The Association of Biomolecular Resource Facilities Proteomics Research Group 2006 Study

Abstract

The determination of differences in relative protein abundance is a critical aspect of proteomics research that is increasingly used to answer diverse biological questions. The Association of Biomolecular Resource Facilities Proteomics Research Group 2006 study was a quantitative proteomics project in which the aim was to determine the identity and the relative amounts of eight proteins in two mixtures. There are numerous methodologies available to study the relative abundance of proteins between samples, but to date, there are few examples of studies that have compared these different approaches. For the 2006 Proteomics Research Group study, there were 52 participants who used a wide variety of gel electrophoresis-, HPLC-, and mass spectrometry-based methods for relative quantitation. The quantitative data arising from this study were evaluated along with several other experimental details relevant to the methodologies used. The determination of differences in relative protein abundance is a critical aspect of proteomics research that is increasingly used to answer diverse biological questions. The Association of Biomolecular Resource Facilities Proteomics Research Group 2006 study was a quantitative proteomics project in which the aim was to determine the identity and the relative amounts of eight proteins in two mixtures. There are numerous methodologies available to study the relative abundance of proteins between samples, but to date, there are few examples of studies that have compared these different approaches. For the 2006 Proteomics Research Group study, there were 52 participants who used a wide variety of gel electrophoresis-, HPLC-, and mass spectrometry-based methods for relative quantitation. The quantitative data arising from this study were evaluated along with several other experimental details relevant to the methodologies used. An important aspect of current biological research is the design of experimental systems that can yield quantitative results. Therefore, it is not surprising that there is keen interest in the quantitative analysis of proteins because proteins are of such fundamental importance in all cellular processes. The results obtained from large scale quantitative analyses of protein expression will undoubtedly play an important role in advancing our understanding of metabolic events, cellular systems, and pathogenic mechanisms of disease. The drive toward quantitative studies has been greatly facilitated by the development of new technologies and chemistries. A large number of methods have been developed for the determination of differences in protein expression, including methods based on gel electrophoresis, MS, immunochemistry, and others. Some of these methods have been in use for many years, whereas others have been implemented or modified more recently (1Glish G.L. Vachet R.W. The basics of mass spectrometry in the twenty-first century.Nat. Rev. Drug Discov. 2003; 2: 140-150Crossref PubMed Scopus (257) Google Scholar). There are two general quantitative approaches: absolute and relative. The present study deals only with the latter, mainly due to the fact that the majority of current proteomics studies are concerned with relative measurements among two or more samples and not absolute quantities. This approach is frequently referred to as comparative proteomics. Although it was not called comparative proteomics at the time, relative quantitative measurements of proteins on a large scale have been conducted ever since the development of two-dimensional (2D) 1The abbreviations used are: 2D, two-dimensional; ABRF, Association of Biomolecular Resource Facilities; PRG, Proteomics Research Group; 1D, one-dimensional. gel electrophoresis (2Klose J. Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals.Humangenetik. 1975; 26: 231-243PubMed Google Scholar, 3O'Farrell P. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar). Following the establishment of sensitive and high throughput mass spectrometric methods over 10 years ago (4Fenn J.B. Mann M. Meng C.K. Wong S.F. Whitehouse C.M. Electrospray ionization for mass spectrometry of large biomolecules.Science. 1989; 246: 64-71Crossref PubMed Scopus (6378) Google Scholar, 5Karas M. Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons.Anal. Chem. 1988; 60: 2299-2301Crossref PubMed Scopus (4850) Google Scholar), strategies for the relative quantitation of proteins based on stable isotope labeling techniques were implemented in the field of quantitative proteomics (6Ong S.E. Mann M. Mass spectrometry-based proteomics turns quantitative.Nat. Chem. Biol. 2005; 1: 252-262Crossref PubMed Scopus (1321) Google Scholar). These methods have been complemented recently by label-free mass spectrometry approaches that are based on spectral ion currents (7Lu P. Vogel C. Wang R. Yao X. Marcotte E.M. Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation.Nat. Biotechnol. 2007; 25: 117-124Crossref PubMed Scopus (916) Google Scholar, 8Mallick P. Schirle M. Chen S.S. Flory M.R. Lee H. Martin D. Ranish J. Raught B. Schmitt R. Werner T. Kuster B. Aebersold R. Computational prediction of proteotypic peptides for quantitative proteomics.Nat. Biotechnol. 2007; 25: 125-131Crossref PubMed Scopus (573) Google Scholar). Currently there is a considerable choice of experimental approaches to study the proteome in a quantitative manner, but there have been few studies in which these techniques have been compared and contrasted for a given sample set (9Kolkman A. Dirksen E.H.C. Slijper M. Heck A.J.R. Double standards in quantitative proteomics. Direct comparative assessment of difference in Gel electrophoresis and metabolic stable isotope labeling.Mol. Cell. Proteomics. 