WO2001007920A2 - Reperage automatique pour electrophorese bidimensionnelle - Google Patents

Reperage automatique pour electrophorese bidimensionnelle Download PDF

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Publication number
WO2001007920A2
WO2001007920A2 PCT/US2000/020268 US0020268W WO0107920A2 WO 2001007920 A2 WO2001007920 A2 WO 2001007920A2 US 0020268 W US0020268 W US 0020268W WO 0107920 A2 WO0107920 A2 WO 0107920A2
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Prior art keywords
proteins
sample
marker
protein spots
marker proteins
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PCT/US2000/020268
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WO2001007920A3 (fr
Inventor
Quan Nguyen
Jon Randall
Diana Davis
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Bio Rad Laboratories Inc
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Bio Rad Laboratories Inc
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Priority to AU66085/00A priority Critical patent/AU6608500A/en
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Publication of WO2001007920A3 publication Critical patent/WO2001007920A3/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules

Definitions

  • This invention relates to areas of technology involving two-dimensional electrophoresis, imaging of two-dimensional electropherograms, identification and quantitation of protein spots or bands in electrophoresis, and the compilation and processing of data in series of multiple two-dimensional electrophoresis gels.
  • Two-dimensional electrophoresis is widely used as a means of identifying, monitoring and quantifying the compositions of complex biological mixtures. Separations performed in two dimensions enable the biochemist to detect and distinguish a large number of components that would not be distinguishable in a linear separation. While the identification and analysis of the separated components in a two-dimensional electrophoresis gel is a complex and difficult task, methods have been developed to simplify, standardize and automate the procedure. Among these methods are those designed for analyzing and comparing each gel in a series of two-dimensional electropherosis gels. This finds application in situations where each gel in the series represents a successive stage of a clinical, diagnostic or analytical procedure or successive stages a life cycle.
  • Certain biological cells for example, have life cycles that are defined by changes in their morphology and physiology, such as changes in the expression of particular genes.
  • the developmental stages of these cells can be defined and monitored by their global patterns of gene expression and the progressive changes that occur in these global patterns and in particular genes. Accelerated cell growth, reductions in cell growth, mutations and other transformations, patterns and processes can be monitored as a means of detecting and monitoring the response of the cell to chemical factors such as drug and hormone treatments, nutrient availability, and other growth conditions, and environmental factors such as heat shock, pH, and other stresses. Tumor growth can be monitored by monitoring the DNA content of a cell.
  • Information of this type can be used to classify tumors in terms of high or low malignancy, therapies can be evaluated and adjusted, and other types of information useful in the diagnosis, treatment and prognosis of cancers can be obtained.
  • cells in which genes have been inserted can be monitored to address questions such as whether or not the gene is expressing a particular protein, whether or not the cell's progeny is expressing the protein, whether the protein is being secreted, and whether mutations in the gene have occurred.
  • These are examples of the many types of information that can be obtained by analyses of multiple two-dimensional electrophoresis gels taken in sequence. Other examples are well known among biochemists.
  • the manner in which the spots are distributed across a two-dimensional gel will depend on the separation parameters used in the first and second dimension stages of the separation. Two parameters that are commonly used in these separations are isoelectric point and molecular weight, although any parameter that can serve as the basis for an electrophoretic separation can be used. Whatever the parameter(s), the number of spots in a two-dimensional gel may be as high as several hundred, and each is detected by means of a signal derived from the individual spot. Various types of signals have been used, examples of which are optical density, radioactive emissions, fluorescence emissions, and colorimetric signals. Detection of the spots is commonly achieved by the use of imaging devices that convert these signals into digital data that is electronically readable data. The conversion is performed by imaging systems that store the information on computer storage media.
