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Washburn Lab
Michael Washburn, Ph.D.
Director of Proteomics Center
mpw@stowers.org		  
Research Description

     Proteomics is loosely defined as the analysis of the protein complement of a protein complex, organelle, cell, tissue, or organism. Traditionally, proteomics is associated with the analysis of highly complex mixtures of proteins by coupling separation techniques like two-dimensional gel electrophoresis or chromatography with mass spectrometry. Such analyses have generated and continue to generate ever-increasing insight into the biology of many organisms including S. cerevisiae, P. falciparum and H. sapiens. The emerging discussions and debates surrounding “systems biology” would not be possible without the dramatic technological advances made in the field of proteomics in recent years.

MudPIT
     We utilize a method named Multidimensional Protein Identification Technology (MudPIT) to analyze the proteomes of organisms. MudPIT is a chromatography-based proteomic technique where a complex peptide mixture is prepared from a sample and loaded directly onto a biphasic microcapillary column packed with reversed phase and strong cation exchange HPLC grade materials. Once the complex peptide mixture is loaded onto the biphasic microcapillary column, this column is placed directly in-line with a tandem mass spectrometer. The tandem mass spectrometry data generated from a MudPIT run is then analyzed to determine the protein content of the original proteomic sample. MudPIT has been proven to be an excellent tool for both qualitative and quantitative proteomic analyses.

Quantitative Proteomics
     The dynamic changes of a proteome or fractions of a proteome; i.e., organelles and protein complexes, can be analyzed via quantitative proteomic methods. In quantitative proteomics, the mass of the same protein from the same organism but from two different growth conditions is modified through a variety of means. In a mass spectrometer, the relative abundances of the same protein, but from two different samples and with two unique masses, can then be measured. In our laboratory, we use metabolic labeling strategies where the mass of proteins is modified by growing an organism, such as S. cerevisiae, in media that has “heavy” or “light” nutrients.

     More recently, we have adopted label-free quantitative proteomic analyses of cells and multiprotein complexes. This approach, based on normalized spectral counting, takes into account the sample-to-sample variation that is obtained when carrying out replicate MudPIT analyses of a sample. Examples of the application of the normalized spectral abundance factor (NSAF) approach to quantitative proteomic analysis include recently described work on the expression changes of membrane proteins in S. cerevisiae and on the human transcriptional regulatory complex, Mediator.

mRNA and Protein Expression
     When an organism such as S. cerevisiae is subjected to different cellular conditions, the complex protein expression changes can be directly measured via quantitative proteomic approaches, and the mRNA expression changes can be directly measured by cDNA or oligonucleotide arrays. By combining global analysis of mRNA expression and global quantitative proteomic analyses, one can gain novel insight into the biology of an organism and begin to discern the interplay between mRNA and protein expression levels of a given locus in an organism’s genome. Projects in the laboratory include the development of methods to increase the coverage of a proteome for quantitative proteomic analysis and interrogating the systems biology of S. cerevisiae subjected to various stimuli.

Dynamics of Multiprotein Complexes
     Increasingly, our research is focusing on multiprotein complexes. Using affinity purification coupled with MudPIT and NSAF, we analyze complexes using different tagged subunits from the same multiprotein complex. This allows us to determine the relative abundance of particular proteins in a complex in a bait-dependent fashion and leads to the analysis of distinct forms of multiprotein complexes that can have important functional insights. In addition, current projects in the lab are using these approaches to determine the impact of different stimuli on multiprotein complexes.

