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Evaluation of Multidimensional Chromatography Coupled with Tandem Mass Spectrometry (LC/LC−MS/MS) for Large-Scale Protein Analysis:  The Yeast Proteome

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Department of Cell Biology, and Taplin Biological Mass Spectrometry Facility, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115
Cite this: Journal of Proteome Research 2003, 2, 1, 43–50
Publication Date (Web):October 15, 2002
Copyright © 2003 American Chemical Society

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    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

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     Department of Cell Biology.

     Taplin Biological Mass Spectrometry Facility.


     To whom correspondence should be addressed. E-mail:  steven_gygi@

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