affinity enrichment (e.g., affinity chromatography), immunoaffinity enrichment, or combinations thereof to produce a purified or enriched sample.
  • a method includes purifying a sample using liquid chromatography.
  • Liquid chromatography is a process of selective retardation of one or more components in a fluid solution as the fluid moves through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (z.e., mobile phase), as this fluid moves relative to the stationary phase(s).
  • Any suitable method can be used for liquid chromatography including reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), high turbulence liquid chromatography (HTLC), hydrophilic interaction liquid chromatography (HILIC), and normal phase liquid chromatography (NPLC).
  • RPLC reverse phase liquid chromatography
  • HPLC high performance liquid chromatography
  • HTLC high turbulence liquid chromatography
  • HILIC hydrophilic interaction liquid chromatography
  • NPLC normal phase liquid chromatography
  • the terms HILIC and NPLC are used interchangeably
  • a method may include analyzing a chromatographically separated analyte (e.g, 14- 3-3 proteins) by mass spectrometry to determine the presence or amount of the analyte in the sample.
  • Mass spectrometry may include filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "in/z.”
  • one or more analytes of interest are ionized, and the ions are subsequently introduced into a mass spectrometer where, due to a combination of electric fields, the ions follow a path in space that is dependent upon mass (“m”) and charge (“z”).
  • the precursor ion is selected following ionization, and that precursor ion is subjected to fragmentation to generate product (z.e., fragment) ions, whereby one or more product ions are selected for detection.
  • product z.e., fragment
  • Each precursor ion is known as a transition, and monitoring of one or more transitions is known as selected reaction monitory (SRM) or multiple reaction monitoring (MMR).
  • SRM reaction monitory
  • MMR multiple reaction monitoring
  • the MS/MS analysis comprises at least one of SRM or MMR.
  • the mass spectrometer uses a quadrupole or quadrupole ion trap system.
  • ions in an oscillating radio frequency (RF) field experience a force proportional to the direct current (DC) potential applied between electrodes and the amplitude of the RF signal.
  • the voltage and amplitude can be selected so that only ions having a particular m/z travel the length of the quadrupole, while all other ions are deflected.
  • quadrupole instruments can act as both a “mass filter” and as a “mass detector” for the ions injected into the instrument.
  • an MS method comprises tandem mass spectrometry or MS/MS.
  • an MS methods or systems herein comprises use of a triple quadrupole MS/MS.
  • Triple quadrupole MS/MS instruments typically consist of two quadrupole mass filters separated by a fragmentation means.
  • a triple quadrupole MS/MS instrument may comprise a quadrupole mass filter operated in the RF only mode as an ion containment or transmission device.
  • a quadrupole may further comprise a collision gas at a pressure of between 1 and 10 millitorr.
  • Many other types of “hybrid” tandem mass spectrometers are contemplated for use in the methods and systems of the present invention including various combinations of magnetic sector analyzers and quadrupole filters.
  • a tandem MS/MS may be operated in a variety of modes.
  • ions can be produced using a variety of methods, nonlimiting examples of which include electron ionization, chemical ionization, fast atom bombardment, field desorption, and matrix-assisted laser desorption ionization (“MALDI”), surface enhanced laser desorption ionization (“SELDI”), photon ionization, electrospray ionization (“ESI”), atmospheric pressure ionization (“ACPI”), nanospray, and inductively coupled plasma.
  • MALDI matrix-assisted laser desorption ionization
  • SELDI surface enhanced laser desorption ionization
  • ESI electrospray ionization
  • ACPI atmospheric pressure ionization
  • nanospray and inductively coupled plasma.
  • a tandem MS/MS spectrometer is operated in a positive or negative ion electrospray ionization mode.
  • a mass spectrometry may isolate precursor ions for further fragmentation.
  • CID collision-induced dissociation
  • precursor ions gain energy through collisions with an inert gas and subsequently fragment by a process referred to as “unimolecular decomposition.”
  • MS comprises 2D-LC-MS/MS.
  • 2D- LC-MS/MS comprises a multiplex system comprising staggered multiplexed LC and MS sample inlet systems.
  • the methods and systems of the present invention may comprise multiple column switching protocols, and/or heart-cutting (LC-LC or 2D-LC) techniques, and/or LC separations prior to MS detection.
  • the methods and systems of the present invention may include a multiplexed two-dimensional liquid chromatographic system coupled with a tandem mass spectrometer (MS/MS) system, for example a triple quadrupole MS/MS system. Such embodiments provide for staggered, parallel sample input into the MS system.
  • An analyte of interest may be quantified based upon an amount of the characteristic transitions measured by a mass spectrometer.
  • the tandem mass spectrometer comprises a triple quadrupole mass spectrometer.
  • the tandem mass spectrometer is operated in an electrospray ion (ESI) mode.
  • the electrospray is operated in a positive ion mode.
  • a nanospray emitter is used.
  • the quantification of an analyte and internal standards is performed in a selected reaction monitoring mode (SRM).
  • SRM selected reaction monitoring mode
  • LC -MS/MS and 2D-LC-MS/MS methods can be applied to the quantification of an analyte of interest in a complex sample biological matrices, including, but not limited to. blood, serum, plasma, urine, saliva, and the like.
  • a complex sample biological matrices including, but not limited to. blood, serum, plasma, urine, saliva, and the like.
  • the methods and systems of the present invention are suitable for clinical applications and/or clinical trials.
  • the systems and methods of the present invention provide approaches for addressing isobaric interferences, varied sample content, including hemolyzed and lipemic samples, while attaining low ng/mL limits of detection (LOD) of target analytes. Accordingly, embodiments of the methods and systems of the present invention may provide for the quantitative, sensitive, and specific detection of clinical analytes used in the clinical diagnosis of disorders.
  • LOD limits of detection
  • a method includes purifying or enriching one or more surrogate peptides of a 14-3-3q protein, e.g., using immunoprecipitation and/or liquid chromatography, ionizing the one or more surrogate peptides and detecting the one or more surrogate peptides of by mass spectrometry.
  • the presence or amount of a 14-3-3q protein in a sample is determined according to the presence or amount of the one or more surrogate peptides detected by mass spectrometry.
  • one or more ions of a surrogate peptide comprise a precursor ion with a mass to charge ratio of 408.7 ⁇ 0.5 and/or one or more fragment ions selected from the group of ions having a mass to charge ratio of 703.3 ⁇ 0.5, 632.2 ⁇ 0.5, 503.3 ⁇ 0.5, 375.2 ⁇ 0.5, 304.2 ⁇ 0.5, 175.1 ⁇ 0.5, I85.1 ⁇ 0.5, 314.2 ⁇ 0.5, and 442.2 ⁇ 0.5.
  • a method of detecting or determining an amount of a 14-3-3q protein in a biological sample includes purifying a sample comprising one or more surrogate peptides of 14-3-3q protein using liquid chromatography, ionizing the one or more surrogate peptides of 14-3-3q protein to generate one or more ions of the surrogate peptides of 14-3-3q protein detectable by mass spectrometry', and determining the amount of the one or more ions of the surrogate peptides of 14-3-3q protein by tandem mass spectrometry' to determine the amount of 14-3-3q protein in the sample.
  • the one or more ions comprise a precursor ion with a mass to charge ratio of 408.7 ⁇ 0.5 and one or more fragment ions selected from the group of ions with mass to charge ratios consisting of 703.3 ⁇ 0.5, 632.2 ⁇ 0.5, 503.3 ⁇ 0.5, 375.2 ⁇ 0.5, 304.2 ⁇ 0.5, 175.1 ⁇ 0.5, 185.1 ⁇ 0.5, 314.2 ⁇ 0.5, and 442.2 ⁇ 0.5.
  • the method for determining the amount of 14-3-3q protein in a sample includes: (a) generating a precursor ion of a surrogate peptide of 14-3-3q protein;
  • step (a) comprises generating one or more precursor ions of the 14-3-3q protein having a mass to charge ratio selected from the group consisting of 408.7 ⁇ 0.5, 516.2 ⁇ 0.5, 739.9 ⁇ 0.5, 454.3 ⁇ 0.5, 308.2 ⁇ 0.5, 452.3 ⁇ 0.5, 433.2.7 ⁇ 0.5. 874.4 ⁇ 0.5. 652.8 ⁇ 0.5, 533.8 ⁇ 0.5, 634.3 ⁇ 0.5, 412.2 ⁇ 0.5, 412.9 ⁇ 0.5, 991.5 ⁇ 0.5, 720.0 ⁇ 0.5, 595.3 ⁇ 0.5, and 708.0 ⁇ 0.5.
  • the method further comprises purifying the sample prior to step (a).
  • the purification step comprises liquid chromatography.
  • a method comprises enriching a sample for one or more 14- 3-3 protein family members prior to generating surrogate peptides of a 14-3-3 r] protein.
  • a sample e.g, a biological sample
  • a method comprises enriching a sample for one or more 14-3-3 protein family members prior to subjecting a sample to LC-MS/MS.
  • a sample can be enriched for one or more 14-3-3 protein family members using solid-phase extraction, precipitation, affinity enrichment, immunoaffmity enrichment, or combinations thereof to produce an enriched sample.
  • a sample is contacted with a binding agent (e.g. , an antibody) that binds specifically to one or more 14-3-3 protein family members.
  • a binding agent e.g. , an antibody
  • the bound 14-3-3 protein family members can then be isolated from a sample using a suitable method.
  • bound complexes of one or more 14-3-3 protein family members are isolated by an affinity purification method.
  • a binding agent comprises a pan-antibody that binds to two, three, four, five or six or more 14-3-3 protein family members in a sample.