  • the data typically stored are data indicating molecular weight, isoelectric point and quantity or concentration. Once stored, the data is processed by the computer in a manner that presents the data in a progressive manner, for example by showing the appearance, disappearance, or growth profile of a particular protein spot throughout the course of the time spanned by the gels.
  • a variety of software programs that perform these functions are commercially available to biochemical laboratories. Examples of such programs are "PDQUEST” and “Melanie” (both available from Bio-Rad Laboratories, Inc., Hercules, California, USA), and “ImageMaster 2-D” (available from Pharmacia Biotech, Cleveland, Ohio, USA),.
  • matchsets Groups of gels that are to be compared to each other in a qualitative and quantitative manner for purposes of observing progressions and other changes in individual protein spots are commonly referred to by the software manufacturers and users as "matchsets.”
  • the software currently in use calls for the inclusion within each matchset of a "standard” that is constructed by the user.
  • the standard is constructed from several electropherograms within the matchset that are selected to collectively contain all of the spots that appear in any of the gels of the matchset. With all of the spots included and identified, the standard serves to identify particular protein spots in any single gel and to locate corresponding spots for particular proteins in different gels.
  • landmarks are actual protein spots that are manually selected by the user, the software then processing the manually selected spots to automatically identify the same spots in all of the member gels of the matchset. These user-selected landmarks are relatively few in number compared to the total number of spots in an individual electropherogram. The selection criteria are that the spots be well- resolved, that they be isolated from other spots, and that they appear in all gels of the matchset. The number of spots to be assigned as landmarks must be large enough that all of the remaining protein spots among the various gels will be successfully matched by the automatic processing.
  • the function of the landmarks is to serve as guideposts in the gel- to-gel comparisons, compensating and correcting for slight differences and distortions among the member gels in the matchset to assure that there will be a proper correspondence of protein spots among different gels in the matchset.
  • each sample protein in the array is determined by the molecular weight and isoelectric point of that protein
  • the molecular weight and isoelectric point of each marker protein will be known prior to their use in the analysis.
  • Other parameters on which electrophoretic separations can be based can be substituted for molecular weight, isoelectric point or both.
  • the signals and the scanning methods are chosen such that the marker protein spots are separately determinable from the sample protein spots, either directly from the scans themselves or upon manipulating the scans by combining them, subtracting one from the other, or similar procedures.
  • the two images that are thus formed are then processed together in a manner that will use the marker protein signals as landmarks for identification of and correspondence between the sample proteins among the various electropherograms.
  • the user must add a sufficient number of marker proteins to achieve successful matching of the protein spots among the various electropherograms in a matchset.
  • marker proteins as a substitute for manual landmarking presents various advantages over the prior art.
  • One advantage is the elimination of the need to select and assign a set of landmark spots each time a new matchset is to be analyzed.
  • Another is the elimination of the need to manually select landmark spots from among the full array of protein spots in an actual sample.
  • a further advantage is the elimination of the requirement that the landmark spots be isolated from other spots so that they can be spatially distinguished on the electropherogram.
  • a still further advantage is the reduction or elimination of errors in landmark selection from one gel to another.
  • Proteins suitable for use as marker proteins can be any proteins that will migrate under the two-dimensional electrophoresis conditions used in the separations to form protein spots in the resulting arrays, and that will not interfere with the migration of the sample proteins.
  • Preferred marker proteins are those that migrate to locations in the array that are well separated from each other and widely dispersed both horizontally and vertically, and yet are close to the locations to which the sample proteins migrate.
  • Particularly preferred marker proteins are those whose spots in the two-dimensional array will appear at locations near the corners of the array, and in or near regions where there is a high concentration of sample protein spots. Marker proteins selected with these considerations will form spots that cover a range of the separation parameters that are used for identification of the sample proteins.
