Antibody Array Analysis with Label-based Detection and Resolution of Protein Size
2008; Elsevier BV; Volume: 8; Issue: 2 Linguagem: Inglês
10.1074/mcp.m800171-mcp200
ISSN1535-9484
AutoresWeiwei Wu, Heidi Slåstad, Daniel de la Rosa Carrillo, Tom Frey, Geir E. Tjønnfjord, Eva Boretti, Hans‐Christian Aasheim, Václav Hořejšı́, Fridtjof Lund‐Johansen,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoElements from DNA microarray analysis, such as sample labeling and micro-spotting of capture reagents, have been successfully adapted to multiplex measurements of soluble cytokines. Application in cell biology is hampered by the lack of mono-specific antibodies and the fact that many proteins occur in complexes. Here, we incorporated a principle from Western blotting and resolved protein size as an additional parameter. Proteins from different cellular compartments were labeled and separated by size exclusion chromatography into 20 fractions. All were analyzed with replicate antibody arrays. The elution profiles of all antibody targets were compiled to color maps that resemble Western blots with bands of antibody reactivity across the size separation range (670–10 kDa). A new solid phase designed for processing in microwell plates was developed to handle the large number of samples. Antibodies were bound to protein G-coupled microspheres surface-labeled with 300 combinations of four fluorescent dyes. Fluorescence from particle color codes and the protein label were measured by high-speed flow cytometry. Cytoplasmic protein kinases were detected as bands near predictable elution points. For proteins with atypical elution characteristics or multiple contexts, two or more antibodies were used as internal references of specificity. Membrane proteins eluted near the void volume, and additional bands corresponding to intracellular forms were detected for several targets. Elution profiles of cyclin-dependent kinases (cdks), cyclins, and cyclin-dependent kinase inhibitors, were compatible with their occurrence in complexes that vary with the cell cycle phase and subcellular localization. A two-dimensional platform circumvents the need for mono-specific capture antibodies and extends the utility of antibody array analysis to studies of protein complexes. Elements from DNA microarray analysis, such as sample labeling and micro-spotting of capture reagents, have been successfully adapted to multiplex measurements of soluble cytokines. Application in cell biology is hampered by the lack of mono-specific antibodies and the fact that many proteins occur in complexes. Here, we incorporated a principle from Western blotting and resolved protein size as an additional parameter. Proteins from different cellular compartments were labeled and separated by size exclusion chromatography into 20 fractions. All were analyzed with replicate antibody arrays. The elution profiles of all antibody targets were compiled to color maps that resemble Western blots with bands of antibody reactivity across the size separation range (670–10 kDa). A new solid phase designed for processing in microwell plates was developed to handle the large number of samples. Antibodies were bound to protein G-coupled microspheres surface-labeled with 300 combinations of four fluorescent dyes. Fluorescence from particle color codes and the protein label were measured by high-speed flow cytometry. Cytoplasmic protein kinases were detected as bands near predictable elution points. For proteins with atypical elution characteristics or multiple contexts, two or more antibodies were used as internal references of specificity. Membrane proteins eluted near the void volume, and additional bands corresponding to intracellular forms were detected for several targets. Elution profiles of cyclin-dependent kinases (cdks), cyclins, and cyclin-dependent kinase inhibitors, were compatible with their occurrence in complexes that vary with the cell cycle phase and subcellular localization. A two-dimensional platform circumvents the need for mono-specific capture antibodies and extends the utility of antibody array analysis to studies of protein complexes. Cellular proteins interact with agonistic and antagonistic regulators, and their functions depend on post-translational modifications and sub-cellular localization. Although it is rational to measure multiple components simultaneously, large scale protein analysis remains a challenge. Mass spectrometry can be used to detect a thousand proteins in a sample and provides unmatched resolution of molecular detail (1Wu L. Han D.K. Overcoming the dynamic range problem in mass spectrometry-based shotgun proteomics.Expert Rev. Proteomics. 2006; 3: 611-619Crossref PubMed Scopus (75) Google Scholar, 2Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2778) Google Scholar, 3Rush J. Moritz A. Lee K.A. Guo A. Goss V.L. Spek E.J. Zhang H. Zha X.M. Polakiewicz R.D. Comb M.J. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.Nat. Biotechnol. 2005; 23: 94-101Crossref PubMed Scopus (954) Google Scholar). The protocols are, however, too laborious and time-consuming to be used for screening purposes. During the past years, efforts have been made to develop platforms for proteins that are analogous to DNA microarrays (4Haab B.B. Applications of antibody array platforms.Curr. Opin. Biotechnol. 2006; 17: 415-421Crossref PubMed Scopus (155) Google Scholar, 5Kingsmore S.F. Multiplexed protein measurement: technologies and applications of protein and antibody arrays.Nat. Rev. Drug Discov. 2006; 5: 310-320Crossref PubMed Scopus (597) Google Scholar, 6MacBeath G. Protein microarrays and proteomics.Nat. Genet. 2002; 32: 526-532Crossref PubMed Scopus (745) Google Scholar, 7Wingren C. Borrebaeck C.A. Antibody microarray analysis of directly labeled complex proteomes.Curr. Opin. Biotechnol. 