WO2020002942A1 - Profilage d'adp-ribosylation - Google Patents

Profilage d'adp-ribosylation Download PDF

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WO2020002942A1
WO2020002942A1 PCT/GB2019/051842 GB2019051842W WO2020002942A1 WO 2020002942 A1 WO2020002942 A1 WO 2020002942A1 GB 2019051842 W GB2019051842 W GB 2019051842W WO 2020002942 A1 WO2020002942 A1 WO 2020002942A1
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adenosine
adp
proteins
ribosylated
alkyne
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Peter DIMAGGIO
Kalesh KARUNAKARAN
Saulius LUKAUSKAS
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Imperial College of London
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91091—Glycosyltransferases (2.4)
    • G01N2333/91142—Pentosyltransferases (2.4.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/40—Post-translational modifications [PTMs] in chemical analysis of biological material addition of nucleotides or derivatives, e.g. adenylation, flavin attachment

Definitions

  • the present invention relates to ADP-ribosylation profiling in systems, in particular, although not exclusively, in cells.
  • Protein ADP-ribosylation is a post-translational modification (PTM) where members of a family of enzymes known as ADP-ribosyltransferases (or PARPs) covalently link ADP-ribose moieties derived from NAD+ to the side chains of a diverse range amino acid residues (glutamate, aspartate, arginine, lysine, cysteine, serine) on target proteins.
  • PTM post-translational modification
  • ADP-ribosyltransferases or PARPs
  • This PTM exists either as a monomer (mono-ADP-ribosylation), consisting of a single ADP-ribose moiety on the target residue, or polymer (poly-ADP-ribosylation) where a branched polymeric chain of ADP-ribose units originates from the target residue.
  • PARPs 1 , 2 and 3 are known to have important roles in DNA regulation, particularly in the base excision repair pathway (Hottiger, 2015), and clinical PARP inhibitors have been developed as cancer therapeutics to exploit the concept of synthetic lethality in patients with germline mutations in BRACA1 or BRACA2 genes (Lord, 2017). Beyond the established roles of these nuclear PARPs in DNA damage response, the broad cellular functions for the majority of the other members of the PARP family remain elusive primarily due to the lack of technologies for the large-scale profiling of intracellular ADP-ribosylation.
  • Mass spectrometry (MS) proteomics studies of ADP-ribosylation have been limited due numerous technical challenges, including the lack of specificity and/or affinity when using recombinant macro domains (Jungmichel, 2013) ⁇ (Martello, 2016), cis-diol binding boronate beads (Zhang, 2013), antibodies (Gagne, 2008) or adapted phosphoproteome enrichment strategies (Daniels, 2014) to enrich for this low abundance PTM against complex protein backgrounds. Additionally, most methods reported to date require a priori knockdown of poly- ADP-ribose glycohydrolase (PARG) activity to sufficiently increase the baseline level of poly- ADP-ribosylation for MS detection.
  • PARG poly- ADP-ribose glycohydrolase
  • “clickable” NAD+ can be metabolically produced by treating cells with the corresponding“clickable” adenosine precursor (Westcott, 2017).
  • 6- alkyne adenosine (6YnAd), a compound that was previously demonstrated to be suitable for labelling poly(A) tails of mRNAs in mammalian cells (Grammel, 2012), was utilised for highly sensitive fluorescence detection and Label Free Quantification (LFQ) proteomic profiling of ADP-ribosylated proteins in live cells under stress induction 10 .
  • LFQ Label Free Quantification
  • the present invention provides a method of identifying one or more adenosine diphosphate (ADP) ribosylated proteins comprising applying 2-substituted adenosine, for example 2-alkynyl adenosine, say 2-alkyne adenosine, to a first system for the ribosylation of proteins.
  • ADP adenosine diphosphate
  • the first system for the ribosylation of proteins is a cell, such as a cell in vitro.
  • the methods of the invention further comprise applying a 6-substituted adenosine, for example 6-alkynyl adenosine, say 6-alkyne adenosine, to a second system for the ribosylation of proteins.
  • a 6-substituted adenosine for example 6-alkynyl adenosine, say 6-alkyne adenosine
  • the second system for the ribosylation of proteins is a cell, such as a cell in vitro.