  • a pan antibody may specifically bind to a 14-3-3q protein, a 14-3-3a protein, and a 14-3-3y protein, or fragments thereof.
  • a pan antibody specifically binds to a 14-3-3q protein and at least one other 14-3-3 protein family member or a fragment thereof.
  • a pan antibody comprises a polyclonal antibody, or a binding fragment thereof.
  • a pan antibody comprises a monoclonal antibody, or binding fragment thereof.
  • a sample is contacted with a binding agent (e.g. , an antibody) that binds specifically to a 14-3-3q protein.
  • a binding agent e.g. , an antibody
  • the bound 14-3-3q protein can then be isolated from a sample using a suitable method.
  • a bound protein is isolated by an affinity purification method.
  • an antibody -bound 14-3-3q protein can be immunoprecipitated using protein G beads, followed by washing, and optionally resuspension in a buffer suitable for protease digestion.
  • an antibody that binds specifically to a 14-3-3rj protein comprises a monoclonal antibody, or binding fragment thereof.
  • unbound proteins can be removed from a sample (e.g., using a washing step) to obtain a sample enriched for a desired protein or peptide.
  • a sample enriched for one or more 14-3-3 family member proteins can be digested with a proteolytic enzyme to produce one or more surrogate peptide of a 14-3-3q protein.
  • an enriched sample comprises one or more 14-3-3 proteins bound to one or more binding agents (z.e., referred to as bound proteins, e.g., antibody-bound proteins) that are attached to a suitable substrate (e.g., a bead or a surface).
  • binding agents z.e., referred to as bound proteins, e.g., antibody-bound proteins
  • a process of enriching a sample comprising increasing a concentration or purity of one or more 14-3-3 protein family members or surrogate peptides in a sample.
  • a method of enriching may increase the concentration and/or purity of a 14-3-3 protein family member or surrogate peptide in a sample 2-fold to a 1000-fold.
  • a method of enriching may increase the concentration and/or purity of a 14-3-3 protein family member or surrogate peptide in a sample greater than 2-fold, greater than 10- fold, greater than 100-fold, or greater than 1000-fold compared to the concentration or purity of the 14-3-3 protein family member or surrogate peptide prior to enrichment.
  • a binding agent comprises or consists of a suitable antibody, an antibody fragment and/or an antigen binding portion thereof (e.g., a binding fragment).
  • An antibody can refer to a natural antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, a chimeric antibody, an antibody binding fragment (e.g.. an antigen binding portion of an antibody), a CDR-grafted antibody, a humanized antibody, a human antibody, or portions thereof.
  • a binding agent comprises or consists of one or more suitable antigen binding portions of an antibody, non-limiting examples of which include Fab, Fab', F(ab')2, Fv fragment, single-chain Fv (scFv), diabody (Dab), synbody, the like and/or a combination or portion thereof.
  • a binding agent comprises TandAbs, aptamers, nanobodies. BiTEs.
  • a binding agent comprises a single-chain polypeptide comprising one or more antigen binding portions of an antibody.
  • a binding agent specifically binds to at least one 14-3-3 protein or to at least one surrogate peptide of a 14-3-3 protein (e.g., a 14-3-3 p protein).
  • a binding agent is coated or bound to a suitable substrate.
  • a substrate is a solid-phase substrate.
  • a solid-phase substrate can comprise a bead, particle or a surface.
  • a solid-phase substrate comprises a magnetic particle.
  • a solid-phase substrate comprises a coating.
  • a bead can be coated with streptavidin.
  • a binding agent is attached to a magnetic bead.
  • a binding agent can be biotinylated and conjugated to magnetic beads coated with streptavidin.
  • bound complexes of one or more 14-3-3 protein family members are bound to a suitable substrate.
  • antibody-bound 14-3-3 protein family members can be immunoprecipitated using protein G beads, followed by washing, and optionally resuspension in a buffer suitable for protease digestion.
  • a binding agent attached to a substrate can be washed to purify and/or enrich a bound protein (e.g., a 14-3-3 protein or surrogate protein) using a suitable washing method.
  • a protein bound to a binding agent that is attached to a substrate can be eluted using a suitable method to provide a purified or enriched protein.
  • surrogate peptides are produced by digesting proteins of a sample with a proteolytic reagent, thereby producing a proteolytic digest. In some embodiments, surrogate peptides are produced by contacting a sample with a proteolytic reagent, thereby producing a proteolytic digest.
  • proteolytic reagents include a protease and proteolytic chemicals such as cyanogen bromide and acids.
  • any suitable proteolytic reagent or method can be used for a method herein with a requirement that (1) at least one of the surrogate peptides produced by the proteolytic digestion process is defined by a specific peptide sequence, and (2) the proteolytic digestion process is reproducible in that the at least one surrogate peptide can be reliably be produced upon repeating the proteolytic digestion process with another identical or similar sample.
  • surrogate peptides are produced prior to an enrichment method. In certain embodiments, surrogate peptides are produced after an enrichment method.
  • surrogate peptides of a 14-3-3 protein are produced by enzymatic digestion (e.g.. protease digestion) of a sample using a suitable proteolytic enzyme.
  • enzymatic digestion e.g.. protease digestion
  • a proteolytic enzyme that can be used for a proteolytic digestion process herein include trypsin, LysN, LysC, Glu-C, Asp-N, ArgC, pepsin, proteinase K. elastase, thermolysin, papain or subtilisin. or any combination thereof.
  • a proteolytic enzyme comprises trypsin, or an isozyme thereof
  • surrogate peptides of a 14-3-3 protein are produced by chemical digestion.
  • a method of generating surrogate peptides includes contacting a sample or a sample enriched for one or more 14-3-3 proteins, with one or more suitable chemical reagents, non-limiting examples of which include an acid, cyanogen bromide, the like or combinations thereof.
  • a 14-3-3 protein e.g., 14-3-3i]
  • a chemical reagent can be any suitable chemical that produces reproducible and predicable fragments from a 14-3-3 protein.
  • a protein sequence can be cleaved with a specific chemical reagent such at least one predetermined peptide is generated.
  • Surrogate peptides produced in such a manner can be isolated and/or separated from a substrate using a suitable method.
  • bound proteins of an enriched sample are contacted with a proteolytic reagent to produce surrogate peptides.
  • a 14-3-3 protein e.g., 14-3- 3q
  • an antibody can be bound to an antibody and immunoprecipitated using protein G beads, followed by contacting the immunoprecipitated complexes with a proteolytic reagent.
  • This method often does not require elution of a 14-3-3 protein from a binding agent or from a substrate (e.g., protein G beads).
  • an enriched sample comprising a bound 14-3-3 protein is contacted with a proteolytic enzyme thereby producing surrogate peptide.
  • Surrogate peptides produced in such a manner can be isolated and/or separated from a substate using a suitable method.
  • a sample is not enriched for one or more 14-3-3 proteins and the sample is digested with a protease or chemical reagent to generate surrogate peptides prior to mass spectrometry.
  • a sample including all proteins, can be digested with a proteolytic enzyme or chemical reagent to produce a digest that includes a plurality of peptides.
  • the peptides in the digest may include one or more surrogate peptides of a 14-3-3q and/or other 14-3-3 protein family members.
  • a proteolytic digest can be enriched for one or more surrogate peptides.
  • a proteolytic digest can be enriched for one or more surrogate peptides derived from 14-3 -3 i or another 14-3-3 protein family members by contacting a proteolytic digest with a binding agent that specifically binds to a surrogate peptide of interest.
  • a sample can be enriched for one or more surrogate peptides using solid-phase extraction, precipitation, affinity enrichment, immunoaffinity enrichment, or combinations thereof to produce an enriched sample.
  • a sample is contacted with a binding agent (e.g., an antibody) that binds specifically to one or more surrogate peptides.
  • a sample is contacted with a binding agent (e.g., an antibody) that binds specifically to a surrogate peptide generated by proteolytic digestion of a 14-3-3q protein.
  • Bound surrogate peptides can then be isolated from a proteolytic digest using a suitable method.
  • bound complexes of one or more surrogate peptides are isolated by an affinity purification method.
  • an antibody-bound surrogate peptide can be immunoprecipitated using protein G beads, followed by washing, and optionally elution using a suitable method.
  • an antibody that binds specifically to a surrogate peptide comprises a monoclonal antibody, or binding fragment thereof.
  • surrogate peptides may include one or more tryptic peptides of 14-3-3 protein family members.
  • the surrogate peptide(s) when using trypsin to conduct a digestion, may be selected from try ptic peptides derived uniquely from 14-3-3q to differentiate 14-3-3 iq from other 14-3-3 protein family members in a sample, as well as from other serum proteins.
  • SEQ ID NOs: 2, 3, 7-15, and 17 are examples of surrogate peptides derived uniquely from 14-3-3q.
  • a presence or amount of a 14-3-3q protein in a biological sample can be determined by using LC-MS/MS following enrichment and/or enzymatic digestion.
  • one or more 14-3-3q surrogate peptides that are unique to 14-3-3q can be measured using MS (e.g., LC-MS/MS), and the amount of surrogate peptides of the 14-3-3i] protein may be used to quantify the amount of 14-3-3q protein in a biological sample.
  • a presence or amount of 14-3-3n in a sample is determined by determining a presence or amount of one or more of SEQ ID NOs: 2, 3, 7-15, and 17 in a sample (e.g., an enriched proteolytic digest) by mass spectrometry'.
  • a method may include measuring surrogate peptides shared among one or more 14-3-3 protein family member or fragments thereof.
  • a surrogate peptide may be shared among one or more 14-3-3 protein family members.