2005; 4: 255-266Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). One reason for this is that many of these techniques are relatively expensive to implement, and a given laboratory may only have access to a limited number of approaches. Carrying out a comprehensive comparison of these techniques by one laboratory is, therefore, quite challenging. Since its creation in 2001, the Association of Biomolecular Resource Facilities (ABRF) Proteomics Research Group (PRG) has carried out research studies that have addressed various topics relevant to the field of proteomics. The study topics have included the identification of components in a protein mixture (10Arnott D.P. Gawinowicz M.A. Grant R.A. Lane W.S. Packman L.C. Speicher K. Stone K. Proteomics in mixtures: study results of ABRF-PRG02.J. Biomol. Tech. 2002; 13: 179-186PubMed Google Scholar), determination of the sites of phosphorylation in a protein (11Arnott D. Gawinowicz M.A. Grant R.A. Neubert T.A. Packman L.C. Speicher K.D. Stone K. Turck C.W. ABRF-PRG03: phosphorylation site determination.J. Biomol. Tech. 2003; 14: 205-215PubMed Google Scholar), differentiation of protein isoforms (12Arnott D. Gawinowicz M.A. Kowalak J.A. Lane W.S. Speicher K.D. Turck C.W.K.A.W. Neubert T.A. ABRF-PRG04: differentiation of protein isoforms.J. Biomol. Tech. 2007; 18: 124-134PubMed Google Scholar), and de novo peptide sequencing. 2C. W. Turck, A. M. Falick, J. A. Kowalak, W. S. Lane, T. A. Neubert, B. S. Phinney, S. T. Weintraub, and K. A. West, poster presented at ABRF 2005, Savannah, GA (February 5–8, 2005). In 2006, the PRG organized a quantitative proteomics study that was designed to evaluate methodologies that are available for relative quantitation of proteins among experimental samples. This study was considered to be particularly timely in that an increasing number of proteomics facilities were being asked to provide large scale quantitative proteomics measurements for their user groups. It was the expectation of the PRG that proteomics facilities would use a variety of strategies to carry out the experiments needed for the study, thereby providing a more comprehensive dataset generated from more diverse experimental approaches than would be possible by a single group. In addition, it was anticipated that the study samples would aid laboratories in gaining experience with methods for carrying out such analyses. All proteins were purchased from Sigma-Aldrich. Deionized water (18.2 megaohms; Milli-Q Gradient A10 from Millipore, Bedford, MA) was used to prepare all solutions. For proteins supplied in small quantities (carbonic anhydrase I, glycogen phosphorylase, horseradish peroxidase, and lactoperoxidase), the requisite volume of water was added to each vial to generate the individual stock solutions. Samples of the remaining proteins (β-casein, BSA, catalase, and ribonuclease A) were weighed on an analytical balance and separately dissolved in water. The exact protein content of each individual stock solution was assessed by amino acid analysis. Three stock mixtures were then prepared: 1) a mixture containing the four proteins that were present at the same concentration in both samples, 2) the remaining proteins for sample A, and 3) the remaining proteins for sample B. Each sample, A and B, received an equal volume of stock mixture 1 and a volume of stock mixture 2 or 3 as appropriate to achieve the same total protein amount in both samples. Each vial was dried in a vacuum centrifuge and stored at room temperature for no longer than 1 week prior to mailing. The study sample consisted of two protein mixtures, each of which contained the same eight proteins. Four of the proteins were present at a 1:1 ratio in both samples, whereas the others were present at varying ratios over a range of approximately 2 orders of magnitude. The participating laboratories were asked to identify the proteins and determine their relative quantities within the two samples. In addition to comparing the ability of different techniques to identify and provide relative quantitation of proteins, other goals of the 2006 PRG study included assessment of the participants’ level of confidence and consistency in the quantitative data and evaluation of the ability of software to determine quantitative differences between samples. The PRG also hoped to provide participating laboratories with a means to evaluate their proficiency in identifying and quantifying proteins in a mixture. From a comparison of the results obtained by different strategies, participants would be able to gauge their own capabilities and establish realistic expectations for the approaches that were used. Because this was the first study of its kind, the PRG wanted it to be suitable for as many different quantitative methods as possible. In preparation for the study, the PRG evaluated 12 proteins for possible inclusion in the study. A one-dimensional (1D) SDS-PAGE separation of the 12 proteins is shown in Fig. 1. All proteins were obtained from a commercial source and showed various degrees of heterogeneity as evidenced by the minor components detected in each lane in addition to the band for the expected protein. No efforts were made to characterize or remove any of the protein impurities and/or isoforms. Eight proteins that showed the least heterogeneity and that had a range of molecular weights and isoelectric points were then selected for the study: β-casein, BSA, carbonic anhydrase I, catalase, glycogen phosphorylase, lactoperoxidase, horseradish peroxidase, and ribonuclease A. Sample mixtures were prepared using the ratios listed in Table I as described