2008; 19: 55-61Crossref PubMed Scopus (58) Google Scholar). Assays based on multiplexed capture antibodies and matched detection reagents are available from several sources. The sandwich format provides dual specificity, but available assays are mainly limited to covering cytokines and a few selected signaling proteins. Moreover, signal-to-noise ratios decline when the number of detection reagents exceeds 20–40 (4Haab B.B. Applications of antibody array platforms.Curr. Opin. Biotechnol. 2006; 17: 415-421Crossref PubMed Scopus (155) Google Scholar, 5Kingsmore S.F. Multiplexed protein measurement: technologies and applications of protein and antibody arrays.Nat. Rev. Drug Discov. 2006; 5: 310-320Crossref PubMed Scopus (597) Google Scholar, 6MacBeath G. Protein microarrays and proteomics.Nat. Genet. 2002; 32: 526-532Crossref PubMed Scopus (745) Google Scholar, 7Wingren C. Borrebaeck C.A. Antibody microarray analysis of directly labeled complex proteomes.Curr. Opin. Biotechnol. 2008; 19: 55-61Crossref PubMed Scopus (58) Google Scholar). High throughput analysis of signaling proteins is possible with the reverse protein arrays described by Petricoin and colleagues (8VanMeter A. Signore M. Pierobon M. Espina V. Liotta L.A. Petricoin 3rd, E.F. Reverse-phase protein microarrays: application to biomarker discovery and translational medicine.Expert Rev. Mol. Diagn. 2007; 7: 625-633Crossref PubMed Scopus (68) Google Scholar) In these assays, the samples rather than the affinity reagents are multiplexed, and each array is labeled with a single or two antibodies.The platform that currently allows detection of the highest number of proteins in a single sample is based on the use of protein labels for detection. All the proteins in the sample are reacted with haptens or fluorescent dyes, and the total amount of protein associated with each capture reagent is measured (9Haab B.B. Dunham M.J. Brown P.O. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions.Genome Biol. 2001; 2RESEARCH0004Crossref PubMed Google Scholar, 10Ghatnekar-Nilsson S. Dexlin L. Wingren C. Montelius L. Borrebaeck C.A. Design of atto-vial based recombinant antibody arrays combined with a planar wave-guide detection system.Proteomics. 2007; 7: 540-547Crossref PubMed Scopus (28) Google Scholar). Sample labeling is adopted from DNA microarray technology and could in principle allow proteome-wide analysis. For cytokine measurement, the specificity and sensitivity is comparable with that of sandwich assays (11Wingren C. Ingvarsson J. Dexlin L. Szul D. Borrebaeck C.A. Design of recombinant antibody microarrays for complex proteome analysis: choice of sample labeling-tag and solid support.Proteomics. 2007; 7: 3055-3065Crossref PubMed Scopus (95) Google Scholar). Extending the principle to cellular proteins is, however, complicated by the lack of mono-specific capture reagents. The high content of protein complexes in cell lysates complicates analysis further (6MacBeath G. Protein microarrays and proteomics.Nat. Genet. 2002; 32: 526-532Crossref PubMed Scopus (745) Google Scholar, 12Gavin A.C. Aloy P. Grandi P. Krause R. Boesche M. Marzioch M. Rau C. Jensen L.J. Bastuck S. Dumpelfeld B. Edelmann A. Heurtier M.A. Hoffman V. Hoefert C. Klein K. Hudak M. Michon A.M. Schelder M. Schirle M. Remor M. Rudi T. Hooper S. Bauer A. Bouwmeester T. Casari G. Drewes G. Neubauer G. Rick J.M. Kuster B. Bork P. Russell R.B. Superti-Furga G. Proteome survey reveals modularity of the yeast cell machinery.Nature. 2006; 440: 631-636Crossref PubMed Scopus (2104) Google Scholar).In Western blotting, antibody reactivity is resolved against protein size, to detect specific binding and cross-reactivity as discrete bands. Measurement with antibody arrays and label-based detection is comparable with Western blotting where the lanes have been compressed into a single band. The fact that non-specific bands in Western blots are frequent and sample-dependent is a strong argument against the current analysis platform (6MacBeath G. Protein microarrays and proteomics.Nat. Genet. 2002; 32: 526-532Crossref PubMed Scopus (745) Google Scholar). On the other hand, the principle of resolving antibody reactivity against protein size could be adapted to array-based analysis. Here, we fractionated proteins by size exclusion chromatography and analyzed a series of fractions with replicate arrays to resolve protein size as a second parameter. Native separation yields liquid fractions of proteins in their functional context. A given protein may elute in non-overlapping fractions depending on its occurrence as a monomer or part of a complex. To compensate for the unpredictable elution profiles, we produced arrays containing two or more antibodies to each target as internal references of specificity.Bead suspension arrays can be processed in microwell plates and are therefore well suited for analyzing a large number of sample fractions. A drawback is limited multiplexing capacity (5Kingsmore S.F. Multiplexed protein measurement: technologies and applications of protein and antibody arrays.Nat. Rev. Drug Discov. 2006; 5: 310-320Crossref PubMed Scopus (597) Google Scholar). The most sophisticated platform resolves 100 populations with different emissions of two fluorescent dyes and requires a dedicated instrument (13Schwenk J.M. Lindberg J. Sundberg M. Uhlen M. Nilsson P. Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics.Mol. Cell. Proteomics. 