  • Measurements derived from surrogate peptides shared among one or more 14-3-3 protein family members may produce a measurement that is a composite measurement of the corresponding 14-3-3 protein family members and not from 14-3-31] protein alone.
  • SEQ ID NOs: 1, 4-6, and 16 in Table 1 provide surrogate peptides shared among one or more 14-3-3 protein family members.
  • measurements derived from a shared surrogate peptide may effectively measure solely 14-3-3q in serum when 14-3-3q is substantially more abundant than other 14-3-3 protein family members.
  • enrichment steps may be performed prior to LC-MS/MS.
  • the methods described herein and in further detail below' may be adapted to use any one of the surrogate peptides in Table 1.
  • a method may comprise ionizing one or more surrogate peptides of a 14-3-3q protein to generate one or more ions of the surrogate peptides of the 14-3-3q protein detectable by mass spectrometry 7 ; and determining the amount of the one or more ions of the surrogate peptides of the 14-3-3q protein by tandem mass spectrometry 7 .
  • generating one or more ions of the surrogate peptides of 14-3-31] protein detectable by mass spectrometry may include (a) generating a precursor ion of a surrogate peptide of 14- 3 -31 protein; (b) generating one or more fragment ions of the precursor ion; and (c) detecting the amount of one or more ions in step (a) or (b) or both to determine the amount of 14-3-31] protein in the sample.
  • the precursor ion of a surrogate peptide of a 14-3-3 protein family member has a m/z ratio of 408.7 ⁇ 0.5 and the product ions produced from the precursor ion have a m/z ratio of 703.3 ⁇ 0.5, 632.3 ⁇ 0.5, 503.3 ⁇ 0.5, 375.2 ⁇ 0.5, 304.2 ⁇ 0.5, 175.1 ⁇ 0.5, 185.1 ⁇ 0.5, 314.2 ⁇ 0.5, 442.2 ⁇ 0.5, or combinations thereof.
  • the product ions produced from the precursor ion have a m/z ratio selected from the group consisting of 703.3 ⁇ 0.5, 632.3 ⁇ 0.5, 503.3 ⁇ 0.5, and 185. l ⁇ 0.5.
  • the precursor ion of a surrogate peptide of a 14-3-3 protein family member has a m/z ratio of 516. ⁇ 0.5 and the product ions produced from the precursor ion have a m/z ratio of 868.4 ⁇ 0.5, 753.3 ⁇ 0.5, 638.3 ⁇ 0.5, 507.3 ⁇ 0.5, 436.2 ⁇ 0.5, 279.1 ⁇ 0.5. 394.1 ⁇ 0.5, 136.1 ⁇ 0.5, 251.1 ⁇ 0.5, or combinations thereof.
  • the product ions produced from the precursor ion have a m/z ratio selected from the group consisting of 868.4 ⁇ 0.5, 753.3 ⁇ 0.5, 507.3 ⁇ 0.5, and 279.1 ⁇ 0.5.
  • the precursor ion of a surrogate peptide of a 14-3-3 protein family member has a m/z ratio of 452.3. ⁇ 0.5 and the product ions produced from the precursor ion have a m/z ratio of 804.4 ⁇ 0.5, 691.4 ⁇ 0.5, 604.3 ⁇ 0.5, 517.3 ⁇ 0.5, 404.2 ⁇ 0.5, 275.2 ⁇ 0.5, 213.2 ⁇ 0.5, 258.1 ⁇ 0.5, 185.2 ⁇ 0.5, or combinations thereof. In some embodiments, the product ions produced from the precursor ion have a m/z ratio selected from the group consisting of 804.4 ⁇ 0.5, 691.4 ⁇ 0.5, 213.2 ⁇ 0.5, and 185.2 ⁇ 0.5.
  • the precursor ion of a surrogate peptide of a 14-3-3 protein family member has a m/z ratio of 433.2 ⁇ 0.5 and the product ions produced from the precursor ion have a m/z ratio of 764.3 ⁇ 0.5, 633.3 ⁇ 0.5, 562.2 ⁇ 0.5, 447.2 ⁇ 0.5, 276.2 ⁇ 0.5, 233.1 ⁇ 0.5, 373.2 ⁇ 0.5, 205.1 ⁇ 0.5, or combinations thereof.
  • the product ions produced from the precursor ion have a m/z ratio selected from the group consisting of 633.3 ⁇ 0.5, 562.2 ⁇ 0.5, 233.1 ⁇ 0.5, and 205.1 ⁇ 0.5.
  • the precursor ion of a surrogate peptide of a 14-3-3 protein family member has a m/z ratio of 634.3 ⁇ 0.5 and the product ions produced from the precursor ion have a m/z ratio of 1066.5 ⁇ 0.5, 967.5 ⁇ 0.5, 868.4 ⁇ 0.5, 739.4 ⁇ 0.5, 668.3 ⁇ 0.5. 202.1 ⁇ 0.5, 301.2 ⁇ 0.5, or combinations thereof.
  • the product ions produced from the precursor ion have a m/z ratio selected from the group consisting of 1066.5 ⁇ 0.5, 967.5 ⁇ 0.5, 868.4 ⁇ 0.5, and 301 ,2 ⁇ 0.5.
  • the precursor ion of a surrogate peptide of a 14-3-3 protein family member has a m/z ratio of 412.2 ⁇ 0.5 and the product ions produced from the precursor ion have a m/z ratio of 623.3 ⁇ 0.5. 476.3 ⁇ 0.5, 347.2 ⁇ 0.5, 234. l ⁇ 0.5, 147. l ⁇ 0.5, 312.2 ⁇ 0.5, 459.2 ⁇ 0.5, or combinations thereof.
  • the product ions produced from the precursor ion have a m/z ratio selected from the group consisting of 623.3 ⁇ 0.5, 476.3 ⁇ 0.5, 347.2 ⁇ 0.5, and 234.1 ⁇ 0.5.
  • the mass spectrometry is tandem mass spectrometry, liquid chromatography tandem mass spectrometry (LC-MS/MS), or 2 dimensional LC-MS/MS.
  • the sample is subjected to a purification step prior to mass spectrometry'.
  • the purification step may comprise liquid-liquid extraction of the sample, protein precipitation or dilution of the sample prior to mass spectrometry.
  • the sample is diluted into a solvent or solvent mixture that may be used for LC and/or MS (e.g. LC-MS/MS or 2D-LC-MS/MS).
  • a further purification step may comprise liquid chromatography, such as high-performance liquid chromatography (HPLC).
  • HPLC high-performance liquid chromatography
  • the chromatography comprises extraction and analytical liquid chromatography.
  • high turbulence liquid chromatography (HTLC) also known as high throughput liquid chromatography
  • HTLC high turbulence liquid chromatography
  • the liquid chromatography separation technique may include reverse phase separation or hydrophilic interaction liquid chromatography separation (HILIC).
  • the method may comprise purifying one or more surrogate peptides of a 14-3-3q protein using liquid chromatography; ionizing the one or more surrogate peptides of the 14-3-3q protein to generate one or more ions of the surrogate peptides of 14-3-3q protein detectable by mass spectrometry; and determining the amount of the one or more ions of the surrogate peptides of the 14-3 -3 r protein by tandem mass spectrometry.
  • generating one or more ions of the surrogate peptides of 14-3-3q protein detectable by mass spectrometry may include (a) generating a precursor ion of a surrogate peptide of 14-3-3 q protein; (b) generating one or more fragment ions of the precursor ion; and (c) detecting the amount of one or more ions in step (a) or (b) or both to determine the amount of 14-3-3q protein in the sample.
  • FIG. 1 In one embodiment of method 100 for determining the presence or amount of at least one biomarker of interest in a biological sample may be represented by FIG. 1 .
  • the method may include first providing a biological sample comprising a plurality of proteins including at least one biomarker of interest.
  • the biomarker of interest may be at least 14-3-3ty protein.
  • the biomarker of interest may be 14-3-3q protein and at least one other 14-3-3 protein family member.
  • the biological sample may be serum or plasma.
  • the method may further include adding a binding agent that binds specifically to 14-3-3q protein in the biological sample.
  • the binding agent is an antibody or an aptamer.
  • the biological sample, comprising at least one of the biomarkers of interest is treated with an internal standard.
  • the internal standard may be a stable isotope of 14-3-3q protein or a surrogate peptide of 14-3-31 ⁇ protein.
  • the biological sample may be subsequently treated with a proteolytic enzyme to produce a proteolytic digest comprising a plurality of peptides.
  • the plurality of peptides may include at least one surrogate peptide derived from 14-3-3p protein.
  • the peptides produced may include surrogate peptides derived from other 14-3- 3 protein family members.
  • the proteolytic enzy me may be trypsin.
  • a substitute proteolytic enzyme may be used in the methods described herein. In such an event, the cleavage of the 14-3-3q peptides in the biological sample may be different and thus alternate antibodies or aptamers may be employed.
  • the surrogate peptides derived from 14-3-3q protein are separated from other peptides in the proteolytic digest to produce an enriched sample.
  • the proteolytic digest can be enriched in the surrogate peptides derived from 14-3-3p protein using solid-phase extraction, precipitation, affinity enrichment, immunoaffinity enrichment, or combinations thereof to produce an enriched sample.
  • the antibodies used for immunoaffinity enrichment can specifically bind to 14-3-3 protein family members or fragments thereof.
  • the separation may include separating, based upon the antibody, via techniques such as precipitation or solid phase extraction.
  • separating the surrogate peptides derived from 14-3-3q protein bound to the binding agent prior to liquid chromatography may include (1) binding the binding agent to a solid phase substrate; (2) washing unbound peptides from the solid phase substrate; and (3) eluting the surrogate peptides derived from 14-3-3q protein bound to the binding agent from the solid phase substrate.