Selected publications

Pavelka N, Fournier ML, Swanson SK, Pelizzola M, Ricciardi-Castagnoli P, Florens L, Washburn MP. Statistical Similarities between Transcriptomics and Quantitative Shotgun Proteomics Data. Mol Cell Proteomics. 2008; 7(4): 631-644. Abstract

Sardiu ME, Cai Y, Jin J, Swanson SK, Conaway RC, Conaway JW, Florens L, Washburn MP. Probabilistic assembly of human protein interaction networks from label-free quantitative proteomics. Proc Natl Acad Sci U S A. 2008;105:1454-1459. Abstract

Liu WL, Coleman RA, Grob P, King DS, Florens L, Washburn MP, Geles KG, Yang JL, Ramey V, Nogales E, Tjian R. Structural Changes in TAF4b-TFIID Correlate with Promoter Selectivity. Mol Cell. 2008;29:81-91. Abstract

Xiang Y, Takeo S, Florens L, Hughes SE, Huo LJ, Gilliland WD, Swanson SK, Teeter K, Schwartz JW, Washburn MP, Jaspersen SL, Hawley RS. The inhibition of polo kinase by matrimony maintains G2 arrest in the meiotic cell cycle. PLoS Biol. 2007;5:e323. Abstract

Lee JS, Shukla A, Schneider J, Swanson SK, Washburn MP, Florens L, Bhaumik SR, Shilatifard A. Histone crosstalk between H2B monoubiquitination and H3 methylation mediated by COMPASS. Cell. 2007;131:1084-1096. Abstract

Cai Y, Jin J, Yao T, Gottschalk AJ, Swanson SK, Wu S, Shi Y, Washburn MP, Florens L, Conaway RC, Conaway JW. YY1 functions with INO80 to activate transcription. Nat Struct Mol Biol. 2007;14:872-874. Abstract

Fournier ML, Gilmore JM, Martin-Brown SA, Washburn MP. Multidimensional separations-based shotgun proteomics. Chem Rev. 2007;107:3654-3686. Abstract

Hrecka K, Gierszewska M, Srivastava S, Kozaczkiewicz L, Swanson SK, Florens L, Washburn MP, Skowronski J. Lentiviral Vpr usurps Cul4-DDB1[VprBP] E3 ubiquitin ligase to modulate cell cycle. Proc Natl Acad Sci U S A. 2007;104:11778-11783. Abstract

Camahort R, Li B, Florens L, Swanson SK, Washburn MP, Gerton JL. Scm3 is essential to recruit the histone h3 variant cse4 to centromeres and to maintain a functional kinetochore. Mol Cell. 2007;26:853-865. Abstract

Litovchick L, Sadasivam S, Florens L, Zhu X, Swanson SK, Velmurugan S, Chen R, Washburn MP, Liu XS, DeCaprio JA. Evolutionarily conserved multisubunit RBL2/p130 and E2F4 protein complex represses human cell cycle-dependent genes in quiescence. Mol Cell. 2007;26:539-551. Abstract

Zybailov BL, Florens L, Washburn MP. Quantitative shotgun proteomics using a protease with broad specificity and normalized spectral abundance factors. Mol Biosyst. 2007;3:354-360. Abstract

Paoletti AC, Parmely TJ, Tomomori-Sato C, Sato S, Zhu D, Conaway RC, Conaway JW, Florens L, Washburn MP. Quantitative proteomic analysis of distinct mammalian Mediator complexes using normalized spectral abundance factors. Proc Natl Acad Sci U S A. 2006;103:18928-18933. Abstract

Florens L, Carozza MJ, Swanson SK, Fournier M, Coleman MK, Workman JL, Washburn MP. Analyzing Chromatin Remodeling Complexes Using Shotgun Proteomics and Normalized Spectral Abundance Factors. Methods. 2006;40:303-311. Abstract

Zybailov B, Mosley AL, Sardiu ME, Coleman MK, Florens L, Washburn MP. Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae. J Proteome Res. 2006;5:2339-2347. Abstract

Yao T, Song L, Xu W, Demartino GN, Florens L, Swanson SK, Washburn MP, Conaway RC, Conaway JW, Cohen RE. Proteasome recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1. Nat Cell Biol. 2006;8:994-1002. Abstract

Florens L, Washburn MP. Proteomic Analysis by Multidimensional Protein Identification Technology. Methods in Molecular Biology: New and Emerging Proteomics Techniques. Totowa, NJ: Humana Press Inc.; 2006;328:159-175.