2007; 6: 125-132Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To extend multiplexing, we examined whether polymer particles could be colored by surface labeling. A wide range of available dyes could potentially be used to generate complex color codes and allow the use of standard flow cytometers. We also determined whether Fc-binding affinity reagents could be used to anchor antibodies to bead suspension arrays. Affinity-based coupling would be compatible with small amounts of crude antibody preparations but could potentially lead to crossover of antibodies between the particles.DISCUSSIONAntibody array measurement with label-based detection was first published in 2001, and later studies have verified its utility for large scale measurement of cytokines (9Haab B.B. Dunham M.J. Brown P.O. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions.Genome Biol. 2001; 2RESEARCH0004Crossref PubMed Google Scholar). Yet, small scale assays with matched detection antibodies are by far dominating commercial products. Most likely, this reflects the difficulties associated with obtaining mono-specific antibodies and validating their performance. In an unfractionated sample, the proportion of the signal that derives from the intended target is not assessed. Because antibody cross-reactivity is frequent and sample-dependent, assumption of mono-specificity must be based on very extensive testing. Resolving protein size as a second parameter provides a solution to the specificity problem because different proteins binding to the same antibody are detected independently. The advantage is significant because very few antibodies have been shown to capture single target from a cell lysate. For example, MacBeath (6MacBeath G. Protein microarrays and proteomics.Nat. Genet. 2002; 32: 526-532Crossref PubMed Scopus (745) Google Scholar) reported that only 5% of commercial antibodies were useful for antibody array analysis of cell lysates.In principle, antibody array analysis is a multiplexed form of immunoprecipitation. Provided that the intended target can be resolved from cross-reactivity, any antibody capable of immunoprecipitating its target should be useful. Still, we found that many reagents recommended by the manufacturer for use in immunoprecipitation failed to capture the labeled target (see Fig. 5B). It is possible that the labels used for detection interfere with the binding of some antibodies. The amount of amine-reactive label used here could theoretically modify 30% of the lysines. On the other hand, it is worth noting that immunoprecipitation is not usually tested very extensively by antibody manufacturers. When test results are available, captured proteins are typically detected by staining Western blots with antibodies that are specific for the intended target. Comparative results for different reagents are rarely shown. The need for better and more systematic quality control of antibodies is underscored by the experience from the Protein Atlas project led by Mathias Uhlen (13Schwenk J.M. Lindberg J. Sundberg M. Uhlen M. Nilsson P. Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics.Mol. Cell. Proteomics. 2007; 6: 125-132Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Only 35% of the commercial reagents they tested performed satisfactory in standard applications such as immunohistochemistry and Western blotting (23Blow N. Antibodies: the generation game.Nature. 2007; 447: 741-744Crossref PubMed Scopus (38) Google Scholar). Antibody arrays may contribute significantly to better validation by allowing parallel testing of large numbers of antibodies under identical conditions.New and elegant techniques for printing antibody arrays on slides allow far more multiplexing than the particles used here (10Ghatnekar-Nilsson S. Dexlin L. Wingren C. Montelius L. Borrebaeck C.A. Design of atto-vial based recombinant antibody arrays combined with a planar wave-guide detection system.Proteomics. 2007; 7: 540-547Crossref PubMed Scopus (28) Google Scholar). Slides cannot be handled in microwell plates, but it is certainly possible to print replicate arrays on a single slide to simplify sample processing. The most important advantage of particles is wider access to multiplexing technology. Most likely, antibody arrays will have to be tailored for specific applications, and few laboratories are equipped to produce high quality planar arrays. The particle format is flexible and simple to scale up. The production and use of a 300-plex may be challenging for new users and requires access to a three-laser flow cytometer. However, even a 30-plex would represent a significant improvement compared with measuring proteins one at a time. This level of multiplexing should be achievable in most laboratories with access to a flow cytometer, either by use of colored particles on the market or by producing arrays as described here.Ideally, antibody array analysis would be analogous to single-chip DNA microarray measurement. Sample fractionation is time-consuming, and algorithms for analyzing array results are designed for a single data point per target (17Eisen M.B. Spellman P.T. Brown P.O. Botstein D. Cluster analysis and display of genome-wide expression patterns.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14863-14868Crossref PubMed Scopus (13137) Google Scholar). It has been suggested that a new generation of affinity reagents dedicated for antibody array analysis will solve the specificity problem (7Wingren C. Borrebaeck C.A. Antibody microarray analysis of directly labeled complex proteomes.Curr. Opin. Biotechnol. 2008; 19: 55-61Crossref PubMed Scopus (58) Google Scholar). Encouraging data have been published for antibodies to several inflammatory mediators in serum (11Wingren C. Ingvarsson J. Dexlin L. Szul D. Borrebaeck C.A. Design of recombinant antibody microarrays for complex proteome analysis: choice of sample labeling-tag and solid support.Proteomics. 