We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.

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Large-scale analysis of the yeast proteome by multidimensional protein identification technology.

We describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT), which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry. The multidimensional liquid chromatography method integrates a strong cation-exchange (SCX) resin and reversed-phase resin in a biphasic column. We detail the improvements over a system described by Link et al. (Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676−682) that separates and acquires tandem mass spectra for thousands of peptides. Peptides elute off the SCX phase by increasing pI, and elution off the SCX material is evenly distributed across an analysis. In addition, we describe the chromatographic benchmarks of MudPIT. MudPIT was reproducible within 0.5% between two analyses. Furthermore, a dynamic range of 10 000 to 1 between the most abundant and least abundant proteins/pep...

An Automated Multidimensional Protein Identification Technology for Shotgun Proteomics

Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control

4. Automated Interpretation of CID Spectra 282 5. Accurate Mass Tags 282 C. Protein Identification in Complex Mixtures 282 D. Analysis of Protein Expression 284 III. Proteomes and Post-Translational Modifications 285 A. Proteomes 285 1. The Analytical Challenge 285 2. Analysis of Protein−Protein Complexes 286 B. Post-Translational Modifications 286 1. Background 286 2. Detection and Purification of Phosphoproteins 288

Mass Spectrometry in Proteomics

he genomics revolution has changed the paradigm for the comprehensive analysis of biological processes and systems. It is now hypothesized that biological processes and systems can be described based on the comparison of global, quantitative gene expression patterns from cells or tissues representing different states. To test this hypothesis, it is essential that methods for the precise measurement of gene expression be developed and applied. Several methods, including serial analysis of gene expression, oligonucleotide and cDNA microarrays, and large-scale se- quencing of expressed sequence tags have been developed to globally and quantitatively measure gene expression at the mRNA level (1, 2). The discovery of posttranscriptional mech- anisms that control rate of synthesis and half-life of proteins (3) and the ensuing nonpredictive correlation between mRNA and protein levels expressed by a particular gene (4, 5) indicate that direct measurement of protein expression also is essential for the analysis of biological processes and systems. Global analysis of gene expression at the protein level is now also termed proteomics. The standard method for quantitative proteome analysis combines protein separation by high- resolution (isoelectric focusingySDS-PAGE) two-dimensional gel electrophoresis (2DE) with mass spectrometric (MS) or tandem MS (MSyMS) identification of selected protein spots. Important technical advances related to 2DE and protein MS have increased sensitivity, reproducibility, and throughput of proteome analysis while creating an integrated technology. By using 2DE with extended pH range and high-sensitivity protein identification by electrospray ionization and MSyMS, we have evaluated the potential of the 2DE-MS strategy to serve as the technology base for comprehensive and quantitative pro- teome analysis.

https://www.pnas.org/content/pnas/97/17/9390.full.pdf

Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology

By proving the opportunity to visualize several hundred proteins at a time, two-dimensional (2-D) gel electrophoresis is an important tool for proteome research. In order to take advantage of the full potential of this technique for yeast studies, we have undertaken a systematic identification of yeast proteins resolved by this technique. We report here the identification of 92 novel protein spots on the yeast 2-D protein map. These identifications extend the number of protein spots identified on our yeast reference map to 401. These spots correspond to the products of 279 different genes. They have been essentially identified by three methods: gene overexpression, amino acid composition and mass spectrometry. These data can be accessed on the Yeast Protein Map server (htpp://www.ibgc.u-bordeaux2.fr/YPM).

Mathias Mann

Papers

Two-dimensional gel protein database of Saccharomyces cerevisiae (update 1999).