  • the 6-substituted adenosine e.g. 6-alkynyl adenosine such as 6- propargyl adenosine
  • 2-alkynyl adenosine such as 2-ethynyl adenosine
  • 6-substituted adenosine may, for example, be applied in a parallel or duplicate system to the 2-substituted adenosine, for example to a collection of cells in vitro that have been obtained, prepared and cultured under the same conditions as the cells to which the 2-substituted adenosine is applied.
  • the 6-substituted adenosine may, for example, be applied to a different system to the 2-substituted adenosine, for example the first system can be a particular cancer cell line in vitro and the second system can be a different cancer cell line in vitro or the same cancer cell line in vitro in a different state (e.g. different passage, different stage of growth, etc).
  • the adenosine analogues used in the invention preferably include alkynyl groups, and therefore allow the use of Click chemistry to manipulate, process and identify the proteins in which the analogues are incorporated following ribosylation.
  • the substituted adenosines used in the invention are preferably 2-alkynyl adenosine and/or 6-alkynyl adenosine.
  • the alkynyl groups of the 2- and 6- substituted adenosines are preferably independently selected from a C2 - C10 alkynyl, and further preferably independently selected from a C2-C5 alkynyl.
  • the alkynyl groups of the 2- and 6- substituted adenosines may be independently selected from ethynyl, butynyl (e.g. 1- or 3-butynyl), 1-propynyl, propargyl, and pentynyl.
  • the 2-substituted adenosine will have a terminal alkyne moiety at the 2-position (e.g. 2-propynyl, 3-butynyl etc.).
  • the 2-substituted adenosine may be 2-ethynyl adenosine (see Figure 2A) and the 6-substituted adenosine may be 6-propargyl adenosine (see Figure 2B).
  • Other alkynyl substituents may be deployed at the 2 and 6 positions respectively.
  • the methods of the invention may further comprise using a capture reagent to selectively enrich for the ribosylated proteins, for example to enrich for or extract the ribosylated proteins from other system components.
  • the capture reagent may be added to the system after the 2-substituted adenosine, such as 2-alkynyl adenosine, and/or the 6- substituted adenosine, such as 6-alkynyl adenosine, and the capture reagent may be specifically added to ribosylated proteins through the use of click chemistry and the alkyne group of the adenosine analogues.
  • the capture reagent will have an affinity for a or the capture matrix.
  • the capture reagent preferably comprises a clickable group, for example an azide moiety.
  • the capture reagent may be a di- or tri-functional capture reagent.
  • the capture reagent may comprise- one or more of biotin, a biotin analogue (for example desthiobiotin) or an immunotherapy tag (e.g. an immunotherapy fluorescent tag).
  • the capture reagent may comprise 5- carboxytetramethylrhodamine (TAMRA) and/or polyethylene glycol.
  • TAMRA 5- carboxytetramethylrhodamine
  • the capture reagent may be selected from 5/6-TAMRA-LysAzide Biotin, 5/6-TAMRA-Azide Biotin (both available from Jena BioScience of Jena Germany, Cat. No. CLK 1048) or Azide-PEG3-Biotin (CAS Number 875770-34-6).
  • the enrichment, isolation or extraction of the ribosylated proteins occurs through the use of an interactor molecule, a molecule that specifically interacts with, preferably binds, the capture reagent.
  • the enrichment or extraction occurs through the use of, for example, as an interactor molecule one or more of avidin, NeutrAvidin or streptavidin, which is able to interact with and/or bind to, say, biotin present in the capture reagent, such as through the use of interactor molecule- coated stationary medium, such as beads, which stationary medium will consequently bind, say, biotin that is attached to the ribosylated proteins due to the inclusion of the capture reagent in the proteins; the non-ribosylated proteins, which do not incorporate the adenosine analogue, can be washed away from the stationary medium leaving the ribosylated proteins selectively attached.
  • Preferred methods of the invention further comprise reducing the size of ADP-ribose modifications added to the proteins. Said reduction of the size may occur, for example, through cleavage by chemical treatment or enzyme treatment.
  • the enzyme treatment may be Nudix treatment or PARG treatment.
  • one or more labels may be added to the ADP-ribosylated proteins in the methods of the invention.
  • the labels may be added to the ribosylated proteins via the alkyne group of the incorporated adenosine analogues, using click chemistry, and/or the labels may be added by other means, for example following enrichment, isolation or extraction of the ribosylated proteins.
  • the labels added to the ribosylated proteins are Tandem Mass Tag (TMT) labels and/or SI LAC labels.