under “Materials and Methods.” Although four of the eight protein pairs were present in identical amounts in the two mixtures, participants were informed about the identity of only one of them (BSA) so that they could use this protein for normalization. The relative protein ratios between the two samples did not exceed 1:100 because it was thought that the technologies currently available could not accurately measure higher ratios. The most challenging case was that of glycogen phosphorylase with a ratio of 1:76 between the two samples. In sample A this protein was only present at 3 pmol. The total amount of protein in each sample was ∼80 μg. Sufficient amounts of the individual proteins were provided so that participants using less sensitive methods such as gel electrophoresis in combination with Coomassie staining would be able to carry out the analysis more than once. The study was designed such that the total amount of protein in each sample was the same. Fig. 2 is an image from the analysis of the combined samples by DIGE (13Unlu M. Morgan M.E. Minden J.S. Difference gel electrophoresis. A single gel method for detecting changes in protein extracts.Electrophoresis. 1997; 18: 2071-2077Crossref PubMed Scopus (1859) Google Scholar). This analysis also demonstrated protein heterogeneity due to the presence of protein isoforms (in some cases) as well as unknown impurities.Table IProteins in PRG2006 study samplesProteinaProteins were purchased from Sigma-Aldrich: β-casein, catalog number C-6905; bovine serum albumin, A-0281; carbonic anhydrase I, C-6653; catalase, C-40; glycogen phosphorylase, P-1261; lactoperoxidase, L-8257; horseradish peroxidase, P-6782; ribonuclease, R-4875.Molecular massQuantityRatio (A/B)ABkDapmolβ-Casein24.05981504:1Bovine serum albumin66.61951951:1Carbonic anhydrase I28.928901:3Catalase57.5299595:1Glycogen phosphorylase97.432351:76Lactoperoxidase77.53003001:1Horseradish peroxidase43.32982981:1Ribonuclease A13.72962961:1a Proteins were purchased from Sigma-Aldrich: β-casein, catalog number C-6905; bovine serum albumin, A-0281; carbonic anhydrase I, C-6653; catalase, C-40; glycogen phosphorylase, P-1261; lactoperoxidase, L-8257; horseradish peroxidase, P-6782; ribonuclease, R-4875. Open table in a new tab Fig. 2Differential in-gel electrophoresis of the two mixtures prepared for the PRG2006 study sample. Sample A was labeled with Cy3 dye, and sample B was labeled with Cy5 dye. Proteins from both samples were then combined and separated in a 3–10 pH non-linear gradient IPG strip in the first dimension and a 12% SDS gel in the second dimension.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The PRG provided the following information to the participants. 1.The sample set consists of two mixtures, A and B.2.Both mixtures contain eight major proteins, each present in various amounts.3.In some cases, isoforms and/or contaminant(s) are present (as typically encountered for real life samples).4.The total amount of protein in each tube is ∼80 μg. The individual proteins are present on average at 300 pmol, ranging from ∼3 to 600 pmol.5.The ratios of proteins between mixtures A and B vary up to 1:100.6.Four of the eight proteins are present at a 1:1 ratio with BSA being one of them.7.The proteins were mixed from aqueous solutions that also contained small amounts of salt and then dried. The protein identifications and quantitative results generated by the participants were collected using a web-based survey system (SurveyMonkey) that has proven in previous studies to be a quick and easy way for web-based data input. Each participant was also asked to provide information about the methods they used for sample preparation and data analysis as well as any other details relevant to the study. The use of this web-based questionnaire greatly facilitated interpretation and consolidation of the submitted data by the PRG. As in the past, all steps of data collection, evaluation, and presentation were conducted in an anonymous manner. Of the 92 laboratories requesting study samples, 52 returned analysis results. This represents a very good response rate compared with previous studies and reflects the great interest in quantitative proteomics in the community. Data were submitted by both ABRF members and non-members from academia, government, and industry (including instrument manufacturers and biotechnology and pharmaceutical companies). For determining the relative quantities of the proteins in the two samples, a little over one-third of the participants used gel electrophoresis-based methods, whereas approximately two-thirds of the laboratories used MS-based techniques (Table In the case of gel electrophoresis, protein included Coomassie and such as For gel electrophoresis, in addition to and protein approaches in one S. W. C. C. W. A. A. R. R. M.A. analysis of by isoelectric focusing in pH gradient 2005; 4: PubMed Scopus Google were used. of the participants who used MS-based techniques used stable isotope labeling for the analysis. These included protein A. J. F. A novel for quantitative proteomics using protein 2005; PubMed Scopus Google Scholar), for relative and absolute quantitation S. S. S. S. S. P. Martin S. M. F. A. protein quantitation in using Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar), J.A. the of peptide in mass Mass 14: PubMed Scopus Google Scholar), and labeling X. A. J. C. labeling for comparative studies with two of Chem. PubMed Scopus Google Scholar). The remaining of the participants who used MS-based techniques a label-free approach based on ion current or spectral evaluate the the of the expected and ratios for each of the eight proteins was assessed using the following of ratio ratio used by