Carrozza MJ, Li B, Florens L, Suganuma T, Swanson SK, Lee KK, Shia WJ, Anderson S, Yates J, Washburn MP, Workman JL. Histone H3 Methylation by Set2 Directs Deacetylation of Coding Regions by Rpd3S to Suppress Spurious Intragenic Transcription. Cell. 2005;123:581-592. Abstract

Prochasson P, Florens L, Swanson SK, Washburn MP, Workman JL. The HIR corepressor complex binds to nucleosomes generating a distinct protein/DNA complex resistant to remodeling by SWI/SNF. Genes Dev. 2005;19:2534-2539. Abstract

Zybailov B, Coleman MK, Florens L, Washburn MP. Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum counting for quantitative proteomic analysis using stable isotope labeling. Anal Chem. 2005;77:6218-6224. Abstract

Schneider J, Wood A, Lee JS, Schuster R, Dueker J, Maguire C, Swanson SK, Florens L, Washburn MP, Shilatifard A. Molecular regulation of histone H3 trimethylation by COMPASS and the regulation of gene expression. Mol Cell. 2005;19:849-856. Abstract

Swanson SK, Washburn MP. The continuing evolution of shotgun proteomics. Drug Discov Today. 2005;10:719-725. Abstract.

Kusch T, Florens L, Macdonald WH, Swanson SK, Glaser RL, Yates Iii JR, Abmayr SM, Washburn MP, Workman JL. Acetylation by Tip60 Is Required for Selective Histone Variant Exchange at DNA Lesions. Science. 2004;306:2084-2087. Abstract.

Sato S, Tomomori-Sato C, Parmely TJ, Florens L, Zybailov B, Swanson SK, Banks CA, Jin J, Cai Y, Washburn MP, Conaway JW, Conaway RC. A set of consensus Mammalian mediator subunits identified by multidimensional protein identification technology. Mol Cell. 2004;14:685-691. Abstract.

Washburn MP. Soft landing for protein chips. Nat Biotech. 2003;21:1156-1157. Abstract.

Washburn MP, Koller A, Oshiro G, Ulaszek RR, Plouffe D, Deciu C, Winzeler E, Yates JR III. Protein pathway and complex clustering of correlated mRNA and protein expression analyses in Saccharomyces cerevisiae. Proc Natl Acad Sci USA. 2003;100:3107-3112. Abstract.

Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, Moch JK, Muster N, Sacci JB, Tabb DL, Witney AA, Wolters D, Wu Y, Gardner MJ, Holder AA, Sinden RE, Yates, JR III, Carucci DJ. A proteomic view of the Plasmodium falciparum life cycle. Nature. 2002;419:520-526. Abstract.

Koller A, Washburn MP, Lange M, Andon NL, Deciu C, Haynes PA, Hays L, Schieltz D, Ulaszek R, Wei J, Wolters D, Yates JR III. Proteomic survey of metabolic pathways in rice. Proc Natl Acad Sci USA. 2002;99:11969-11974. Abstract.

MacCoss MJ, McDonald WH, Saraf A, Sadygov R, Clark JM, Tasto JT, Gould KL, Wolters D, Washburn MP, Weiss A, Clark JI, Yates JR III. Shotgun identification of protein modifications from protein complexes and lens tissue. Proc Natl Acad Sci USA. 2002;99:7900-7905. Abstract.

Washburn MP, Ulaszek R, Deciu C, Schieltz DM, Yates JR III. Analysis of quantitative proteomic data generated via multi-dimensional protein identification technology. Anal Chem. 2002;74:1650-1657. Abstract.

Washburn MP, Wolters D, Yates JR III. Large-scale analysis of the yeast proteome via multidimensional protein identification technology. Nat Biotech. 2001;19:242-247. Abstract.

Wolters D, Washburn MP, Yates JR III. An automated multidimensional protein identification technology for shotgun proteomics. Anal Chem. 2001;73:5683-5690. Abstract.

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