  • TMT Tandem Mass Tag
  • SI LAC labels quantitative proteomic methodology
  • iTRAQ dimethyl labelling and so on.
  • We prefer to use TMT labels because that methodology provides a signal enhancement effect in MS2 that uniquely enables detection of even low-abundant modified proteins.
  • one or more of the ADP ribosylated proteins are characterized or identified using mass spectrometry (MS) and/or liquid chromatography- mass spectrometry (LC-MS).
  • MS mass spectrometry
  • LC-MS liquid chromatography- mass spectrometry
  • Methods of the invention may further comprise using a debiased linear analysis to analyse the data output from the LC-MS/MS.
  • aspects of the invention may provide a method of identifying one or more adenosine diphosphate (ADP) ribosylated proteins comprising applying a first adenosine analogue and a second, different, adenosine analogue to a system, e.g. a cell in vitro or in vivo.
  • the first and second adenosine analogues may comprise adenosine with substituents at different positions.
  • the first adenosine analogue may be a 2-substituted adenosine, e.g.
  • 2-alkynyl adenosine for example 2-alkyne adenosine
  • the second adenosine analogue may be a 6-substituted adenosine, e.g. 6-alkynyl adenosine, such as 6-alkyne adenosine.
  • compositions for identifying one or more adenosine diphosphate (ADP) ribosylated proteins comprising a 2-substituted adenosine, e.g. 2-alkyne adenosine and a kit of parts for identifying one or more adenosine diphosphate (ADP) ribosylated proteins comprising a first composition comprising a 2-substituted adenosine, e.g. 2-alkynyl adenosine or 2-alkyne adenosine, and a second composition comprising a 6- substituted adenosine, e.g.
  • ADP adenosine diphosphate
  • composition of the invention includes two differently substituted adenosines for identifying ADP ribosylated proteins.
  • the compositions of the invention and kit of parts of the invention may be for separate, simultaneous or sequential administration of different adenosines, e.g. 2-substituted adenosine and 6-substituted adenosine, to first and second systems, for example in accordance with the methods of the invention.
  • FIG 1 shows an overview of ADP-ribosylation by PARP
  • FIG. 2 shows further details of structures involved in the methods of the invention
  • FIG. 3 shows a schematic overview of a method in accordance with the invention
  • Figure 4 shows general structures of labelled ribosylated proteins generated in methods of the invention, using the 2YnAd probe
  • Figure 5 shows general structures of labelled ribosylated proteins generated in methods of the invention, using the 6YnAd probe.
  • Figure 6 shows chemical structures of click capture reagents that may be used in methods of the invention
  • Figure 7 shows metabolic labelling of MDA-MB-231 breast cancer cells by 6Yn-Ad and 2Yn- Ad.
  • Figure 8 shows a schematic overview of quantitative profiling of global ADP-ribosylome in MDA-MB-231 breast cancer cell line using a combination of metabolic incorporation of the clickable NAD precursors (6YnAd and 2YnAd) and TMTsixplex isobaric mass tagging.
  • MDA-MB-231 cells were metabolically labelled with 3 different concentrations of 6YnAd and 2YnAd in parallel. After cell lysis and click reaction with a biotin-containing capture reagent, the labelled proteins are affinity enriched on NeutrAvidin-Agarose resins. Stringent washing at this stage removes non-modified proteins from the beads. After on-bead reduction, alkylation and tryptic digestion, the eluted peptides are desalted and each sample is labelled using a unique TMTsixplex channel.
  • Figure 9 shows a comparison of metabolic labelling of 6YnAd and 2YnAd, according to the schematic overview of Figure 8.
  • Figure 10 shows experimental workflow for the dual metabolic labelling method, with a schematic illustrating the workflow involving parallel treatment of PARP inhibitors Olaparib and Rucaparib followed by dual metabolic labelling of 6YnAd and 2YnAd, click chemistry, affinity enrichment of the labelled proteins on NeutrAvidin-Agarose beads and TMT10plex-based quantification of global changes in the cellular ADP-ribosylome.
  • Figure 1A shows the ADP-ribosylation of a target protein wherein adenosine binds to a target protein by use of a PARP. More than 15 different PARP enzymes have been identified ( Figure 1 B), although the majority are poorly characterized and their targets unknown.
  • the present invention provides simple methods to profile post translational modification (PTM) events that are taking place in a particular system.