participants in PRG2006 for relative and absolute protein for relative and absolute protein Open table in a new tab of the results submitted by the study participants is shown in 3 and for of ratio for proteins study proteins BSA, which was to study participants as being present at a 1:1 and average both for proteins or proteins glycogen phosphorylase, the most challenging protein to be in this are shown for all methods that were used by the participants. Fig. an assessment of the of each method in quantifying the relative of the proteins present at unknown ratios in the two of ratio results for relative quantitation of the study proteins by analysis A, B, MS-based The number of for each method is shown in for relative and absolute protein Large Image Figure ViewerDownload Hi-res image Download (PPT) It is important to that the PRG evaluation of data received from this study was to or any analytical method or of The number of submitted was to yield any measure of the ability of any method to point is that the results of this study are not only on the absolute capabilities of the methods used but also on the experience of the who the analyses because some of the participating laboratories were these analyses for the first The study was with the of laboratories to both and the range of their own capabilities and to provide them with a means of testing the techniques that they It is only with these in that any can be from the 52 that were most of the protein identifications were conducted by one laboratory used in combination with to the relatively large quantities of most were able to identify all eight proteins not This that identification by of proteins that are present in amounts has In to the in protein there was in the of the quantitation results. The differences between the expected and protein ratios were to be and For the majority of methods, the of ratio was for glycogen phosphorylase ratio of 3 and using ion current or spectral 1) and gel electrophoresis using 1) ratios to the expected for glycogen methods showed of of ratio than MS-based approaches for this study the results obtained using electrophoresis were to the expected for proteins present at a 1:1 ratio than for proteins present at other ratios A and For electrophoresis, the of ratio was for DIGE 3) and whereas Coomassie and 1) relatively high for of ratio electrophoresis showed of of ratio than MS-based obtained by ion current or spectral 1) were as to the expected as obtained by stable isotope labeling 3 and with stable isotope labeling was the of ratio was for all proteins glycogen phosphorylase 3 and whereas using ion current or spectral showed an of the of ratio for all proteins including glycogen phosphorylase by was not by prior separation of proteins by electrophoresis not This have been different a more protein mixture had been analysis of the samples results that were to the expected as compared with analyses only This was particularly for electrophoresis-based for the MS-based methods, analyses and did not yield of the The majority of participants that they did not on software for data analysis but of their results not This is in with of participants in previous PRG studies with other of proteomics (12Arnott D. Gawinowicz M.A. Kowalak J.A. Lane W.S. Speicher K.D. Turck C.W.K.A.W. Neubert T.A. ABRF-PRG04: differentiation of protein isoforms.J. Biomol. Tech. 2007; 18: 124-134PubMed Google Scholar). 2C. W. Turck, A. M. Falick, J. A. Kowalak, W. S. Lane, T. A. Neubert, B. S. Phinney, S. T. Weintraub, and K. A. West, poster presented at ABRF 2005, Savannah, GA (February 5–8, 2005). It as proteomics software that was available at the of the 2006 study was not of data for quantitative proteomics. As the study participants on evaluation and of the results by to the results of the quantitative proteomics was being as a in about of the participants’ The great majority of that there is a for relative protein quantitation for submitted to their by the participants’ about the of the study and their confidence in their quantitation the level of experience with quantitative proteins was of major in determining the of the analysis. The PRG2006 study is the first of its and has interest not only by members of the ABRF but also by other and K. ABRF proteomics quantitation and Google Scholar, A. J. J. S. H. M. J. T. proteomics analysis of the Full Text Full Text PDF PubMed Scopus Google Scholar, mass well in PRG quantitation Scholar). As it is important to that there were not submitted to about the capabilities of various methods for relative quantitation. In addition, it can be anticipated that the results obtained by the participants have been quite different a more sample or protein quantities had been the eight proteins that were provided in the samples were to be appropriate for all of the different approaches used in this study. It is that some of the techniques used by participants would only be with this of sample. aspect that be considered is that the of a quantitation method greatly on both the and the design of the particularly the number of The study results the range of capabilities of a variety of different methods for quantitative proteomics. It is that in a number of are In addition, there is no in the proteomics on to quantitative data with to there is a for comparative of methods for quantitative proteomics. This study and others that will will undoubtedly provide the proteomics with information on methods and approaches that can be used for relative protein quantitation. The PRG of at for the study sample mixtures and gel electrophoresis, Packman of for amino acid of for DIGE analysis of the study sample, of for as a study and and of for with the data analysis.