  • the selection of marker proteins will be governed in many cases by the particular separation parameter(s) used in the two- dimensional electrophoresis.
  • the separation parameter(s) and the manner in which the second dimension separation differs from the first dimension separation can vary widely. Two of the most prominent and widely used separation parameters are molecular weight, isoelectric point, and a combination of both, and accordingly, these are preferred separation parameters in the practice of this invention.
  • the marker proteins may be identical to some of the sample proteins except labeled in a distinguishable manner. Alternatively, the marker proteins may be distinct from the sample proteins.
  • marker proteins is used herein in the specification and claims to denote the proteins whose spots that will serve as landmarks
  • sample proteins is used herein in the specification and claims to denote the proteins that were present in the sample(s) before the marker proteins were introduced.
  • marker proteins and the particular proteins to be used as marker proteins are not critical to this invention and may vary widely. Considerations governing the number and choice of marker proteins are the same as those governing the number and choice of reference spots used as landmarks according to procedures of the prior art. Thus, any number can be used that is sufficient to achieve successful matching of sample protein spots among each of the various gels in a matchset. Preferably, at least five marker proteins will be used, more preferably from 5 to 25, and most preferably from 9 to 16. The optimal number of marker proteins for any given gel will be the number that provides good mapping that will compensate for inter-gel distortion, and yet still allow unambiguous automatic matching of marker spots.
  • Labeling of the marker proteins can be achieved by any labeling technique that will permit detection in a manner that is selective relative to the sample proteins. Many labeling methods are known and can be used for the marker proteins. Examples of conventional labels are fluorescent labels, chemiluminescent labels, colorimetric labels, and radioisotope labels.
  • Differentiation between the marker proteins and the sample proteins can be achieved by labeling the sample proteins with labels that are distinguishable from the labels of the marker proteins, or by labeling only one of the two groups of proteins and staining the gels containing the two-dimensional protein spot arrays in a manner that will respond to the marker proteins differently than to the sample proteins.
  • signal differentiation can be achieved by having the two groups of proteins are labeled with different labels, or by labeling one and not the other.
  • labels that can be differentiated from each other are fluorescent dyes with different emission maxima. Differential imaging of the two fluorescent dyes can then be performed by focusing on the maxima.
  • cyanine dyes Cy3TM and Cy5.5TM, both products of Amersham Life Science, Inc., Arlington Heights, Illinois, USA.
  • Other cyanine dyes can be used, as well as fluorescein dyes, rhodamine dyes such as TAMRA (N,N,N',N'-tetramethyl-6-carboxyrhodamine) and Alexa dyes (sulfonated rhodamines), and phenanthridine dyes such as Texas Red. Stains that can be used with these dyes include SYPRO Ruby, SYPRO Rose, and SYPRO Orange. Examples of other stains are silver stain, copper stain, zinc stain, and Coomassie Brilliant Blue.
  • radioactive labeling can be achieved, for example, by using different radioisotopes whose signals can be distinguished from each other.
  • 32 P can be used as the label for one group of proteins and 35 S as the label for the other.
  • the signals can be separated using an attenuation material such as a thin copper layer.
  • Other combinations will be readily apparent to those skilled in radioisotope labeling.
  • Differentiation can also be obtained by using different types of labels, such as a fluorescent label on one group and staining the other group with silver stain. Other combinations and methods of differentiation will be readily apparent to those skilled in the art of protein labeling and detection.
  • the combining of the marker protein spots with the sample protein spots in the two-dimensional electropherograms may be achieved by simply adding the marker proteins to the samples prior to electrophoresis. Labeling of the marker proteins, sample proteins, or both can be performed prior to adding the marker proteins to the samples. In certain applications, it is preferable to leave the sample proteins unlabeled and to thus add the labeled marker proteins to unlabeled sample proteins. After electrophoresis, the gel can be stained with a fluorescent dye that is distinguishable from the fluorescent label that was pre-attached to the marker proteins. By not pre-attaching a label to the sample proteins, one avoids the risk of affecting the electrophoretic migration of the sample proteins due to changes in the mass and/or isoelectric point.