  • PTM post translational modification
  • Figure 2A the use of the adenosine analogue 2-alkyne adenosine
  • Figure 1A provides the ability to profile live cells to identify the ADP-ribosylation of proteins
  • adenosine analogues which are then incorporated into NAD+ within the cell, surprisingly overcomes the difficulties of introducing NAD+ analogues into cells. Therefore, the invention provides the ability to generate a profile of the ADP-ribosylation that is occurring, for example in different cells types and/or under different conditions. As well as enabling further elucidation of normal cellular pathways and their control, this allows, in particular, the assessment and identification of new disease- associated pathways and drug targets.
  • methods in which say 2-alkynyl adenosine is used provide information about additional ADP-ribosylated proteins, compared to information from methods in which, say, 6-alkynyl adenosine is used in the same experimental circumstances and conditions. Also, by providing methods in which both 2- substituted adenosine and 6-substituted adenosine are used, the invention enables the profiling of a wider group of ribosylated proteins in one series of experiments, compared to just using one of the adenosine analogues on their own, for example 6-alkynyl adenosine.
  • the different analogues may be added to the same system, e.g. cells, at the same time, or they may be added to the same system, e.g.
  • the 2-substituted adenosine and 6-substituted adenosine may be added to different systems, for example, the first system may be cells from a particular cancer cell line and the second system may be cells from a different cancer cell line or the same cancer cell line in a different state (e.g. different passage, different stage of growth, etc).
  • the combination of the dual metabolic incorporation strategy with TMT-based isobaric labelling approach enables comprehensive profiling of cellular ADP-ribosylation.
  • This novel combination provides unprecedented enhancement in the sensitivity of detection of the PTM and provides an opportunity to derive more information about many ADP-ribosylated proteins.
  • the TMT-labelling approach is able to be used to increase the sensitivity of detection.
  • PARG knockdown (which is a requirement for existing methods to overcome poor sensitivity) is known to cause alterations in cellular physiology. Also, induction of cellular stress (which is a requirement of the existing methods) leads to preferential in-cell activation of PARP1 which masks activity of other members of the PARP family and severely undermine their study.
  • the invention because of its high sensitivity, requires neither PARG knockdown/knockout nor induction of cellular stress and as such provides a way for an unbiased comprehensive quantitative profiling of endogenous ADP-ribosylated proteins.
  • the invention provides methods comprising adding 2-substituted adenosine, such as 2-alkynyl adenosine ( Figure 2A), and optionally 6-substituted adenosine, such as 6-alkynyl adenosine ( Figure 2B) to systems.
  • 2-substituted adenosine such as 2-alkynyl adenosine ( Figure 2A)
  • 6-substituted adenosine such as 6-alkynyl adenosine ( Figure 2B)
  • adenosine analogues can be synthesised using methods known to those skilled in the art; see, for example (Jiang, 2009) and (Wang, 2014), which are incorporated herein by reference.
  • the adenosine analogues are metabolically incorporated into Nicotinamide Adenosine Dinucleotide (NAD) in the systems used in the methods of the invention, for example in the live cells, such that NAD + analogues are produced in the systems, which have the alkynyl group, or“clickable handle” of the corresponding adenosine analogue precursor molecule.
  • NAD + analogues can then be used in the ADP-ribosylation of target protein residues by PARP enzymes ( Figures 1A and 1 B), resulting in ADP-ribosylated proteins with the alkynyl group“clickable handle” (see Figure 3).
  • the PARP enzymes may add mono ADP-ribose modifications to their protein target(s), or they may add poly ADP-ribose modifications to their protein target(s) (Figure 1A).
  • Figure 1B The currently understood preferences with regard to individual PARP enzyme ability to add mono or poly groups, is shown in Figure 1 B.
  • the methods of the invention may comprise the addition of mono ADP-ribose groups having a 2-alkynyl handle or 6-alkynyl handle, leading to corresponding labelled proteins (see Figures 4A and 5A, respectively, for example), and/or may comprise the addition of poly ADP-ribose groups having a 2-alkynyl handle or 6-alkynyl handle, leading to corresponding labelled proteins (see Figures 4B and 5B, respectively, for example).
  • the alkyne bonds of the analogues allow the use of click chemistry in methods of the invention.
  • Click chemistry is a term generally understood in the art.
