Approaching complete peroxisome characterization by gas‐phase fractionation

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 GPFm/z and GPFRI. 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 GPFm/z from 400–800, 800–1200 and 1200–1800 produced the following results: (i) 1.3× more proteins were identified by GPFm/z for an equal amount of effort (i.e., 3 νLC/ESI‐MS/MS) and (ii) proteins identified by GPFm/z had a lower average codon bias value. Use of GPFRI identified more proteins per m/z unit scanned than GPFm/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 GPFm/z, but only 73% using a standard wide m/z range survey scan.

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