Analysis of murine natural killer cell microsomal proteins using two-dimensional liquid chromatography coupled to tandem electrospray ionization mass spectrometry.
This study describes the application of a single tube sample preparation technique coupled with multidimensional fractionation for the analysis of a complex membrane protein sample from murine natural killer (NK) cells. A solution-based method that facilitates the solubilization and tryptic digestion of integral membrane proteins is conjoined with strong cation exchange (SCX) liquid chromatography (LC) fractionation followed by microcapillary reversed-phase (microRP) LC tandem mass spectrometric analysis of each SCXLC fraction in second dimension. Sonication in buffered methanol solution was employed to solubilize, and tryptically digest murine NK cell microsomal proteins, allowing for the large-scale identification of integral membrane proteins, including the mapping of the membrane-spanning peptides. Bioinformatic analysis of the acquired tandem mass spectra versus the murine genome database resulted in 11,967 matching tryptic peptide sequences, corresponding to 5782 unique peptide identifications. These peptides resulted in identification of 2563 proteins of which 876 (34%) are classified as membrane proteins.
Blonder
Josip
J
Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc, Frederick, Maryland 21702-1201, USA.
Rodriguez-Galan
Maria C
MC
Chan
King C
KC
Lucas
David A
DA
Yu
Li-Rong
LR
Conrads
Thomas P
TP
Issaq
Haleem J
HJ
Young
Howard A
HA
Veenstra
Timothy D
TD
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D015395:Histocompatibility Antigens Class I / Q000032:analysis / Q000737:chemistry; D007694:Killer Cells, Natural / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008861:Microsomes / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization
D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins; D010455:Peptides; D020543:Proteome