  • the alkyne bonds provide that the ADP-ribosylation added during the methods of the invention can be conjugated to desirable labels, such as capture reagents, in particular using a Cu(l)-catalyzed azide-alkyne cycloaddition (CuAAC).
  • 2-substituted adenosine is added to a first system for the ribosylation of proteins and, optionally, 6-substituted adenosine is added to a second system for the ribosylation of proteins.
  • a system for the ribosylation of proteins comprises the elements necessary to incorporate one of the substituted adenosine analogues of the methods of the invention into NAD + , and for one or more PARP enzymes to use the NAD + to ADP- ribosylate one or more target proteins.
  • the first, and where present, second systems are cells, preferably live cells. The methods of the invention are preferably carried out in vitro.
  • both 2-substituted adenosine and 6-substituted adenosine may be added to the same systems such that the first and second systems are one and the same.
  • the two adenosine analogues may be added to the same tissue culture of cells. In this way, the addition of both analogues allows the detection of a wider range of ADP-ribosylated proteins than would be detected by the use of just one of the analogues on their own, in otherwise similar circumstances.
  • the first and second systems may be different but deliberately similar, such that the only differences identified in the systems using the method are due to whether it was the 2-substituted adenosine, such as 2-alkynyl adenosine, or 6-substituted adenosine, such as 6- alkynyl adenosine, that was added to the system.
  • 2-substituted adenosine such as 2-alkynyl adenosine
  • 6-substituted adenosine such as 6- alkynyl adenosine
  • the first and second systems may be cultures of the same type(s) of cell, such as HeLa or MB-231 breast cancer cells, where the culture has and is being grown under identical conditions, so that any difference in ribosylation pattern can be associated with a preference within the system for either the 2- substituted adenosine or 6-substituted adenosine analogue.
  • the methods may comprise performing replicates, such that the 2-substituted adenosine and/or 6-substituted adenosine are added to multiple systems, and some of the systems do not differ with respect to the conditions of the systems, including which and how much analogue is added to them (see Figure 8).
  • experiments may be carried out to investigate the effect of different conditions on ADP-ribosylation patterns, so that most of the conditions of the systems are kept the same, but particular conditions, such as the concentration of the analogue or a particular drug or reagent in the systems, are deliberately altered in a measured way (see Figures 8 and 9).
  • Conditions that may be assessed in this way, to analyse the effects on ADP- ribosylation include PARP inhibitor treatments (see Figure 10), oxidative stress induction, cytotoxic/chemotherapy agent treatment, viral/bacterial infection, and hypoxia induction.
  • ADP adenosine diphosphate
  • identification should be interpreted broadly in the context of the invention. Therefore, for example, identification may simply comprise noting the presence of ribosylated proteins, e.g. seeing proteins in which the alkyne handle is incorporated following the addition of an adenosine analogue ( Figure 7A). Alternatively, identification in accordance with the invention may comprise analyzing and characterizing specific ribosylated proteins, for example by assigning the correct name to the proteins ( Figure 9).
  • Methods of the invention may comprise adding one or more labels or tags to the ADP- ribosylated proteins.
  • the one or more labels or tags may be any suitable label or tag, for example for visualising, identifying, or capturing one or more of the ADP-ribosylated proteins.
  • a label or tag is added to the ADP-ribosylated protein using click chemistry and, say, an alkyne clickable handle.
  • the label or tag may be added to the proteins though the incorporation of a capture reagent.
  • click enrichment may be carried out in methods of the invention by the addition of di- or tri-functional capture reagent carrying azide chemical functionality and biotin (and a fluorescent dye unit in the case of the tri-functional capture reagent) to the treated cells.
  • the click reaction ensures chemical tagging/labelling of the ADP-ribosylated proteins/peptides with the capture reagent, which can be selectively enriched (i.e. purified) using its biotin group on modified Avidin-based beads (exploiting the strong biotin-Avidin interaction so that extensive wash steps can remove unmodified proteins).
  • Suitable capture reagents therefore include 5/6-TAMRA-LysAzide-Biotin, 5/6-TAMRA-Azide-Biotin, and Azide-PEG3-Biotin ( Figure 6).
  • Methods of the invention may comprise the addition of T andem Mass T ag (TMT) labels and/or the use of Stable Isotope Labeling with Amino acids in Cell culture (SI LAC) (Harsha, 2008).