  • liquid chromatography may be optionally performed on the separated sample to purify the sample.
  • the liquid chromatography may include high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the chromatography comprises extraction and analytical liquid chromatography.
  • HTLC high turbulence liquid chromatography
  • the liquid chromatography separation technique may include reverse phase separation or hydrophilic interaction liquid chromatography separation (H1LIC).
  • the amount or quantify of 14-3-3q protein in the sample is measured using mass spectrometry.
  • the analytical technique used to measure the biomarker may be mass spectrometry, but the analytical technique used to measure the internal standard may not be mass spectrometry.
  • the internal standard may be measured by immunometric methods, colorimetric methods, electrochemical methods, or fluorometric methods.
  • the analytical technique used to measure the biomarker may not be mass spectrometry (but may be one of the other methods disclosed herein).
  • the mass spectrometry is tandem mass spectrometry, liquid chromatography tandem mass spectrometry (LC-MS/MS), or 2 dimensional LC-MS/MS.
  • the step of measuring the amount of 14-3-3q protein in the sample using mass spectrometry' may include the steps of (1) generating at least one precursor ion of the surrogate peptide of 14-3-3q protein; (2) generating one or more fragment ions of the precursor ion; and (3) detecting the presence or amount of the precursor ion generated in step (1) and/or the at least one or more fragment ions generated in step (2), or both, and relating the detected ions to the presence or amount of the 14-3-3q protein in the biological sample.
  • the precursor ions and/or fragment ions may be selected from any one of the ions listed in Table 2.
  • the at least one precursor ion is formed by methods such as atmospheric pressure chemical ionization (APCI) mode or Electrospray Ionization (ESI).
  • APCI atmospheric pressure chemical ionization
  • ESI Electrospray Ionization
  • the mode may be selected from either negative ion mode or positive ion mode.
  • positive ion mode ESI is utilized to produce the at least one precursor ion.
  • tandem mass spectrometry would generally include electrospray ionization of the liquid chromatography eluent to generate gas-phase precursor ions of the 14- 3-3q surrogate peptides.
  • the precursor ions would be isolated in the first stage of mass analysis and subsequently fragmented (or dissociated) to derive product ions (fragment ions), that would be subjected to a second stage of mass analysis.
  • Isolation of the precursor ion in the first stage of mass analysis can be performed using a quadrupole mass analyzer or ion trap mass analyzer. Fragmentation of the isolated precursor ion can be performed in a number of ways, such as by collision-induced dissociation (CID) or Electron-capture dissociation (ECD).
  • the second stage of mass analysis can be performed using any number of mass analyzers such as a quadrupole, ion trap, time-of-flight, or orbitrap.
  • Precursor and product ions which may be suitable for tandem mass spectrometry analysis of the 14-3-3q surrogate peptides are listed in Table 2.
  • the methods described herein may include a method according to FIG. 2.
  • the method 200 described in FIG. 2 may be a different workflow than the method described in FIG. 1.
  • the method includes providing a biological sample that includes a plurality of proteins including one or more 14-3-3 protein family members.
  • the one or more 14-3-3 protein family members can be 14-3-3i] protein and at least one other family member of 14-3-3 protein (e.g, 14-3-3
  • the biological sample may be subsequently treated with a proteolytic enzyme to produce a proteolytic digest comprising a plurality of peptides.
  • the plurality of peptides may include at least one surrogate peptide derived from the 14-3-3 protein family.
  • the plurality of peptides may include at least one surrogate peptide derived from 14-3-3q protein.
  • the peptides may include surrogate peptides derived from other 14-3-3 protein family members.
  • the proteolytic enzyme may be trypsin.
  • the biological sample is treated with an internal standard prior to proteolysis, wherein the internal standard contains proteolytic sites as well as stable isotopes of any one of the SEQ ID NOs: 1-17, such that once proteolyzed produce stable isotopes of the corresponding SEQ ID NOs: 1-17.
  • the method may further include adding a binding agent to the proteolytic digest.
  • the binding agent binds specifically to surrogate peptides of 14-3-3q protein and/or 14-3-3 protein family members.
  • the binding agent may bind specifically to 14-3-3q protein and at least one other 14-3-3 protein family member.
  • the binding agent is an anti-peptide antibody or an aptamer.
  • the anti-peptide antibody may bind with any surrogate peptide with at least 80 % identical to any one of SEQ ID NOs.: 1-17 provided in Table 1 (e.g, at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %. or 99 % Identity).
  • any surrogate peptide with at least 80 % identical to any one of SEQ ID NOs.: 1-17 provided in Table 1 (e.g, at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %,
  • the method may further include adding an internal standard to the biological sample prior to digestion of the sample.
  • the internal standard may be a stable isotope of 14-3-3q protein and/or other 14-3-3 protein family members.
  • the proteolytic digest comprising the plurality of peptides may include surrogate peptides of the stable isotope of 14-3-3q.
  • the method may comprise adding an internal standard to the enriched sample, wherein the internal standard includes a stable isotope of the surrogate peptide.
  • the stable isotope of the surrogate peptide can be proteolyzed to form a stable isotope surrogate peptide.
  • the method may comprise adding an internal standard to the enriched or proteolyzed sample, wherein the internal standard is a stable isotope of the surrogate peptide.
  • the method may include using two or more different stable isotope labeled internal standards.
  • two different stable isotope internal standards are used, that allow for the target analyte, first stable isotope labeled internal standard and second stable isotope internal standard to be distinguished from one other using mass spectrometry.
  • one stable isotope labeled internal standard could include a single label (e.g..
  • the stable isotope labeled internal standards are the same as the target analyte, but for the presence of the stable isotope label(s).
  • the first stable isotope labeled molecule can be a 14-3-3 protein family member containing a stable isotope and the second stable isotope labeled molecule can be a 14-3-3 protein family member containing a stable isotope distinguishable from the stable isotope on the first stable isotope labeled 14-3-3 protein family member.
  • a peptide is the target (e.g., used as a surrogate for detecting the presence of a protein)
  • the same principles apply.
  • the target analyte is 14-3-3q protein
  • the peptide used to determine the presence of 14-3-3q protein is YDDMASAMK (SEQ ID NO: 2)
  • the first stable isotope labeled molecule can be a YDDMASAMK (SEQ ID NO: 2) containing a stable isotope
  • the second stable isotope labeled molecule can be YDDMASAMK (SEQ ID NO: 2) containing a stable isotope distinguishable from the stable isotope on the first stable isotope labeled 14-3-3q protein.
  • the YDDMASAMK (SEQ ID NO: 2) containing the first stable isotope could have a stable isotope on a single amino acid
  • the YDDMASAMK (SEQ ID NO: 2) containing the second stable isotope could have a stable isotope on two amino acids or have a single amino acid with two stable isotopes. Variations of this labeling are possible, if the first and second stable isotope labeled molecules are distinguishable from one another and from the native molecules via mass spectrometry.
  • the method may include the step of enriching the proteolytic digest with surrogate peptides of 14-3-3q protein, and optionally one or more other surrogate peptides of 14-3-3 protein family members, in the proteolyzed digest to produce a sample enriched with surrogate peptides of l4-3-3q protein.
  • the enrichment methods may include immunoaffinity enrichment, affinity enrichment, and abundant protein depletion.
  • the sample enriched with surrogate peptides of 14-3-3q protein, and optionally one or more other surrogate peptides of 14-3-3 protein family members, can be purified.
  • liquid chromatography may be performed on the enriched sample to purify the sample.
  • Liquid chromatography may include high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • liquid chromatography comprises extraction and analytical liquid chromatography.
  • HTLC high turbulence liquid chromatography
  • the liquid chromatography separation technique may include reverse phase separation or hydrophilic interaction liquid chromatography separation (HILIC).
  • the amount of 14-3-3 protein in the sample is measured using mass spectrometry.
  • the amount of surrogate peptides of 14-3-3q protein can be measured using mass spectrometry to quantify the amount of 14-3-3q protein in the sample.
  • the step of measuring the amount of 14-3-3q protein and/or other 14- 3-3 protein family members in the sample using mass spectrometry 7 may include the steps of (1) generating at least one precursor ion of a surrogate peptide of 14-3-3q protein and optionally 14-3-3 protein family members; (2) generating one or more fragment ions of the precursor ion; and (3) detecting the presence or amount of the precursor ion generated in step (1) and/or the one or more fragment ions generated in step (2), or both, and relating the detected ions to the presence or amount of the 14-3-3q protein in the biological sample.
  • the precursor ions and/or fragment ions may be selected from any one of the ions listed in Table 2.
  • the analytical technique used to measure the biomarker may be mass spectrometry, but the analytical technique used to measure the internal standard may not be mass spectrometry.
  • the internal standard may be measured by immunometric methods, colorimetric methods, electrochemical methods, or fluorometric methods.
  • the analytical technique used to measure the biomarker may not be mass spectrometry (but may be one of the other methods disclosed herein).
  • the mass spectrometry is tandem mass spectrometry, liquid chromatography tandem mass spectrometry (LC-MS/MS), or 2 dimensional LC-MS/MS.
  • the native 14-3-3q protein in a biological sample is measured.
  • the method for detecting 14-3-3q protein in a biological sample may include generating a precursor ion of the 14-3-3q protein and detecting the amount of precursor ion of the 14-3-3q protein to determine the amount of 14-3-3q protein in the sample by mass spectrometry'.
  • a surrogate peptide of 14-3-3q protein is not used.
  • the method may include purifying the sample prior to mass spectrometry, for example, using liquid chromatography.