  • the amounts of marker proteins can vary and are not critical to the invention, but optimal results will generally be obtained if the amounts are selected such that clear detection of both the marker proteins and the sample proteins can be achieved.
  • the marker proteins will typically be added to the samples to concentrations ranging from about 5 nanograms to about 500 nanograms for each marker protein. Differences in signal intensities between the two groups of protein spots can be accommodated by adjustments in the scanning techniques.
  • the two-dimensional electrophoresis portion of the method of this invention can be performed according to known procedures, which may vary widely. In a typical procedure, the sample is first given a linear separation in an elongate or rod- shaped gel, with an electric potential imposed along the length of the gel.
  • Migration and separation thus occur along the gel axis until the proteins in the sample are distributed among zones positioned along the length of the gel. This is followed by placement of the elongate gel along one edge of a slab gel, and the imposition of an electric potential in a direction lying within the plane of the slab gel and perpendicular to the edge where the elongate gel is placed.
  • the proteins from each zone of the elongate gel migrate into the slab gel in the direction transverse to the axis of the elongate gel. The result is a two- dimensional array of protein spots in the slab gel.
  • the first mode may be one based on charge, such as isoelectric focusing
  • the second may be based on molecular weight.
  • the two dimensions of the separation may be based on the same parameter but may differ in the compositions of the two gels.
  • the two gels may differ in gel concentration or chemical components.
  • the two dimensions of the separation may be based on the same parameter and performed in gels of the same composition and concentration, but differ in a separation condition, such as a stepwise difference in pH, for example.
  • Still further alternatives are the use of a homogeneous gel in one of the two dimensions and a gradient gel in the other, the use of two different protein solubilizers in the two dimensions, or two different concentrations of the same protein solubilizer, and the use of a nonchanging buffer system in one dimension and a changing (gradient, for example) buffer system in another dimension.
  • each gel is scanned twice, once to obtain an image of the marker protein spots and once to obtain an image of the sample protein spots.
  • the order of the scans is not critical; either one can be performed first.
  • the two scans are performed in such a manner that they permit differentiation of the marker protein spots from the sample protein spots. This can be achieved by selectively scanning the marker protein spots in one scan and selectively scanning the sample protein spots in the other scan, or selectively scanning either the marker protein spots or the sample protein spots in one scan, scanning all spots in the other scan, and subtracting the signals from the first scan from those of the second scan.
  • each of the two scans is a selective scan, one detecting only the marker protein spots and the other only the sample protein spots.
  • This invention is directed to two-dimensional electrophoresis in general, although the most common separation media, and those that are preferred in the practice of this invention, are gels.
  • the type and composition of the gels used for either of the two dimensions are not critical to the invention and may vary.
  • Agarose gels and polyacrylamide gels are two prominent and common examples, and analogs of these gels such as formamide and urea gels can also be used.
  • the preferred gels are polyacrylamide gels. Scanning of the gels can be achieved by known techniques with commercially available scanning equipment. Examples of such equipment are multicolor imaging charge coupled devices and laser imaging systems. Other systems will be readily apparent to those skilled in the art. Selection of the appropriate scanning system will depend on the types of labels used and the manner in which the marker protein signals are differentiated from the sample protein signals.
  • the scanning data can be processed into digital form by conventional computer software of the type that is currently used for processing data of this kind, such as the various commercially available programs listed above or simple variations on these programs.
  • the two scans for each electropherogram in the matchset can be processed together by methods that use the marker protein data as landmarks and achieve the same or equivalent results as the prior art systems which rely on landmarking.
  • the data can be processed to identify individual proteins according to the parameters used as bases for the separations, match corresponding protein data among the various gels in the matchset, and compile and present the progression or other changes of the amounts of each protein throughout the entire sequence of gels.
  • MALDI-TOF Microx-Assisted Laser Desorption Ionization — Time of Flight