  • TMT T andem Mass T ag
  • SI LAC Stable Isotope Labeling with Amino acids in Cell culture
  • More than one tag or label may be added to the ADP-ribosylated proteins in the methods of the invention, and different labels may be added at different stages of methods of the invention, according to need.
  • a capture reagent may be added to the proteins using click chemistry, in order to extract or enrich for the ribosylated proteins, and the capture reagent may be removed following the isolation or enrichment and a TMT label added instead to assist with characterisation of the ribosylated proteins by MS and/or LC-MS.
  • the label or tag may be added to the proteins after preprocessing to provide additional information about the protein and its ribosylation.
  • the system used in the methods of the invention may be a live cell and the label or tag, for example a TMT label, may be added after cell fractionation of the system(s). In this way, it may be possible to identify the locations of the ADP-ribosylated protein(s) in the live cell.
  • the methods of the invention may comprise extracting the ADP-ribosylated proteins.
  • the extraction may be absolutely specific or near absolutely specific, so that substantially no non- ribosylated proteins remain with the ADP-ribosylated proteins, or the extraction may be relative, leading to an enrichment of the ADP-ribosylated proteins, so that the concentration of the ADP-ribosylated proteins significantly increases but some non-ribosylated proteins remain.
  • the extraction occurs using a capture reagent, as discussed above. Thus, specific affinity enrichment can be carried out.
  • Suitable methods for reducing the size of the modification(s) by cleavage include chemical cleavage, for example using the method of (Zhang, 2013) , or enzymatic cleavage, for example using Nudix treatment or PARG treatment. In particularly preferred methods, all three cleavage strategies (chemical, Nudix and PARG) may be used to ensure comprehensive cleavage.
  • the cleavage may occur immediately following extraction of the ADP-ribosylated proteins, for example whilst the proteins are still attached to avidin beads used for extraction so that the cleavage occurs“on-bead”.
  • the cleavage results in the presence of modified residues in the proteins, which residues can be detected and identified using MS, so that the site of addition of the ADP-ribose chain can be identified within one or more of the proteins.
  • MS and in particular quantitative liquid chromatography-tandem mass-spectrometry (LC-MS/MS) is used to identify the ADP- ribosylated proteins and their sites of modification.
  • LC-MS/MS quantitative liquid chromatography-tandem mass-spectrometry
  • MDA-MB-231 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% foetal bovine serum (FBS) (Gibco) without antibiotics in a humidified incubator with 5% CO2 at 37°C and passages at every 2-3 days at 80-90% confluency.
  • DMEM Modified Eagle Medium
  • FBS foetal bovine serum
  • the cells were then washed with PBS three times and lysed using 4% SDS lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCI, 4% SDS and 500 U Benzonase) and whole-cell lysates were collected using a cell scraper. Lysates were cleared at 16,000g for 5 min and protein concentrations were estimated using Bio-Rad DCTM protein assay.
  • SDS lysis buffer 50 mM HEPES pH 7.4, 150 mM NaCI, 4% SDS and 500 U Benzonase
  • the cell lysates at 1 mg/ml concentration were treated with freshly premixed click chemistry reaction cocktail (100 mM capture reagent, 1 mM CuS0 4 solution, 1 mM tris(2-carboxyethyl) phosphine hydrochloride) (TCEP), 100 mM [tris(1 -benzyl-1 H-1 , 2, 3-trazol-4-yl)methyl]amine (TBTA)) for 3 h at room temperature.
  • TCEP tris(2-carboxyethyl) phosphine hydrochloride
  • TBTA tris(1 -benzyl-1 H-1 , 2, 3-trazol-4-yl)methyl]amine
  • Capture reagent (Azido-TAMRA-Biotin or Azide-PEG3-Biotin (Jena Bioscience)): 10 mM in DMSO; CuS0 4 : 50 mM in MilliQ water; TCEP: 50 mM in MilliQ water; TBTA: 10 mM in DMSO. Proteins were precipitated by adding ice-cold methanol (4 volumes), chloroform (1.5 volumes) and water (3 volumes) and the precipitates were collected by centrifugation at 16,000g for 5 min. The protein precipitates were then washed twice with ice- cold methanol (10 volumes) and the supernatants were discarded.