  • one or more precursor ions of the 14-3-3q are generated.
  • the method comprises enriching the sample in the 14-3-3q protein prior to generating precursor ions.
  • inventions comprise systems.
  • the disclosure herein provides a system for determining the presence and/or amount of 14-3-3q protein in a biological sample, the system comprising: a device for providing a test sample comprising a body fluid; and a station for analyzing the body fluid by mass spectrometry to determine the presence or amount of 14-3-3q protein in the biological sample.
  • the system may comprise: a station for providing a biological sample believed to contain 14-3-3q protein wherein the biological sample comprises serum or plasma; a station for at least partially purify ing the 14-3-3q protein from the biological sample; a station for chromatographically separating the 14-3-3q protein from the biological sample; and a station for measuring the 14-3-3q protein by mass spectrometry to determine the presence or amount of the at least one biomarker of interest in the biological sample.
  • the mass spectrometry' is operated in an atmospheric pressure chemical ionization (APCI) mode.
  • APCI atmospheric pressure chemical ionization
  • at least one of the stations is automated and/or controlled by a computer.
  • at least some of the steps are automated such that little to no manual intervention is required.
  • the station for chromatographic separation comprises at least one apparatus to perform liquid chromatography (LC).
  • the station for liquid chromatography comprises a column for extraction chromatography.
  • the station for liquid chromatography comprises a column for analytical chromatography.
  • the column for extraction chromatography and analytical chromatography comprises a single station or single column.
  • liquid chromatography is used to purify the biomarker of interest from other components in the sample that co-purify with the biomarker of interest after extraction or dilution of the sample.
  • the separation technique used in the liquid chromatography may include reverse phase separation or HILIC.
  • the system may also include a station for analyzing the chromatographically separated one or more biomarkers of interest by mass spectrometry to determine the presence or amount of the one or more biomarkers in the test sample.
  • tandem mass spectrometry is used (MS/MS).
  • the station for tandem mass spectrometry comprises an Applied Biosystems API4000 or API5000 or thermo quantum or Agilent 7000 triple quadrupole mass spectrometer or an Applied Biosystems API5500 or API6500 triple quadrupole or thermo Q-Exactive mass spectrometer.
  • the system may also comprise a station for partially purifying at least one biomarker of interest from the biological sample and/or diluting the sample.
  • the station for purifying comprises a station for supported liquid extraction (SLE) and/or liquidliquid extraction (e.g, for smaller molecules and/or lipids).
  • the station for liquid-liquid extraction may comprise equipment and binding agents for addition of solvents to the sample and removal of waste fractions.
  • the station for purifying may comprise protein precipitation.
  • the station for purifying may comprise immunoaffinity enrichment (e.g., for proteins or peptides).
  • the methods and systems of the present invention may comprise multiple liquid chromatography steps.
  • a two- dimensional liquid chromatography (LC) procedure is used.
  • the method and systems of the present invention may comprise transferring the sample, or surrogate peptides derived from the sample, from a LC extraction column to an analytical column.
  • the transferring from the extraction column to an analytical column is done by a heart-cuting technique.
  • transfer from the extraction column to an analytical column by a chromatofocusing technique.
  • transfer from the extraction column to an analytical column may be done by a column switching technique. These transfer steps may be done manually or may be part of an on-line system.
  • the column used for extraction liquid chromatography may be varied depending on the biomarker of interest.
  • the extraction column is a functionalized silica or polymer-silica hybrid or polymeric particle or monolithic silica stationary phase, such as a Poroshell SBC- 18 column.
  • the column used for analytical liquid chromatography may be varied depending on the analyte and/or the column that was used for the extraction liquid chromatography step.
  • the analytical column comprises particles having an average diameter of about 5 pm.
  • the analytical column is a functionalized silica or polymer-silica hybrid, or a polymeric particle or monolithic silica stationary phase, such as a phenyl-hexyl functionalized analytical column.
  • reverse phase separation may include a column including particles functionalized with hydrocarbon chains (Cl 2 to Cl 8) or aromatic groups such as phenyl or bi-phenyl.
  • the weak mobile phase may include predominately water and the strong mobile phase may include one or more organic solvents such as acetonitrile and methanol.
  • the mass spectrometer may comprise a tandem mass spectrometer (MS/MS).
  • the tandem MS/MS spectrometry comprises a triple quadrupole tandem mass spectrometer.
  • the tandem mass spectrometer may be a hybrid mass spectrometer, such as a quadrupole-orbit rap or a quadrupole-time-of-flight mass spectrometer.
  • the tandem MS/MS may be operated in a variety of modes.
  • the tandem MS/MS spectrometer is operated in an atmospheric pressure chemical ionization (APCI) mode or Electrospray Ionization (ESI).
  • APCI atmospheric pressure chemical ionization
  • ESI Electrospray Ionization
  • the electrospray ionization maybe performed in either negative ion mode or positive ion mode.
  • the quantification of the analytes and internal standards is performed in the selected reaction monitoring mode (SRM).
  • SRM reaction monitoring mode
  • the systems and methods of the present invention may. in certain embodiments, provide for a multiplexed or high throughput assay.
  • certain embodiments may comprise a multiplexed liquid chromatography tandem mass spectrometry (LC-MS/MS) or two-dimensional or tandem liquid chromatography -tandem mass spectrometry (LC)-LC- MS/MS) methods for the proteomic analysis.
  • LC-MS/MS multiplexed liquid chromatography tandem mass spectrometry
  • LC two-dimensional or tandem liquid chromatography -tandem mass spectrometry
  • MS liquid chromatography tandem mass spectrometry
  • a tandem MS/MS system is used.
  • the precursor ion is selected following ionization, and that precursor ion is subjected to fragmentation to generate product ions (z.e., fragment), whereby one or more product ions are subjected to a second stage of mass analysis for detection.
  • the second stage of mass analysis could be performed using any number of mass analyzers such as quadrupole, ion trap, time-of-flight, or orbitrap.
  • Precursor and product ions which may be suitable for tandem mass spectrometry analysis of 14-3-3q surrogate peptides are listed in Table 2.
  • a first step may include electrospray ionization of the liquid chromatography eluent to generate gas-phase precursor ions of the 14-3-3q surrogate peptides.
  • Those precursor ions may be isolated in the first stage of mass analysis and subsequently fragmented (or dissociated) to derive product ions, that would be subjected to a second stage of mass analysis.
  • Isolation of the precursor ion in the first stage of mass analysis may be performed using a quadrupole mass analyzer or ion trap mass analyzer. Fragmentation of the isolated precursor ion may be performed in a number of ways including collision-induced dissociation (CID) or electron capture dissociation (ECD).
  • the second stage of mass analysis may be performed using any number of mass analyzers including quadrupole, ion-trap, time- of-flight, or orbitrap.
  • the analyte of interest may then be detected and/or quantified based upon the amount of the characteristic transitions measured by tandem MS.
  • the tandem mass spectrometer comprises a triple quadrupole mass spectrometer.
  • the tandem mass spectrometer is operated in a positive ion Atmospheric Pressure Chemical Ionization (APCI) mode.
  • APCI Positive ion Atmospheric Pressure Chemical Ionization
  • the quantification of the analytes is performed in the selected reaction monitoring mode (SRM).
  • SRM reaction monitoring mode
  • an internal standard may be added to the sample and the internal standard and the analytes quantification is performed in SRM.
  • the back-calculated amount of each analyte in each sample may be determined by comparison of unknown sample response or response ratio when employing internal standardization to calibration curves generated by spiking a known amount of purified analyte material into a standard test sample, e.g, charcoal stripped human serum.
  • calibrators are prepared at know n concentrations and analyzed as per the biomarker methodology to generate a response or response ratio when employing internal standardization versus concentration calibration curve.
  • the temperature for heating the sample during ionization may, in alternate embodiments range from 100 °C to about 1000 °C and includes all ranges therein.
  • the dehydration step is performed within the interface of the mass spectrometer employed in APCI or electrospray mode at 500 degrees C ⁇ 100 degrees.
  • the sample is heated for several microseconds at the interface for dehydration to occur.
  • the heating step is done for less than 1 second, or less than 100 milliseconds (msec), or less than 10 msec, or less than 1 msec, or less than 0.1 msec, or less than 0.01 msec, or less than 0.001 msec.
  • FIG. 3 shows an embodiment of a system (302) of the present invention.
  • the system may comprise a station for processing a sample comprising serum or plasma (304) that may comprise a biomarker of interest into sampling containers (e.g.. 96 well microtiter assay wells).
  • the station for processing the sample may include components for adding at least one of a quantity control (QC) and/or calibration standard to the bodily fluid.
  • the station for processing the sample may include performing a proteolytic digest and adding anti-peptide antibodies to the bodily fluid.
  • the sample is aliquoted into a container or containers to facilitate extraction at an extraction station (306) of the biomarker of interest.
  • the station for aliquoting may comprise receptacles to discard the portion of the biological sample that is not used in the analysis.
  • the extraction station (306) may optionally comprise a station for adding an internal standard to the sample.
  • the extraction station (306) may comprise a station for adding anti-peptide antibodies to the bodily fluid to purify or enrich the sample.
  • the internal standard comprises the biomarker of interest labeled with a nonnatural isotope.
  • the station for extraction and/or adding an internal standard may comprise safety features to facilitate adding an isotopically labeled internal standard solutions to the sample.
  • the system may comprise a station for aliquoting portions of the extracted sample (308) for measurement of a normalization marker and measurement of the biomarker of interest.
  • the system may also comprise a station (310) for measuring the normalization marker with a technique that is independent of mass spectrometry.
