Proteomic investigation of natural killer cell microsomes using gas-phase fractionation by mass spectrometry.
We have explored the utility of gas-phase fractionation by mass spectrometry (MS) in the mass-to-charge (m/z) dimension (GPF(m/z)) for increasing the effective number of protein identifications in cases where sample quantity limits the use of multi-dimensional chromatographic fractionation. A peptide digestate from proteins isolated from the membrane fraction of natural killer (NK) cells was analyzed by microcapillary reversed-phase liquid chromatography coupled online to an ion-trap (IT) mass spectrometer. Performing GPF(m/z) using eight narrow precursor ion scan m/z ranges enabled the identification of 340 NK cell proteins from 12 microg of digestate, representing more than a fivefold increase in the number of proteins identified as compared to the same experiment employing a standard precursor ion survey scan m/z range (i.e., m/z 400-2000). The results show that GPF(m/z) represents an effective technique for increasing protein identifications in global proteomic investigations especially when sample quantity is limited.
Blonder
Josip
J
Biomedical Proteomics Program, Laboratory of Proteomics and Analytical Technologies, SAIC Frederick, Inc., National Cancer Institute at Frederick, PO Box B, Frederick, MD 21702, USA.
Rodriguez-Galan
Maria Cecilia
MC
Lucas
David A
DA
Young
Howard A
HA
Issaq
Haleem J
HJ
Veenstra
Timothy D
TD
Conrads
Thomas P
TP
D000595:Amino Acid Sequence; D000818:Animals; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007694:Killer Cells, Natural / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*
D027682:Cation Transport Proteins; D008565:Membrane Proteins; D010455:Peptides; D020543:Proteome; C424902:Sfxn1 protein, mouse