  • the protein pellets were air dried for 20 min and suspended in resuspension buffer (4% SDS, 50 mM HEPES pH 7.4, 150 mM NaCI) to 3 mg/ml concentration. Equal volumes of 2X SDS loading buffer (Bio-Rad) containing 10% b-mercaptoethanol were added and the samples were boiled at 95 °C for 10 min and allowed to cool to room temperature. 30 pg of protein was loaded per gel lane (12% SDS Tris-HCI gels) and resolved by SDS-PAGE. The gels were scanned for fluorescence labelling using GE typhoon 5400 gel scanner.
  • the dried protein pellets obtained after click chemistry and protein precipitation were suspended in resuspension buffer (4% SDS, 50 mM HEPES pH 7.4, 150 mM NaCI) to 10 mg/ml concentration.
  • resuspension buffer 4% SDS, 50 mM HEPES pH 7.4, 150 mM NaCI
  • 500 pg of whole-cell lysate after click chemistry and protein precipitation was resuspended in 50 pi of the resuspension buffer.
  • the samples were subsequently diluted 20 fold with HEPES buffer (50 mM HEPES pH 7.5, 150 mM NaCI) so that the final SDS amount is 0.2%.
  • the beads after affinity enrichment were resuspended in 100 pi of 50 mM ammonium bicarbonate buffer and treated with 3 mM dithiothreitol (DTT) (100 mM stock solution in water) solution for 30 min at room temperature.
  • DTT dithiothreitol
  • the beads were washed once with 50 mM ammonium bicarbonate buffer and resuspended in 100 pi of 50 mM ammonium bicarbonate buffer and treated with 10 mM iodoacetamide (IAA) (500 mM stock solution in water) in dark for 30 min at room temperature.
  • IAA mM iodoacetamide
  • the beads were again washed with 50 mM ammonium bicarbonate buffer and resuspended in 100 pi of fresh 50 mM ammonium bicarbonate buffer and treated with 2 pg of trypsin (Promega, Product Code V51 13) at 37 °C overnight.
  • the samples were acidified to pH 3 using formic acid and allowed to stand for 5 min, centrifuged and collected the supernatant.
  • the beads were washed with 0.1 % formic acid solution in water, centrifuged and the supernatant were mixed with the previous supernatant.
  • the collected tryptic peptides were desalted on C18 Empore disks on micropipette tip format and the desalted peptide mixture were dried on a speedvac. TMTsixplex labelling.
  • MDA-MB-231 cells were fed with three different concentrations (1 , 0.5 and 0.25 mM) of 6YnAd and 2YnAd in parallel. After 1 h of incubation, whole-cell lysates were prepared and proteins were quantified. 500 pg of proteins from each condition were subjected to click chemistry using the capture reagent Azido-TAMRA-Biotin. Proteins were precipitated, re-solubilised and subjected to affinity enrichment on NeutrAvidin-Agarose beads. After extensive washings, the beads were subjected to DTT treatment, IAA treatment and overnight trypsinisation.
  • the peptide digests were collected, desalted, dried and re-dissolved in 100 mI of 100 mM triethylammonium bicarbonate (TEAB) buffer (pH 8.5). Each sample was subjected to TMT labelling with a unique TMTsixplex reagent for 1 h at room temperature. After quenching the TMT reactions with hydroxylamine (10 pL of 5% hydroxylamine per reaction for 15 min at room temperature) treatment, the six samples were mixed together, concentrated to complete dryness and re-dissolved in 0.1 % formic acid. The sample was desalted on C18 Empore disks on micropipette tip format and the desalted sample was dried on a speedvac.
  • TEAB triethylammonium bicarbonate
  • Peptides were reconstituted in 0.1 % trifluoroacetic acid and chromatographically resolved using an Ultimate 3000 RSLCnano (Dionex) UHPLC. Peptides were first loaded onto an Acclaim PepMap 100 C18, 3 pm particle size, 100A pore size, 20 mm x 75 pm ID (Thermo Scientific) trap column heated to 40°C, using a loading buffer (2% acetonitrile (MeCN) and 0.05 % trifluoroacetic acid in 97.05 % H20) with a flow rate of 7 pL/minute.
  • a loading buffer 2% acetonitrile (MeCN) and 0.05 % trifluoroacetic acid in 97.05 % H20
  • Chromatographic separation was achieved using an EASY-Spray column, PepMap C18, 2 pm particles, 100 A pore size, 500 mm x 75 pm ID (Thermo Scientific) heated to 40 °C.
