Linking protein fractionation with multidimensional monolithic reversed-phase peptide chromatography/mass spectrometry enhances protein identification from complex mixtures even in the presence of abundant proteins

Stefanie Wienkoop, Mirko Glinski, Nobuo Tanaka, Vladimir Tolstikov, Oliver Fiehn, Wolfram Weckwerth

Research output: Contribution to journalArticle

80 Scopus citations

Abstract

Recently, multidimensional shotgun proteomics has proven to be an alternative technology able to identify hundreds of proteins from single samples. Two major limitations of the technology are the presence of high abundance proteins (e.g. RUBISCO in plant leaf tissue) and the enormous number of co-eluting peptides that overstrain the loading and resolving capacity of conventional particle-packed columns as well as the capacity of electrospray ionisation due to ion suppression. Here, the coupling of fast performance liquid chromatography (FPLC) pre-fractionation of an Arabidopsis leaf protein extract and subsequent two-dimensional liquid chromatography/mass spectrometry with improved resolution using a monolithic silica C18 capillary column allowed the identification of 1032 unique proteins in a single 4 mg total protein plant leaf tissue sample. The reassignment of peptide IDs to distinct FPLC protein fractions enhances the identification procedure, especially in the case of present protein isoforms. The proposed strategy is useful to detect proteins otherwise not seen in conventional multidimensional chromatography/mass spectrometry approaches.

Original languageEnglish (US)
Pages (from-to)643-650
Number of pages8
JournalRapid Communications in Mass Spectrometry
Volume18
Issue number6
StatePublished - 2004
Externally publishedYes

ASJC Scopus subject areas

  • Analytical Chemistry
  • Spectroscopy

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