2007; 7: 3055-3065Crossref PubMed Scopus (95) Google Scholar). It remains, however, to be determined if reagents with similar specificity can be obtained for most intracellular proteins or solve the problem of protein complexes. Several immunologists hold the opinion that polyspecificity is a fundamental feature of immune receptor recognition (24Wucherpfennig K.W. Allen P.M. Celada F. Cohen I.R. De Boer R. Garcia K.C. Goldstein B. Greenspan R. Hafler D. Hodgkin P. Huseby E.S. Krakauer D.C. Nemazee D. Perelson A.S. Pinilla C. Strong R.K. Sercarz E.E. Polyspecificity of T cell and B cell receptor recognition.Semin. Immunol. 2007; 19: 216-224Crossref PubMed Scopus (156) Google Scholar). At best, it will take many years to replace existing antibodies with a new generation of mono-specific reagents, and producing reagents that are specific for all their contexts is beyond current biotechnology. On short term, efforts to improve protein separation techniques and labeling conditions are likely to be more fruitful than attempting to produce truly mono-specific reagents.Another argument in favor of fractionation is that important information may be obtained about subcellular localization and protein context. Cellular proteomes are modular, and measurement of complexes may be more important than assessment of total protein levels (12Gavin A.C. Aloy P. Grandi P. Krause R. Boesche M. Marzioch M. Rau C. Jensen L.J. Bastuck S. Dumpelfeld B. Edelmann A. Heurtier M.A. Hoffman V. Hoefert C. Klein K. Hudak M. Michon A.M. Schelder M. Schirle M. Remor M. Rudi T. Hooper S. Bauer A. Bouwmeester T. Casari G. Drewes G. Neubauer G. Rick J.M. Kuster B. Bork P. Russell R.B. Superti-Furga G. Proteome survey reveals modularity of the yeast cell machinery.Nature. 2006; 440: 631-636Crossref PubMed Scopus (2104) Google Scholar). So far, interactomics has largely been based on protocols that involve transfection of yeast or cell lines with affinity-tagged proteins (25Kiemer L. Cesareni G. Comparative interactomics: comparing apples and pears?.Trends Biotechnol. 2007; 25: 448-454Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). An intriguing aspect of this study is the possibility of screening for protein complexes in primary cells and patient samples. The assay is currently limited to detecting a single component of each complex. Moreover, the chromatography resin only resolves complexes smaller than 600 kDa. This is clearly a limitation, because protein concentration was highest in the early fractions, and a large number of antibodies captured protein near the void volume. A rational next step is to expand the fractionation range and use affinity chromatography downstream. This may facilitate better identification of complexes and candidate interaction partners. The particle platform is compatible with extensive fractionation because samples are handled in microwell plates and each array analyzed within seconds. To characterize all the components of the identified complexes, it will be necessary to develop high throughput protocols for analysis of immunoprecipitates by mass spectrometry. Array-based screening of chromatography fractions followed by mass spectrometry of antibody-isolated targets may provide a powerful combination of throughput and resolution that is lacking in current interactomics. In conclusion, the present study shows that a two-dimensional analysis platform addresses many of the specificity problems associated with array-based proteomics and allows large scale detection of protein complexes. Cellular proteins interact with agonistic and antagonistic regulators, and their functions depend on post-translational modifications and sub-cellular localization. Although it is rational to measure multiple components simultaneously, large scale protein analysis remains a challenge. Mass spectrometry can be used to detect a thousand proteins in a sample and provides unmatched resolution of molecular detail (1Wu L. Han D.K. Overcoming the dynamic range problem in mass spectrometry-based shotgun proteomics.Expert Rev. Proteomics. 2006; 3: 611-619Crossref PubMed Scopus (75) Google Scholar, 2Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2778) Google Scholar, 3Rush J. Moritz A. Lee K.A. Guo A. Goss V.L. Spek E.J. Zhang H. Zha X.M. Polakiewicz R.D. Comb M.J. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.Nat. Biotechnol. 2005; 23: 94-101Crossref PubMed Scopus (954) Google Scholar). The protocols are, however, too laborious and time-consuming to be used for screening purposes. During the past years, efforts have been made to develop platforms for proteins that are analogous to DNA microarrays (4Haab B.B. Applications of antibody array platforms.Curr. Opin. Biotechnol. 2006; 17: 415-421Crossref PubMed Scopus (155) Google Scholar, 5Kingsmore S.F. Multiplexed protein measurement: technologies and applications of protein and antibody arrays.Nat. Rev. Drug Discov. 2006; 5: 310-320Crossref PubMed Scopus (597) Google Scholar, 6MacBeath G. Protein microarrays and proteomics.Nat. Genet. 2002; 32: 526-532Crossref PubMed Scopus (745) Google Scholar, 7Wingren C. Borrebaeck C.A. Antibody microarray analysis of directly labeled complex proteomes.Curr. Opin. Biotechnol. 2008; 19: 55-61Crossref PubMed Scopus (58) Google Scholar). Assays based on multiplexed capture antibodies and matched detection reagents are available from several sources. The sandwich format provides dual specificity, but available assays are mainly limited to covering cytokines and a few selected signaling proteins. Moreover, signal-to-noise ratios decline when the number of detection reagents exceeds 20–40 (4Haab B.B. Applications of antibody array platforms.Curr. Opin. Biotechnol. 2006; 17: 415-421Crossref PubMed Scopus (155) Google Scholar, 5Kingsmore S.F. Multiplexed protein measurement: technologies and applications of protein and antibody arrays.Nat. Rev. Drug Discov. 2006; 5: 310-320Crossref PubMed Scopus (597) Google Scholar, 6MacBeath G. Protein microarrays and proteomics.Nat. Genet. 2002; 32: 526-532Crossref PubMed Scopus (745) Google Scholar, 7Wingren C. Borrebaeck C.A. Antibody microarray analysis of directly labeled complex proteomes.Curr. Opin. Biotechnol. 2008; 19: 55-61Crossref PubMed Scopus (58) Google Scholar). High throughput analysis of signaling proteins is possible with the reverse protein arrays described by Petricoin and colleagues (8VanMeter A. Signore M. Pierobon M. Espina V. Liotta L.A. Petricoin 3rd, E.F. Reverse-phase protein microarrays: application to biomarker discovery and translational medicine.Expert Rev. Mol. Diagn. 2007; 7: 625-633Crossref PubMed Scopus (68) Google Scholar) In these assays, the samples rather than the affinity reagents are multiplexed, and each array is labeled with a single or two antibodies. The platform that currently allows detection of the highest number of proteins in a single sample is based on the use of protein labels for detection. All the proteins in the sample are reacted with haptens or fluorescent dyes, and the total amount of protein associated with each capture reagent is measured (9Haab B.B. Dunham M.J. Brown P.O. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions.Genome Biol. 2001; 2RESEARCH0004Crossref PubMed Google Scholar, 10Ghatnekar-Nilsson S. Dexlin L. Wingren C. Montelius L. Borrebaeck C.A. Design of atto-vial based recombinant antibody arrays combined with a planar wave-guide detection system.Proteomics. 2007; 7: 540-547Crossref PubMed Scopus (28) Google Scholar). Sample labeling is adopted from DNA microarray technology and could in principle allow proteome-wide analysis. For cytokine measurement, the specificity and sensitivity is comparable with that of sandwich assays (11Wingren C. Ingvarsson J. Dexlin L. Szul D. Borrebaeck C.A. Design of recombinant antibody microarrays for complex proteome analysis: choice of sample labeling-tag and solid support.Proteomics. 2007; 7: 3055-3065Crossref PubMed Scopus (95) Google Scholar). Extending the principle to cellular proteins is, however, complicated by the lack of mono-specific capture reagents. The high content of protein complexes in cell lysates complicates analysis further (6MacBeath G. Protein microarrays and proteomics.Nat. Genet. 2002; 32: 526-532Crossref PubMed Scopus (745) Google Scholar, 12Gavin A.C. Aloy P. Grandi P. Krause R. Boesche M. Marzioch M. Rau C. Jensen L.J. Bastuck S. Dumpelfeld B. Edelmann A. Heurtier M.A. Hoffman V. Hoefert C. Klein K. Hudak M. Michon A.M. Schelder M. Schirle M. Remor M. Rudi T. Hooper S. Bauer A. Bouwmeester T. Casari G. Drewes G. Neubauer G. Rick J.M. Kuster B. Bork P. Russell R.B. Superti-Furga G. Proteome survey reveals modularity of the yeast cell machinery.Nature. 2006; 440: 631-636Crossref PubMed Scopus (2104) Google Scholar). In Western blotting, antibody reactivity is resolved against protein size, to detect specific binding and cross-reactivity as discrete bands. Measurement with antibody arrays and label-based detection is comparable with Western blotting where the lanes have been compressed into a single band. The fact that non-specific bands in Western blots are frequent and sample-dependent is a strong argument against the current analysis platform (6MacBeath G. Protein microarrays and proteomics.Nat. Genet. 2002; 32: 526-532Crossref PubMed Scopus (745) Google Scholar). On the other hand, the principle of resolving antibody reactivity against protein size could be adapted to array-based analysis. Here, we fractionated proteins by size exclusion chromatography and analyzed a series of fractions with replicate arrays to resolve protein size as a second parameter. Native separation yields liquid fractions of proteins in their functional context. A given protein may elute in non-overlapping fractions depending on its occurrence as a monomer or part of a complex. To compensate for the unpredictable elution profiles, we produced arrays containing two or more antibodies to each target as internal references of specificity. Bead suspension arrays can be processed in microwell plates and are therefore well suited for analyzing a large number of sample fractions. A drawback is limited multiplexing capacity (5Kingsmore S.F. Multiplexed protein measurement: technologies and applications of protein and antibody arrays.Nat. Rev. Drug Discov. 2006; 5: 310-320Crossref PubMed Scopus (597) Google Scholar). The most sophisticated platform resolves 100 populations with different emissions of two fluorescent dyes and requires a dedicated instrument (13Schwenk J.M. Lindberg J. Sundberg M. Uhlen M. Nilsson P. Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics.Mol. Cell. Proteomics. 2007; 6: 125-132Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To extend multiplexing, we examined whether polymer particles could be colored by surface labeling. A wide range of available dyes could potentially be used to generate complex color codes and allow the use of standard flow cytometers. We also determined whether Fc-binding affinity reagents could be used to anchor antibodies to bead suspension arrays. Affinity-based coupling would be compatible with small amounts of crude antibody preparations but could potentially lead to crossover of antibodies between the particles. DISCUSSIONAntibody array measurement with label-based detection was first published in 2001, and later studies have verified its utility for large scale measurement of cytokines (9Haab B.B. Dunham M.J. Brown P.O. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions.Genome Biol. 2001; 2RESEARCH0004Crossref PubMed Google Scholar). Yet, small scale assays with matched detection antibodies are by far dominating commercial products. Most likely, this reflects the difficulties associated with obtaining mono-specific antibodies and validating their performance. In an unfractionated sample, the proportion of the signal that derives from the intended target is not assessed. Because antibody cross-reactivity is frequent and sample-dependent, assumption of mono-specificity must be based on very extensive testing. Resolving protein size as a second parameter provides a solution to the specificity problem because different proteins binding to the same antibody are detected independently. The advantage is significant because very few antibodies have been shown to capture single target from a cell lysate. For example, MacBeath (6MacBeath G. Protein microarrays and proteomics.Nat. Genet. 2002; 32: 526-532Crossref PubMed Scopus (745) Google Scholar) reported that only 5% of commercial antibodies were useful for antibody array analysis of cell lysates.In principle, antibody array analysis is a multiplexed form of immunoprecipitation. Provided that the intended target can be resolved from cross-reactivity, any antibody capable of immunoprecipitating its target should be useful. Still, we found that many reagents recommended by the manufacturer for use in immunoprecipitation failed to capture the labeled target (see Fig. 5B). It is possible that the labels used for detection interfere with the binding of some antibodies. The amount of amine-reactive label used here could theoretically modify 30% of the lysines. On the other hand, it is worth noting that immunoprecipitation is not usually tested very extensively by antibody manufacturers. When test results are available, captured proteins are typically detected by staining Western blots with antibodies that are specific for the intended target. Comparative results for different reagents are rarely shown. The need for better and more systematic quality control of antibodies is underscored by the experience from the Protein Atlas project led by Mathias Uhlen (13Schwenk J.M. Lindberg J. Sundberg M. Uhlen M. Nilsson P. Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics.Mol. Cell. Proteomics. 2007; 6: 125-132Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Only 35% of the commercial reagents they tested performed satisfactory in standard applications such as immunohistochemistry and Western blotting (23Blow N. Antibodies: the generation game.Nature. 2007; 447: 741-744Crossref PubMed Scopus (38) Google Scholar). Antibody arrays may contribute significantly to better validation by allowing parallel testing of large numbers of antibodies under identical conditions.New and elegant techniques for printing antibody arrays on slides allow far more multiplexing than the particles used here (10Ghatnekar-Nilsson S. Dexlin L. Wingren C. Montelius L. Borrebaeck C.A. Design of atto-vial based recombinant antibody arrays combined with a planar wave-guide detection system.Proteomics. 2007; 7: 540-547Crossref PubMed Scopus (28) Google Scholar). Slides cannot be handled in microwell plates, but it is certainly possible to print replicate arrays on a single slide to simplify sample processing. The most important advantage of particles is wider access to multiplexing technology. Most likely, antibody arrays will have to be tailored for specific applications, and few laboratories are equipped to produce high quality planar arrays. The particle format is flexible and simple to scale up. The production and use of a 300-plex may be challenging for new users and requires access to a three-laser flow cytometer. However, even a 30-plex would represent a significant improvement compared with measuring proteins one at a time. This level of multiplexing should be achievable in most laboratories with access to a flow cytometer, either by use of colored particles on the market or by producing arrays as described here.Ideally, antibody array analysis would be analogous to single-chip DNA microarray measurement. Sample fractionation is time-consuming, and algorithms for analyzing array results are designed for a single data point per target (17Eisen M.B. Spellman P.T. Brown P.O. Botstein D. Cluster analysis and display of genome-wide expression patterns.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14863-14868Crossref PubMed Scopus (13137) Google Scholar). It has been suggested that a new generation of affinity reagents dedicated for antibody array analysis will solve the specificity problem (7Wingren C. Borrebaeck C.A. Antibody microarray analysis of directly labeled complex proteomes.Curr. Opin. Biotechnol. 2008; 19: 55-61Crossref PubMed Scopus (58) Google Scholar). Encouraging data have been published for antibodies to several inflammatory mediators in serum (11Wingren C. Ingvarsson J. Dexlin L. Szul D. Borrebaeck C.A. Design of recombinant antibody microarrays for complex proteome analysis: choice of sample labeling-tag and solid support.Proteomics. 2007; 7: 3055-3065Crossref PubMed Scopus (95) Google Scholar). It remains, however, to be determined if reagents with similar specificity can be obtained for most intracellular proteins or solve the problem of protein complexes. Several immunologists hold the opinion that polyspecificity is a fundamental feature of immune receptor recognition (24Wucherpfennig K.W. Allen P.M. Celada F. Cohen I.R. De Boer R. Garcia K.C. Goldstein B. Greenspan R. Hafler D. Hodgkin P. Huseby E.S. Krakauer D.C. Nemazee D. Perelson A.S. Pinilla C. Strong R.K. Sercarz E.E. Polyspecificity of T cell and B cell receptor recognition.Semin. Immunol. 2007; 19: 216-224Crossref PubMed Scopus (156) Google Scholar). At best, it will take many years to replace existing antibodies with a new generation of mono-specific reagents, and producing reagents that are specific for all their contexts is beyond current biotechnology. On short term, efforts to improve protein separation techniques and labeling conditions are likely to be more fruitful than attempting to produce truly mono-specific reagents.Another argument in favor of fractionation is that important information may be obtained about subcellular localization and protein context. Cellular proteomes are modular, and measurement of complexes may be more important than assessment of total protein levels (12Gavin A.C. Aloy P. Grandi P. Krause R. Boesche M. Marzioch M. Rau C. Jensen L.J. Bastuck S. Dumpelfeld B. Edelmann A. Heurtier M.A. Hoffman V. Hoefert C. Klein K. Hudak M. Michon A.M. Schelder M. Schirle M. Remor M. Rudi T. Hooper S. Bauer A. Bouwmeester T. Casari G. Drewes G. Neubauer G. Rick J.M. Kuster B. Bork P. Russell R.B. Superti-Furga G. Proteome survey reveals modularity of the yeast cell machinery.Nature. 2006; 440: 631-636Crossref PubMed Scopus (2104) Google Scholar). So far, interactomics has largely been based on protocols that involve transfection of yeast or cell lines with affinity-tagged proteins (25Kiemer L. Cesareni G. Comparative interactomics: comparing apples and pears?.Trends Biotechnol. 2007; 25: 448-454Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). An intriguing aspect of this study is the possibility of screening for protein complexes in primary cells and patient samples. The assay is currently limited to detecting a single component of each complex. Moreover, the chromatography resin only resolves complexes smaller than 600 kDa. This is clearly a limitation, because protein concentration was highest in the early fractions, and a large number of antibodies captured protein near the void volume. A rational next step is to expand the fractionation range and use affinity chromatography downstream. This may facilitate better identification of complexes and candidate interaction partners. The particle platform is compatible with extensive fractionation because samples are handled in microwell plates and each array analyzed within seconds. To characterize all the components of the identified complexes, it will be necessary to develop high throughput protocols for analysis of immunoprecipitates by mass spectrometry. Array-based screening of chromatography fractions followed by mass spectrometry of antibody-isolated targets may provide a powerful combination of throughput and resolution that is lacking in current interactomics. In conclusion, the present study shows that a two-dimensional analysis platform addresses many of the specificity problems associated with array-based proteomics and allows large scale detection of protein complexes. Antibody array measurement with label-based detection was first published in 2001, and later studies have verified its utility for large scale measurement of cytokines (9Haab B.B. Dunham M.J. Brown P.O. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions.Genome Biol. 2001; 2RESEARCH0004Crossref PubMed Google Scholar). Yet, small scale assays with matched detection antibodies are by far dominating commercial products. Most likely, this reflects the difficulties associated with obtaining mono-specific antibodies and validating their performance. In an unfractionated sample, the proportion of the signal that derives from the intended target is not assessed. Because antibody cross-reactivity is frequent and sample-dependent, assumption of mono-specificity must be based on very extensive testing. Resolving protein size as a second parameter provides a solution to the specificity problem because different proteins binding to the same antibody are detected independently. The advantage is significant because very few antibodies have been shown to capture single target from a cell lysate. For example, MacBeath (6MacBeath G. Protein microarrays and proteomics.Nat. Genet. 2002; 32: 526-532Crossref PubMed Scopus (745) Google Scholar) reported that only 5% of commercial antibodies were useful for antibody array analysis of cell lysates. In principle, antibody array analysis is a multiplexed form of immunoprecipitation. Provided that the intended target can be resolved from cross-reactivity, any antibody capable of immunoprecipitating its target should be useful. Still, we found that many reagents recommended by the manufacturer for use in immunoprecipitation failed to capture the labeled target (see Fig. 5B). It is possible that the labels used for detection interfere with the binding of some antibodies. The amount of amine-reactive label used here could theoretically modify 30% of the lysines. On the other hand, it is worth noting that immunoprecipitation is not usually tested very extensively by antibody manufacturers. When test results are available, captured proteins are typically detected by staining Western blots with antibodies that are specific for the intended target. Comparative results for different reagents are rarely shown. The need for better and more systematic quality control of antibodies is underscored by the experience from the Protein Atlas project led by Mathias Uhlen (13Schwenk J.M. Lindberg J. Sundberg M. Uhlen M. Nilsson P. Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics.Mol. Cell. Proteomics. 2007; 6: 125-132Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Only 35% of the commercial reagents they tested performed satisfactory in standard applications such as immunohistochemistry and Western blotting (23Blow N. Antibodies: the generation game.Nature. 2007; 447: 741-744Crossref PubMed Scopus (38) Google Scholar). Antibody arrays may contribute significantly to better validation by allowing parallel testing of large numbers of antibodies under identical conditions. New and elegant techniques for printing antibody arrays on slides allow far more multiplexing than the particles used here (10Ghatnekar-Nilsson S. Dexlin L. Wingren C. Montelius L. Borrebaeck C.A. Design of atto-vial based recombinant antibody arrays combined with a planar wave-guide detection system.Proteomics. 