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Abstract

Des gels d'électrophorèse bidimensionnelle sont analysés par ensembles, chaque ensemble renfermant une multitude de gels, lesdits gels représentant généralement des échantillons prélevés à partir de la même source à des moments différents. Ce type d'analyse permet d'observer la progression et les modification séquentielles au niveau des compositions protéiniques des échantillons biologiques. La présente invention concerne des moyens permettant de standardiser, de normaliser, et d'établir des correspondances correctes entre les spots protéiques apparaissant dans les différents gels de l'ensemble, par l'addition de protéines de marquage formant des spots qui peuvent être détectés d'une manière permettant de les distinguer des spots représentant les protéines des échantillons. Les gels sont donc scannés deux fois, la première fois pour détecter les spots protéiques de marquage, la seconde pour détecter les spots protéiques des échantillons, la première étape servant à l'établissement de repères pour le traitement automatique des données du balayage. Cela évite d'avoir à procéder à un repérage manuel, tel que pratiqué dans les systèmes correspondants de l'état de la technique.
PCT/US2000/020268 1999-07-27 2000-07-25 Reperage automatique pour electrophorese bidimensionnelle Ceased WO2001007920A2 (fr)

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AU66085/00A AU6608500A (en) 1999-07-27 2000-07-25 Automated landmarking for two-dimensional electrophoresis

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US36178999A 1999-07-27 1999-07-27
US09/361,789 1999-07-27

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1264839A1 (fr) * 2001-06-07 2002-12-11 ProteoSys AG Méthode pour la séparation de mélanges de protéines
FR2833082A1 (fr) * 2001-12-03 2003-06-06 Centre Nat Rech Scient Detection positionnement et dosage de molecules biologiques separees sur une surface, en utilisant la mesure d'une propriete ou effet electrique
WO2003058230A1 (fr) * 2002-01-11 2003-07-17 Ludesi Ab Procede et programme informatique pour traitement d'image digitale
EP1577823A1 (fr) * 2004-03-16 2005-09-21 Centre National De La Recherche Scientifique (Cnrs) Méthode pour identifier des protéines inconnues par une séparation bi-dimensionelle e l'utilisation de marqueurs
US7781173B2 (en) 2003-09-25 2010-08-24 Life Technologies Corporation Homogeneous populations of molecules
US8012715B2 (en) 1997-01-08 2011-09-06 Life Technologies Corporation Methods for production of proteins

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4507233A (en) * 1981-04-22 1985-03-26 Oriental Yeast Co., Ltd. Colored molecular weight marker
US4592089A (en) * 1983-08-15 1986-05-27 Bio Image Corporation Electrophoretogram analytical image processing system
NZ215967A (en) * 1985-05-01 1988-05-30 Commw Serum Lab Commission Composition of peptides of known weight for determining weight of unknown peptide
WO1986006835A1 (fr) * 1985-05-06 1986-11-20 Wisconsin Alumni Research Foundation Procede d'etude d'expressions de genes et de diagnostic de maladies
JPH05281191A (ja) * 1991-04-25 1993-10-29 Toyobo Co Ltd 多標識系２次元電気泳動法
US5904822A (en) * 1996-11-13 1999-05-18 The University Of Iowa Research Foundation Methods and apparatus for analyzing electrophoresis gels

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8012715B2 (en) 1997-01-08 2011-09-06 Life Technologies Corporation Methods for production of proteins
US8389681B2 (en) 1997-01-08 2013-03-05 Life Technologies Corporation Methods for production of proteins
US9409942B2 (en) 1997-01-08 2016-08-09 Life Technologies Corporation Methods for production of proteins
EP1264839A1 (fr) * 2001-06-07 2002-12-11 ProteoSys AG Méthode pour la séparation de mélanges de protéines
FR2833082A1 (fr) * 2001-12-03 2003-06-06 Centre Nat Rech Scient Detection positionnement et dosage de molecules biologiques separees sur une surface, en utilisant la mesure d'une propriete ou effet electrique
WO2003048757A1 (fr) * 2001-12-03 2003-06-12 Centre National De La Recherche Scientifique (Cnrs) Detection positionnement et dosage de molecules biologiques separees sur une surface, en utilisant la mesure d'une propriete ou effet electrique.
WO2003058230A1 (fr) * 2002-01-11 2003-07-17 Ludesi Ab Procede et programme informatique pour traitement d'image digitale
US7781173B2 (en) 2003-09-25 2010-08-24 Life Technologies Corporation Homogeneous populations of molecules
EP1577823A1 (fr) * 2004-03-16 2005-09-21 Centre National De La Recherche Scientifique (Cnrs) Méthode pour identifier des protéines inconnues par une séparation bi-dimensionelle e l'utilisation de marqueurs
WO2005091199A3 (fr) * 2004-03-16 2005-11-24 Centre Nat Rech Scient Procede pour l'identification de proteines inconnues ou pour l'identification d'evenements de modification chimique subie par des proteines dans un echantillon

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AU6608500A (en) 2001-02-13

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