Evaluation of M ultidimensional Chromatography Coupled w ith Tandem Mass Spectrometry (LC/ LC -MS/ MS) for Large-Scale Protein Analysis: The Yeast Proteome Junmin Peng, † Joshua E. Elias, † Carson C. Thoreen, ‡ Larry J. Licklider, ‡ and Steven P. Gygi* ,†,‡ Departm ent of Cell Biology, and Taplin Biological Mass Spectrom etry Facility, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115 Received July 30, 2002 Highly complex protein mixtures can be directly analyzed after proteolysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this paper, we have utilized the combination of strong cation exchange (SCX) and reversed-phase (RP) chromatography to achieve two-dimensional separation prior to MS/MS. One milligram of whole yeast protein was proteolyzed and separated by SCX chromatography (2.1 mm i.d.) with fraction collection every minute during an 80-min elution. Eighty fractions were reduced in volume and then re-injected via an autosampler in an automated fashion using a vented-column (100 μm i.d.) approach for RP-LC-MS/MS analysis. More than 162 000 MS/MS spectra were collected with 26 815 matched to yeast peptides (7537 unique peptides). A total of 1504 yeast proteins were unambiguously identified in this single analysis. We present a comparison of this experiment with a previously published yeast proteome analysis by Yates and colleagues (Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-7). In addition, we report an in-depth analysis of the false-positive rates associated with peptide identification using the Sequest algorithm and a reversed yeast protein database. New criteria are proposed to decrease false-positives to less than 1% and to greatly reduce the need for manual interpretation while permitting more proteins to be identified. Keywords: proteome • tandem mass spectrometry • LC-MS/MS • vented column • Sequest criteria Introduction One of the greatest challenges facing researchers in the post- genomic era is to identify and quantify all expressed protein components in cells, tissues, and organisms. While two- dimensional gel electrophoresis (2DE) is a powerful technique for protein separation, 1-3 it has a number of limitations that have spawned new technologies as alternatives to 2DE. 4-12 A powerful alternative technique is the so-called MUDPIT (multi- dimensional analysis of proteins identification technology) pioneered by Yates and colleagues. 13-18 This technique is also termed DALPC for direct analysis of large protein com- plexes. 13,19,20 The acquired tandem mass spectrometry (MS/MS) data are used for database searching, 21-26 which leads to identification of peptides and proteins. At the heart of the method is the use of two-dimensional (2D) chromatography to separate a peptide mixture prior to analysis by mass spectrometry. By utilizing peptides’ unique physical properties of charge and hydrophobicity, a complex peptide mixture can be ef- fectively resolved and concentrated prior to sequence analysis by mass spectrometry. Strong cation exchange (SCX) chroma- tography is generally implemented as a primary separation technique due to its potential for increased loading capacity while reversed-phase (RP) chromatography is a perfect compli- ment as a secondary separation technique because of its ability to remove salts and its direct compatibility with mass spec- trometry through electrospray ionization. 2D chromatography can be accomplished by either an online or an offline approach. 27 For the online approach, an acidified complex peptide mixture is applied to an SCXchromatography column and discrete fractions of the adsorbed peptides are sequentially displaced directly onto the RP chromatography column using a salt step gradient. Peptides are then eluted and analyzed by tandem mass spectrometry (MS/MS). This ap- proach can utilize as many as 15 (or more) salt “bumps” to fractionate a peptide mixture. 13,14 Aprincipal advantage to the online technique is nearly complete automation in an un- attended fashion. Essentially, the sample is loaded onto the SCXcolumn and 24 h later the analysis is finished. The offline approach is performed by applying the acidified complex peptide mixture to the SCXchromatography column followed by a binary gradient to high salt to elute the peptides. Fractions are typically collected every minute into a 96-well plate and reduced in volume, and then each fraction is loaded onto a RP chromatography column automatically via an autosampler and analyzed by LC-MS/MS. The offline tech- nique has several advantages over the online technique: (i) *To whom correspondence should be addressed. E-mail: steven_gygi@ hms.harvard.edu. † Department of Cell Biology. ‡ Taplin Biological Mass Spectrometry Facility. 10.1021/pr025556v CCC: $25.00  2003 American Chemical Society Journal of Proteome Research 2003, 2, 43-50 43 Published on Web 10/15/2002