2007; 7: 540-547Crossref PubMed Scopus (28) Google Scholar). Slides cannot be handled in microwell plates, but it is certainly possible to print replicate arrays on a single slide to simplify sample processing. The most important advantage of particles is wider access to multiplexing technology. Most likely, antibody arrays will have to be tailored for specific applications, and few laboratories are equipped to produce high quality planar arrays. The particle format is flexible and simple to scale up. The production and use of a 300-plex may be challenging for new users and requires access to a three-laser flow cytometer. However, even a 30-plex would represent a significant improvement compared with measuring proteins one at a time. This level of multiplexing should be achievable in most laboratories with access to a flow cytometer, either by use of colored particles on the market or by producing arrays as described here. Ideally, antibody array analysis would be analogous to single-chip DNA microarray measurement. Sample fractionation is time-consuming, and algorithms for analyzing array results are designed for a single data point per target (17Eisen M.B. Spellman P.T. Brown P.O. Botstein D. Cluster analysis and display of genome-wide expression patterns.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14863-14868Crossref PubMed Scopus (13137) Google Scholar). It has been suggested that a new generation of affinity reagents dedicated for antibody array analysis will solve the specificity problem (7Wingren C. Borrebaeck C.A. Antibody microarray analysis of directly labeled complex proteomes.Curr. Opin. Biotechnol. 2008; 19: 55-61Crossref PubMed Scopus (58) Google Scholar). Encouraging data have been published for antibodies to several inflammatory mediators in serum (11Wingren C. Ingvarsson J. Dexlin L. Szul D. Borrebaeck C.A. Design of recombinant antibody microarrays for complex proteome analysis: choice of sample labeling-tag and solid support.Proteomics. 2007; 7: 3055-3065Crossref PubMed Scopus (95) Google Scholar). It remains, however, to be determined if reagents with similar specificity can be obtained for most intracellular proteins or solve the problem of protein complexes. Several immunologists hold the opinion that polyspecificity is a fundamental feature of immune receptor recognition (24Wucherpfennig K.W. Allen P.M. Celada F. Cohen I.R. De Boer R. Garcia K.C. Goldstein B. Greenspan R. Hafler D. Hodgkin P. Huseby E.S. Krakauer D.C. Nemazee D. Perelson A.S. Pinilla C. Strong R.K. Sercarz E.E. Polyspecificity of T cell and B cell receptor recognition.Semin. Immunol. 2007; 19: 216-224Crossref PubMed Scopus (156) Google Scholar). At best, it will take many years to replace existing antibodies with a new generation of mono-specific reagents, and producing reagents that are specific for all their contexts is beyond current biotechnology. On short term, efforts to improve protein separation techniques and labeling conditions are likely to be more fruitful than attempting to produce truly mono-specific reagents. Another argument in favor of fractionation is that important information may be obtained about subcellular localization and protein context. Cellular proteomes are modular, and measurement of complexes may be more important than assessment of total protein levels (12Gavin A.C. Aloy P. Grandi P. Krause R. Boesche M. Marzioch M. Rau C. Jensen L.J. Bastuck S. Dumpelfeld B. Edelmann A. Heurtier M.A. Hoffman V. Hoefert C. Klein K. Hudak M. Michon A.M. Schelder M. Schirle M. Remor M. Rudi T. Hooper S. Bauer A. Bouwmeester T. Casari G. Drewes G. Neubauer G. Rick J.M. Kuster B. Bork P. Russell R.B. Superti-Furga G. Proteome survey reveals modularity of the yeast cell machinery.Nature. 2006; 440: 631-636Crossref PubMed Scopus (2104) Google Scholar). So far, interactomics has largely been based on protocols that involve transfection of yeast or cell lines with affinity-tagged proteins (25Kiemer L. Cesareni G. Comparative interactomics: comparing apples and pears?.Trends Biotechnol. 2007; 25: 448-454Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). An intriguing aspect of this study is the possibility of screening for protein complexes in primary cells and patient samples. The assay is currently limited to detecting a single component of each complex. Moreover, the chromatography resin only resolves complexes smaller than 600 kDa. This is clearly a limitation, because protein concentration was highest in the early fractions, and a large number of antibodies captured protein near the void volume. A rational next step is to expand the fractionation range and use affinity chromatography downstream. This may facilitate better identification of complexes and candidate interaction partners. The particle platform is compatible with extensive fractionation because samples are handled in microwell plates and each array analyzed within seconds. To characterize all the components of the identified complexes, it will be necessary to develop high throughput protocols for analysis of immunoprecipitates by mass spectrometry. Array-based screening of chromatography fractions followed by mass spectrometry of antibody-isolated targets may provide a powerful combination of throughput and resolution that is lacking in current interactomics. In conclusion, the present study shows that a two-dimensional analysis platform addresses many of the specificity problems associated with array-based proteomics and allows large scale detection of protein complexes.
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