  • the gradient utilised a flow of 0.3 pL/minute, starting at 98 % mobile A (0.1 % formic acid, 5% dimethyl sulfoxide (DMSO) in H2O) and 2 % mobile B (0.1 % formic acid, 75 % acetonitrile, 5% DMSO and 19.9 % H20).
  • Mobile B was increased to 8 % over 0.5 minutes, to 30 % over 27 minutes, further increased to 40 % in 10 minutes, and finally up to 95 % in 2 minutes and held for 4 minutes. Mobile B was reduced back to 5 % over 1 minute for the rest of the acquisition.
  • High-resolution HCD MS2 spectra were generated in positive mode for precursor masses using a normalised collision energy of 38 %, within a 0.7 m/z isolation window, using quadrupole isolation, in top speed mode over a 3 second cycle time, at 50000 resolution in profile mode, 100 ms maximum injection time and 5e4 AGC target, with an instrument determined scan range beginning at 100 m/z.
  • MDA-MB-231 cells grown to 80-90% confluency in 10 cm plates were treated with 25, 5, 1 , 0.2 and 0 (DMSO control) mM concentrations of the PARP inhibitors Olaparib (10 mM stock solution in DMSO) and Rucaparib (10 mM stock solution in DMSO) in parallel. All treatments were performed in 10 ml DM EM with 10% FBS and the cells were placed in an incubator with 5% CO2 and 37 °C for 1 h.
  • Proteins were precipitated using the methanol/chloroform/water system and air-dried precipitates were resuspended in the pull down buffer (50 mM HEPES, pH 7.4, 0.2% SDS, 150 mM NaCI) and subjected to affinity enrichment on NeutrAvidin-Agarose beads. After extensive washings, the beads were subjected to DTT treatment, IAA treatment and overnight trypsinisation. The peptide digests from the 10 conditions were collected, desalted, dried and re-dissolved in 100 pi of 100 mM triethylammonium bicarbonate (TEAB) buffer (pH 8.5).
  • TEAB triethylammonium bicarbonate
  • Each sample was subjected to TMT labelling with a unique TMH Oplex reagent for 1 h at room temperature. After quenching the TMT reactions with hydroxylamine treatment, the 10 samples were mixed together, concentrated to complete dryness and re-dissolved in 0.1 % formic acid solution in water. The sample was desalted on C18 Empore disks on micropipette tip format and the desalted sample was dried on a speedvac. The sample was dissolved in 300 pi of 0.1 % TFA solution in water and subjected to fractionation using High pH Reverse-Phase Peptide Fractionation Kit (Thermo scientific) to 8 fractions and dried on a speedvac.
  • High pH Reverse-Phase Peptide Fractionation Kit Thermo scientific
  • Cofactor effect i.e. the main measured effect. Encoded by +1 in reporters corresponding to 2YnAd and -1 in reporters corresponding to 6YnAd (e.g. sum-to-one encoding). Positive coefficients to this factor therefore indicate 2YnAd preference, and negative values indicate 6YnAd preference.
  • Inhibition effect This term corresponds to the second major effect in the experiment, which captures the general effect of inhibition to the protein that does not depend on the inhibitor. It was encoded as 1 (in cases where inhibitor is present) and 0 otherwise. This way values below than zero correspond to decrease in intensity upon inhibition.
  • the TMT labelling strategy offers increased sample multiplexing to enable the head-to-head comparison of the labelling between 2YnAd and 6YnAd at different concentrations or conditions, and the isobaric nature of the tag results in the pooling of peptide signal across all experimental conditions, which increases the sensitivity in detecting lower abundance ADP-ribosylated proteins relative to any non-specific background.

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Abstract

La présente invention concerne une méthode d'identification d'une ou de plusieurs protéines ribosylées de l'adénosine diphosphate (ADR), la méthode comprenant l'application d'adénosine 2-substituée, de préférence l'adénosine 2-alcyne, à un premier système pour la ribosylation de protéines. L'invention concerne un kit de pièces et une composition comprenant de l'adénosine 2-alcyne.
PCT/GB2019/051842 2018-06-29 2019-06-28 Profilage d'adp-ribosylation Ceased WO2020002942A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
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US20160299141A1 (en) * 2015-04-08 2016-10-13 The Board Of Regents Of The University Of Texas System Nad analogs and methods of using said nad analogs in determining ribosylation of proteins with parp mutants
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