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Approaching complete peroxisome characterization by gas‐phase fractionation
Author(s) -
Yi Eugene C.,
Marelli Marcello,
Lee Hookeun,
Purvine Samuel O.,
Aebersold Ruedi,
Aitchison John D.,
Goodlett David R.
Publication year - 2002
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200209)23:18<3205::aid-elps3205>3.0.co;2-y
Subject(s) - chromatography , chemistry , fractionation , proteome , lysis , tandem mass spectrometry , electrospray ionization , mass spectrometry , bottom up proteomics , peptide , biochemistry , protein mass spectrometry
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 (νLC/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 νLC/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× more proteins were identified by GPF m/z for an equal amount of effort ( i.e. , 3 νLC/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.