Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome

J Proteome Res. Jan-Feb 2003;2(1):43-50. doi: 10.1021/pr025556v.

Abstract

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 microm 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 (7,537 unique peptides). A total of 1,504 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cations
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange / methods
  • Chromatography, Liquid / methods*
  • Databases as Topic
  • False Positive Reactions
  • Fungal Proteins / analysis
  • Fungal Proteins / chemistry*
  • Ions
  • Mass Spectrometry / methods*
  • Nanotechnology
  • Peptides / chemistry
  • Proteome*
  • Saccharomyces cerevisiae / chemistry*
  • Saccharomyces cerevisiae / metabolism
  • Time Factors

Substances

  • Cations
  • Fungal Proteins
  • Ions
  • Peptides
  • Proteome