  • the technique used will depend on the nature of the normalization (normalizing) standard, but may be one of any of the techniques known to one of skill in the art.
  • the system may also comprise a station for further processing of the biomarker.
  • the system may also, in some embodiments, comprise a station (312) for purification steps such as supported liquid extraction, liquid-liquid extraction, protein precipitation and/or dilution of the sample.
  • the system may also comprise a station for liquid chromatography (e.g., HPLC) of the sample (314).
  • the station for liquid chromatography may comprise an extraction liquid chromatography column.
  • the station for liquid chromatography may comprise a column comprising the stationary phase, as well as containers or receptacles comprising solvents that are used as the mobile phase.
  • the mobile phase comprises a gradient of methanol and water, acetonitrile and water, or other miscible solvents with aqueous volatile buffer solutions.
  • the station may comprise the appropriate lines and valves to adjust the amounts of individual solvents being applied to the column or columns.
  • the station may comprise a means to remove and discard those fractions from the LC that do not comprise the biomarker of interest.
  • the fractions that do not contain the biomarker of interest are continuously removed from the column and sent to a waste receptacle for decontamination and to be discarded.
  • the system may also comprise an analytical LC column (314). The analytical column may facilitate further purification and concentration of the biomarker of interest as may be required for further characterization and quantification.
  • the system may comprise a station for characterization and quantification of the biomarker of interest.
  • the system may comprise a station for mass spectrometry (MS) (316) of the biomarker.
  • the station for mass spectrometry comprises a station for tandem mass spectrometry (MS/MS).
  • the station for characterization and quantification may comprise a station for data analysis (318) for the biomarker and the normalization marker and/or a computer (320) and software for analysis of the results.
  • the analysis comprises both identification and quantification of the biomarker of interest.
  • one or more of the purification or separation steps can be performed “on-line.’'
  • the term “on-line” refers to purification or separation steps that are performed in such a way that the test sample is disposed, e.g. injected, into a system in which the various components of the system are operationally connected and, in some embodiments, in fluid communication with one another.
  • the on-line system may comprise an autosampler for removing aliquots of the sample from one container and transferring such aliquots into another container.
  • an autosampler may be used to transfer the sample after extraction onto an LC extraction column.
  • the on-line system may comprise one or more injection ports for injecting the fractions isolated from the LC extraction columns onto the LC analytical column. Additionally, or alternatively, the on-line system may comprise one or more injection ports for injecting the LC purified sample into the MS system.
  • the on-line system may comprise one or more columns, including but not limited to, an extraction column, including an HTLC extraction column, and in some embodiments, an analytical column.
  • the system may comprise a detection system, e.g., a mass spectrometer system.
  • the on-line system may also comprise one or more pumps; one or more valves; and necessary plumbing. In such “on-line” systems, the test sample and/or analytes of interest can be passed from one component of the system to another without exiting the system, e.g., without having to be collected and then disposed into another component of the system.
  • the on-line purification or separation method can be automated. In such embodiments, the steps can be performed without the need for operator intervention once the process is set-up and initiated.
  • the system, or portions of the system may be controlled by a computer or computers (120).
  • the present invention may comprise software for controlling the various components of the system, including pumps, valves, autosamplers, and the like. Such software can be used to optimize the extraction process through the precise timing of sample and solute additions and flow rate.
  • the computer architecture may be a server computer, workstation, desktop computer, laptop, tablet, network appliance, personal digital assistant (“PDA”), e-reader, digital cellular phone, or other computing device, and may be utilized to execute any aspects of the software components presented herein.
  • PDA personal digital assistant
  • the computer may include a baseboard, or “motherboard.” which is a printed circuit board to which a multitude of components or devices may be connected by way of a system bus or other electrical communication paths.
  • a baseboard or “motherboard.”
  • CPUs central processing units
  • the CPUs may be standard programmable processors that perform arithmetic and logical operations necessary for the operation of the computer.
  • Switching elements may generally include electronic circuits that maintain one of two binary states, such as flip-flops, and electronic circuits that provide an output state based on the logical combination of the states of one or more other switching elements, such as logic gates. These basic switching elements may be combined to create more complex logic circuits, including registers, adders-subtractors, arithmetic logic units, floating-point units, and the like.
  • the chipset provides an interface between the CPUs and the remainder of the components and devices on the baseboard.
  • the chipset may provide an interface to a random access memory' (“RAM”), used as the main memory' in the computer.
  • the chipset may further provide an interface to a computer-readable storage medium such as a read-only memory (“ROM”) or non-volatile RAM (“NVRAM”) for storing basic routines that help to startup the computer and to transfer information between the various components and devices.
  • ROM or NVRAM may also store other software components necessary' for the operation of the computer 1000 in accordance with the embodiments described herein.
  • the computer may operate in a networked environment using logical connections to remote computing devices and computer systems through a network, such as the local area network.
  • the chipset may include functionality for providing network connectivity 7 through a NIC, such as a gigabit Ethernet adapter.
  • the NIC can connect the computer to other computing devices over the network. It should be appreciated that multiple NICs may be present in the computer, connecting the computer to other types of networks and remote computer systems.
  • the computer may be connected to a mass storage device that provides non-volatile storage for the computer.
  • the mass storage device may store system programs, application programs, other program modules, and data, which have been described in greater detail herein.
  • the mass storage device may be connected to the computer through a storage controller connected to the chipset.
  • the mass storage device may consist of one or more physical storage units.
  • the storage controller may interface with the physical storage units through a serial attached SCSI ("SAS") interface, a serial advanced technology attachment (“SATA”) interface, a fiber channel (“FC”) interface, or other type of interface for physically connecting and transferring data between computers and physical storage units.
  • SAS serial attached SCSI
  • SATA serial advanced technology attachment
  • FC fiber channel
  • the computer may store data on the mass storage device by transforming the physical state of the physical storage units to reflect the information being stored.
  • the specific transformation of physical state may depend on various factors, in different implementations of this description. Examples of such factors may include, but are not limited to, the technology used to implement the physical storage units, whether the mass storage device is characterized as primary or secondary storage, and the like.
  • the computer may store information to the mass storage device by issuing instructions through the storage controller to alter the magnetic characteristics of a particular location within a magnetic disk drive unit, the reflective or refractive characteristics of a particular location in an optical storage unit, or the electrical characteristics of a particular capacitor, transistor, or other discrete component in a solid-state storage unit.
  • Other transformations of physical media are possible without departing from the scope and spirit of the present description, with the foregoing examples provided only to facilitate this description.
  • the computer may further read information from the mass storage device by detecting the physical states or characteristics of one or more particular locations within the physical storage units.
  • the computer may have access to other computer-readable storage media to store and retrieve information, such as program modules, data structures, or other data.
  • computer-readable storage media can be any available media that provides for the storage of non-transitory data and that may be accessed by the computer.
  • Computer-readable storage media may include volatile and non-volatile, removable and non-removable media implemented in any method or technology.
  • Computer-readable storage media includes, but is not limited to, RAM, ROM, erasable programmable ROM (“EPROM”), electrically-erasable programmable ROM (“EEPROM”), flash memory or other solid-state memory technology, compact disc ROM (“CD-ROM”), digital versatile disk (“DVD”), high definition DVD (“HD-DVD”), BLU- RAY, or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store the desired information in a non-transitory fashion.
  • the mass storage device may store an operating system utilized to control the operation of the computer.
  • the operating system comprises the LINUX operating system.
  • the operating system comprises the WINDOWS® SERVER operating system from MICROSOFT Corporation.
  • the operating system may comprise the UNIX or SOLARIS operating systems. It should be appreciated that other operating systems may also be utilized.
  • the mass storage device may store other system or application programs and data utilized by the computer. The mass storage device might also store other programs and data not specifically identified herein.
  • the mass storage device or other computer-readable storage media is encoded with computer-executable instructions which, when loaded into the computer, transforms the computer from a general-purpose computing system into a specialpurpose computer capable of implementing the embodiments described herein.
  • These computer-executable instructions transform the computer by specifying how the CPUs transition between states, as described above.
  • the computer has access to computer-readable storage media storing computer-executable instructions which, when executed by the computer, perform the various routines described above in the methods of detecting 14-3-3rp
  • the computer might also include computer-readable storage media for performing any of the other computer-implemented operations described herein.
  • the computer may also include one or more input/output controllers for receiving and processing input from a number of input devices, such as a keyboard, a mouse, a touchpad, a touch screen, an electronic stylus, or other type of input device. Similarly, the input/output controller may provide output to a display, such as a computer monitor, a flat-panel display, a digital projector, a printer, a plotter, or other ty pe of output device. It will be appreciated that the computer may not include all of the components described above, may include other components that are not explicitly described, or may utilize an architecture completely different than that described herein. It should also be appreciated that many computers, might be utilized in combination to embody aspects of the various technologies disclosed herein.
  • the steps in the method and the stations comprising the system may be on-line, in certain embodiments, some or all of the steps may be performed “off-line/’
  • the term “off-line” refers to a purification, separation, or extraction procedure that is performed separately from previous and/or subsequent purification or separation steps and/or analysis steps.
  • the analytes of interest typically are separated, for example, on an extraction column or by liquid/liquid extraction, from the other components in the sample matrix and then collected for subsequent introduction into another chromatographic or detector system.
  • Off-line procedures typically require manual intervention on the part of the operator.
  • Liquid chromatography may, in certain embodiments, comprise high turbulence liquid chromatography or high throughput liquid chromatography (HTLC). See, e.g, Zimmer et al., J. Chromatogr. A 854:23-35 (1999); see also, U.S. Pat. Nos. 5,968.367; 5,919,368;
  • HTLC high turbulence liquid chromatography
  • samples may be extracted using an HTLC extraction cartridge which captures the analyte, then eluted and chromatographed on a second HTLC column or onto an analytical HPLC column prior to ionization.
