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 (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.
Yi
Eugene C
EC
The Institute for Systems Biology, Seattle, WA 98103, USA.
Marelli
Marcello
M
Lee
Hookeun
H
Purvine
Samuel O
SO
Aebersold
Ruedi
R
Aitchison
John D
JD
Goodlett
David R
DR
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods; D010446:Peptide Fragments / Q000302:isolation & purification; D020675:Peroxisomes / Q000737:chemistry*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins