Approaching complete peroxisome characterization by gas-phase fractionation

Electrophoresis. 2002 Sep;23(18):3205-16. doi: 10.1002/1522-2683(200209)23:18<3205::AID-ELPS3205>3.0.CO;2-Y.


We examined the utility of gas-phase fractionation (GPF) in the m/z dimension to increase proteome coverage and reproducibility of peptide ion selection by direct microliquid chromatography/electrospray ionization-tandem mass spectrometry (microLC/ESI-MS/MS) analysis of the peptides produced by proteolytic digestion of unfractionated proteins from a yeast whole-cell lysate and in a peroxisomal membrane protein fraction derived from isolated yeast peroxisomes. We also investigated GPF in the relative ion intensity dimension and propose denoting the two types of GPF as GPF(m/z) and GPF(RI). Comparison of results of direct nuLC/ESI-MS/MS analysis of the unfractionated mixture of peptides from proteolysis of a yeast whole cell lysate by DD ion selection from 400-1800 m/z in triplicate and GPF(m/z) from 400-800, 800-1200 and 1200-1800 produced the following results: (i) 1.3 x more proteins were identified by GPF(m/z) for an equal amount of effort (i.e., 3 microLC/ESI-MS/MS) and (ii) proteins identified by GPF(m/z) had a lower average codon bias value. Use of GPF(RI) identified more proteins per m/z unit scanned than GPF(m/z) or triplicate analysis over a wide m/z range. After tryptic digestion of all the proteins from a discontinuous Nycodenz gradient fraction known to be enriched with yeast peroxisomal membrane proteins we detected 93% (38/41) of known peroxisomal proteins using GPF(m/z), but only 73% using a standard wide m/z range survey scan.

Publication types

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

MeSH terms

  • Chromatography, High Pressure Liquid / methods
  • Electrophoresis, Capillary / methods
  • Peptide Fragments / isolation & purification
  • Peroxisomes / chemistry*
  • Proteomics / instrumentation
  • Proteomics / methods
  • Saccharomyces cerevisiae Proteins / isolation & purification
  • Spectrometry, Mass, Electrospray Ionization / instrumentation
  • Spectrometry, Mass, Electrospray Ionization / methods*


  • Peptide Fragments
  • Saccharomyces cerevisiae Proteins