  • an HTLC extraction cartridge which captures the analyte
  • chromatographed on a second HTLC column or onto an analytical HPLC column prior to ionization Because the steps involved in these chromatography procedures can be linked in an automated fashion, the requirement for operator involvement during the purification of the analyte can be minimized.
  • the use of a high turbulence liquid chromatography sample preparation method can eliminate the need for other sample preparation methods including liquid-liquid extraction.
  • the test sample e.g., a biological fluid
  • the test sample e.g., a biological fluid
  • the test sample e.g., a biological fluid
  • the sample in a typical high turbulence or turbulent liquid chromatography system, may be injected directly onto a narrow (e.g., 0.5 mm to 2 mm internal diameter by 20 to 50 mm long) column packed with large (e.g., > 25 micron) particles.
  • a flow rate e.g., 3-500 mL per minute
  • the relatively narrow width of the column causes an increase in the velocity of the mobile phase.
  • the large particles present in the column can prevent the increased velocity from causing back pressure and promote the formation of vacillating eddies between the particles, thereby creating turbulence within the column.
  • the analyte molecules may bind quickly to the particles and typically do not spread out, or diffuse, along the length of the column. This lessened longitudinal diffusion typically provides better, and more rapid, separation of the analytes of interest from the sample matrix. Further, the turbulence within the column reduces the friction on molecules that typically occurs as they travel past the particles. For example, in traditional HPLC, the molecules traveling closest to the particle move along the column more slowly than those flowing through the center of the path between the particles. This difference in flow rate causes the analyte molecules to spread out along the length of the column. When turbulence is introduced into a column, the friction on the molecules from the particle is negligible, reducing longitudinal diffusion.
  • the liquid chromatography method may comprise reverse phase separation.
  • particles may be functionalized with hydrocarbon chains (C12 or Cl 8) or aromatic groups (phenyl or bi-phenyl).
  • the weak mobile phase may be comprised predominantly of water and the strong mobile phase may be comprised of one or more organic solvents such as acetonitrile and methanol.
  • organic solvents such as acetonitrile and methanol.
  • ion pairing binding agents such as formic acid or acetic acid, may be added to the weak and or strong mobile phases.
  • the liquid chromatography method may include hydrophilic interaction liquid chromatography (HILIC).
  • HILIC separation may include particles functionalized with silanol groups, amide groups, or cyano groups.
  • the weak mobile phase may be comprised predominately of aprotic organic solvents such as acetonitrile or ethyl acetate.
  • the strong mobile phase may be comprised predominately of water and/or protic organic solvents such as methanol.
  • ion pairing binding agents such as ammonium formate or ammonium acetate may be added to the weak and/or strong mobile phase.
  • the ion pairing reagent may be added to both the weak and strong mobile phase.
  • the methods and systems of the present invention may use mass spectrometry to detect and quantify the biomarker of interest.
  • mass spectrometry or “MS” as used herein generally refer to methods of filtering, detecting, and measuring ions based on their mass-to- charge ratio, or “m/z.”
  • MS techniques one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrometer where, due to a combination of electric fields, the ions follow a path in space that is dependent upon mass (“m”) and charge (“z”).
  • the mass spectrometer uses a “quadrupole” system.
  • a “quadrupole” or “quadrupole ion trap” mass spectrometer ions in an oscillating radio frequency (RF) field experience a force proportional to the direct current (DC) potential applied between electrodes, the amplitude of the RF signal, and m/z.
  • the voltage and amplitude can be selected so that only ions having a particular m/z travel the length of the quadrupole, while all other ions are deflected.
  • quadrupole instruments can act as both a “mass filter” and as a “mass detector” for the ions injected into the instrument.
  • tandem mass spectrometry is used. See, e.g., U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus for Tandem Mass Spectrometry,” which is hereby incorporated by reference in its entirety. Further, the selectivity of the MS technique can be enhanced by using “tandem mass spectrometry,” or “MS/MS.” Tandem mass spectrometry (MS/MS) is the name given to a group of mass spectrometric methods wherein “parent or precursor” ions generated from a sample are fragmented to yield one or more “fragment or product” ions, which are subsequently mass analyzed by a second MS procedure.
  • MS/MS methods are useful for the analysis of complex mixtures, especially biological samples, in part because the selectivity of MS/MS can minimize the need for extensive sample clean-up prior to analysis.
  • precursor ions are generated from a sample and passed through a first mass filter to select those ions having a particular mass-to-charge ratio. These ions are then fragmented, typically by collisions with neutral gas molecules in a suitable ion containment device, to yield product (fragment) ions, the mass spectrum of which is recorded by an electron multiplier detector.
  • the product ion spectra so produced are indicative of the structure of the precursor ion, and the two stages of mass filtering can eliminate ions from interfering species present in the conventional mass spectrum of a complex mixture.
  • the methods and systems of the present invention use a triple quadrupole MS/MS (see e.g., Yost, Enke in Ch. 8 of Tandem Mass Spectrometry, Ed. McLafferty. pub. John Wiley and Sons. 1983).
  • Triple quadrupole MS/MS instruments typically consist of two quadrupole mass filters separated by a fragmentation means.
  • the instrument may comprise a quadrupole mass filter operated in the RF only mode as an ion containment or transmission device.
  • the quadrupole may further comprise a collision gas at a pressure of between 1 and 10 millitorr.
  • tandem mass spectrometers are also known and can be used in the methods and systems of the present invention including various combinations of orbitrap analyzers and quadrupole filters.
  • These hybrid instruments often comprise high resolution orbitrap analyzers (see e.g., Hu Q, Noll RJ, Li H, Makarov A. Hardman M. Graham Cooks R. The Orbitrap: a new mass spectrometer. J Mass Spectrum. 2005;40(4):430-443) for the second stage of mass analysis.
  • Use of high-resolution mass analyzer may be highly effective in reducing chemical noise to very low levels.
  • ions can be produced using a variety of methods including, but not limited to. electron ionization, chemical ionization, fast atom bombardment, field desorption, and matrix-assisted laser desorption ionization (‘'MALDI”), surface enhanced laser desorption ionization (“SELDI”), photon ionization, electrospray ionization, and inductively coupled plasma.
  • 'MALDI matrix-assisted laser desorption ionization
  • SELDI surface enhanced laser desorption ionization
  • photon ionization photon ionization
  • electrospray ionization electrospray ionization
  • inductively coupled plasma inductively coupled plasma.
  • CID collision-induced dissociation
  • precursor ions gain energy through collisions with an inert gas and subsequently fragment by a process referred to as “unimolecular decomposition.”
  • Sufficient energy must be deposited in the precursor ion so that certain bonds within the ion can be broken due to increased vibrational energy.
  • ECD electron-capture dissociation
  • disulfide bonds may be specifically cleaved. Fragmentation may be fast and specific.
  • labile post-translational modifications and non-covalent bonds often remain intact after bond dissociation during ECD.
  • ECD may provide a high amount of sequence coverage as compared to CID. For example, at high electron energies, ECD may be able to distinguish between leucine and isoleucine.
  • the presently disclosed 2D-LC-MS/MS methods include multiplexed sample preparation procedures. For example, in certain embodiments dialysis of the sample is performed using a 96 well plate having a dialysis membrane in each well or multiple sample tubes. Additionally, or alternatively, the multiplex system may comprise staggered multiplexed LC and MS sample inlet systems. Also, the methods and systems of the present invention may comprise multiple column switching protocols, and/or heart-cutting (LC-LC or 2D-LC) techniques, and/or LC separations prior to MS detection.
  • multiplex system may comprise staggered multiplexed LC and MS sample inlet systems.
  • the methods and systems of the present invention may comprise multiple column switching protocols, and/or heart-cutting (LC-LC or 2D-LC) techniques, and/or LC separations prior to MS detection.
  • the methods and systems of the present invention may include a multiplexed two-dimensional liquid chromatographic system coupled with a tandem mass spectrometer (MS/MS) system, for example a triple quadrupole MS/MS system.
  • MS/MS tandem mass spectrometer
  • Such embodiments provide for staggered, parallel sample input into the MS system.
  • samples may each be applied to individual extraction columns. Once the samples have each run through the extraction column, they may each be transferred directly (e.g., by column switching) to a second set of analytical columns. As each sample elutes from the analytical column, it may be transferred to the mass spectrometer for identification and quantification.
  • a plurality of analytes can be analyzed simultaneously or sequentially by the presently disclosed LC-MS/MS and 2D-LC-MS/MS methods.
  • Exemplary analytes amenable to analysis by the presently disclosed methods include, but are not limited to, peptides, steroid hormones, nucleic acids, vitamins and the like.
  • One of ordinary skill in the art would recognize after a review of the presently disclosed subject matter that other similar analytes could be analyzed by the methods and systems disclosed herein.
  • the methods and systems may be used to quantify steroid hormones, protein and peptide hormones, peptide and protein biomarkers, drugs of abuse and therapeutic drugs.
  • optimization of key parameters for each analyte can be performed using a modular method development strategy' to provide highly tuned bioanalytical assays.
  • certain steps may be varied depending upon the analyte being measured as disclosed herein.
  • embodiments of the methods and systems of the present invention may provide equivalent sensitivity' attainable for many of the analytes being measured using much less sample. For example, through using this optimization procedure, an LLOQ of about 10 nmol/L for detection of a peptide biomarker for dried plasma corresponding to about 20 pL of liquid plasma. Such small sample sizes render sampling (often by finger-prick) much more accessible.
  • YDDMASAMK SEQ ID No. 6: VISSIEQK
  • SEQ ID No. 7 TMADGNEK
  • SEQ ID No. 12 EAFEISK
  • Stable-isotope labeled peptide w'ere labeled with [ 13 C6, 15 N2]-lysine or with [ 13 C6. 15 N4]-arginine.
  • the purity' of all synthesized peptides was verified to be >95% by high- performance liquid chromatography (215 nm) and concentrations were assigned by amino acid analysis.
  • All peptides were initially reconstituted in water with 0.1% (v/v) formic acid and 0.001% (w/v) ZWITTERGENT® 3-16 detergent at a concentration of 100 pg/mL, then combined and diluted in the same solvent to produce a stable-isotope labeled peptide solution with each at a final, working concentration of 20 ng/mL.
  • Calibrators were prepared for the mass spectrometer.
  • the calibrators were prepared using recombinant 14-3-3p spiked into phosphate buffered saline (PBS) containing 20 mg/mL human serum albumin (HSA) or 20 mg/mL bovine serum albumin (BSA).
  • PBS phosphate buffered saline
  • HSA human serum albumin
  • BSA bovine serum albumin
  • the recombinant 14-3-3p had >98% purity as verified by SDS-PAGE (Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis) and concentrations were determined using a Bradford assay.
  • monoclonal rabbit, anti-14-3-3 pan antibody was used for enrichment of the sample.
  • the monoclonal rabbit, anti-14-3-3 pan antibody was biotinylated and conjugated to magnetic beads coated with streptavidin for enrichment of 14-3-3p and other 14-3-3 protein family members.
  • the immunogen used to produce this antibody was a peptide from the conserved region on the C-terminus of 14-3-3 family members (i.e., SEQ ID NO. 18).
  • Monoclonal mouse anti-peptide antibodies were developed for the four tryptic peptides of 14-3-3p (see FIG. 4, SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 12) and were denoted as anti-YDD (SEQ ID No. 2), anti -VIS (SEQ ID No. 6), anti-TMA (SEQ ID No. 7), and anti-EAF (SEQ ID No. 12) antibodies based on the first 3 amino acids of the corresponding tryptic peptide. Each anti-peptide antibody was conjugated to magnetic Protein G beads for enrichment of the corresponding peptide following trypsin digestion.
  • the mass spectrometer was operated in positive electrospray ionization mode using a spray voltage of 2500 V, curtain gas of 40 psi, source gas 1 and 2 of 60 psi, and source temperature of 500 °C.
  • Matching selected reaction monitoring (SRM) transitions were acquired for each natural and stable-isotope labeled peptide with 5 msec dwell times; unit resolution for both QI and Q3; EP, CXP, and Q0D settings of 10 V; and optimized collision energies at a CAD gas setting of 9.
  • Data analysis and quantitation was performed in Skyline v24.1 using the total area of all transitions that did not have an identifiable chromatographic interferent. External calibration curves were produced with a linear, 1/x-weighted regression.
  • Example 1 (Workflow 1)
  • the following example provides a method of quantifying 14-3-3 proteins using surrogate peptide enrichment.
  • the sample was digested with a digestion aid to produce surrogate peptides of the target analyte followed by enrichment of the target analyte.
  • Serum samples and calibrators (1.58 - 200 ng/mL 14-3-3p) were digested to produce surrogate peptides of 14-3-3 proteins with subsequent enrichment of the resulting 14- 3-3 surrogate peptides using anti-peptide antibodies. Initially, 200 uL of sample was diluted
  • TPCK-treated bovine trypsin 800 pg (trypsin treated with N- tosyl-L-phenylalanine chloromethyl ketone (TPCK) was added to denatured samples and allowed to digest for 30 min at 37 °C followed by addition of soybean try psin inhibitor (1 mg) to terminate the digestion.
  • each anti-peptide antibody coupled to protein G magnetic beads was added to digested serum samples and mixed for 2 hours at room temperature. After removing the unbound fraction, magnetic beads containing the bound peptides were washed 3 times with 200 pL of PBS containing 0.03% (w/v) CHAPS (3-((3- cholamidopropyl) dimethylammonium)-! -propanesulfonate) detergent before elution of the bound peptides in 100 pL of buffer containing 100 mM Glycine, 2% (v/v) formic acid.
  • PBS containing 0.03% (w/v) CHAPS (3-((3- cholamidopropyl) dimethylammonium)-! -propanesulfonate) detergent
  • the following example provides a method of quantifying 14-3-3 proteins using protein enrichment.
  • the sample was enriched in one or more 14-3-3 proteins using immunoaffinity enrichment followed by digestion of 14-3-3 proteins to produce surrogate peptides of 14-3-3 proteins.
  • Serum samples and calibrators (1.58 - 200 ng/mL 14-3-3eta) were enriched for one or more 14-3-3 proteins using anti-14-3-3(pan) antibodies with subsequent on-bead digestion of the enriched 14-3-3 proteins to produce 14-3-3 surrogate peptides.
  • 200 uL of sample was diluted 2-fold with phosphate buffered saline containing 1% (v/v) Tween-20 and 5 mg/mL bovine serum albumin.
  • 4 ug of anti-14-3-3(pan) antibody were coupled to streptavidin coated magnetic beads.
  • the buffered sample solution was added to 4 ug of anti-14-3-3(pan) antibody coupled to streptavidin magnetic beads and mixed for 1 hour at room temperature.
  • the magnetic beads containing the bound 14-3-3 protein were washed 3 times with 200 pL phosphate buffered saline containing 1% (v/v) Tween-20 and 5 mg/mL bovine serum albumin. Subsequently, the washed beads containing the bound 14-3- 3p were resuspended in 350 uL of 50 mM Tris-HCl (pH 8) containing 0.2% (w/v) sodium deoxy cholate and 1 mM dithiothreitol, then denatured for 30 min at 56 °C before addition of 0.4 ng of each stable isotope labeled peptide.
  • TPCK-treated bovine trypsin (62.5 pg) was added and allowed to digest for 30 min at 37 °C followed by the addition 500 uL of buffer containing 100 mM Glycine, 2% (v/v) formic acid, 0.001% (w/v) ZWITTERGENT® and 1% (w/v) heptanesulfonic acid.
  • the digested samples containing residual beads and precipitated deoxy cholate were centrifuged for 10 minutes at 2,200 x g before collecting the resulting clarified supernatant for analysis by LC-MS/MS.
  • FIG. 10 shows a mean and standard deviation box plot of the measured values of surrogate peptides of 14-3-3p (14-3-3 VIS, 14-3-3_TMA, 14-3-3_YDD, and 14-3-3_EAF).
  • 14-3-3 was measured in serum samples from ostensibly healthy donors using the workflow of Example 1 and Example 2. Regardless of surrogate peptide, 14-3-3 results were systematically lower using the workflow of Example 2 compared to the workflow of Example 1 .
  • Serum samples and calibrators (1.58 - 200 ng/mL 14-3-3eta) were enriched for 14- 3-3 protein using anti-14-3-3(pan) antibodies with subsequent on-bead digestion of the enriched 14-3-3 proteins to produce 14-3-3 surrogate peptides.200 uL of sample w as diluted 2-fold with phosphate buffered saline containing 1% (v/v) Tween-20 and 5 mg/mL bovine serum albumin before adding 4 ug of anti-14-3-3(pan) antibody coupled to streptavidin magnetic beads and mixing for 1 hour at room temperature.
  • TPCK-treated bovine trypsin (62.5 pg) w as added and allowed to digest for 30 min at 37 °C followed by the addition soybean try psin inhibitor (1 mg) to terminate the digestion.
  • soybean try psin inhibitor (1 mg) to terminate the digestion.
  • 2 ug of each anti-peptide antibody coupled to protein G magnetic beads were added to the digested samples and mixed for 2 hours at room temperature.
  • the magnetic beads containing the bound peptides w ere w ashed 3 times with 200 pL of PBS containing 0.03% (w/v) CHAPS before elution of the bound peptides in 100 pL of buffer containing 100 mM Glycine, 2% (v/v) formic acid, 0.001% (w/v) Zwittergent 3-16 and 1% (w/v) heptanesulfonic acid for LC- MS/MS analysis.
  • FIGS. 11 A-B and 11C-D show example chromatograms for the 14-3-3 TMA surrogate peptide (FIG. 11 A-B) and 14-3-3 YDD surrogate peptide (FIG. 11C-D) measured according to the workflow of Example 1 (Figs. 11 A and 11C) and Example 3 (Figs. 1 IB and 1 ID).
  • the natural and stable isotope labeled peptides are shown in each case with the indicated transitions, which were suitable for both workflows.
  • Example 3 was devised whereby following 14-3-3 protein enrichment and digestion of the enriched 14-3-3 protein, the resulting 14-3-3 surrogate peptides were further enriched using anti-peptide antibodies. In doing this, >10-fold enhancement in sensitivity was observed. It is expected that other forms of surrogate peptide enrichment, such as solid phase extraction, may be suitable following 14-3-3 protein enrichment and digestion of enriched 14-3-3 protein.

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Abstract

L'invention concerne des procédés et des systèmes utilisant la spectrométrie de masse pour la détection et/ou la quantification de protéines 14-3-3, ou de peptides de substitution de celles-ci, dans un échantillon biologique.
PCT/US2025/032142 2024-06-04 2025-06-03 Procédés et systèmes de détection de biomarqueurs protéiques de la polyarthrite rhumatoïde dans un échantillon biologique à l'aide de lc-ms/ms Pending WO2025255166A2 (fr)

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