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Defining the membrane proteome of NK cells
Author(s) -
Ghosh Dhimankrishna,
Lippert Dustin,
Krokhin Oleg,
Cortens John P.,
Wilkins John A.
Publication year - 2010
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.1696
Subject(s) - chemistry , membrane , proteome , membrane protein , transmembrane protein , peripheral membrane protein , integral membrane protein , trypsin , biochemistry , tandem mass spectrometry , chromatography , mass spectrometry , enzyme , receptor
Abstract The present study was initiated to define the composition of the membrane proteome of the Natural Killer (NK) like cell line YTS. Isolated membranes were treated with reagents that have been reported to remove peripheral membrane proteins. Additional steps involving trifluoroethanol (TFE) were introduced in an effort to remove remaining nonintegral membrane proteins. This treatment resulted in the release of a subset of proteins without any apparent disruption of membrane integrity. The membranes were solubilized and digested with trypsin in 25% TFE. The resulting peptides were separated using an off‐line two‐dimensional reversed phase LC technique at alkaline and acidic pHs. Mass spectrometric analysis identified 1843 proteins with high confidence scores. On the basis of the presence of transmembrane regions or evidence of posttranslational modifications and prediction algorithms, approximately 40% of the identified proteins were predicted as plausible membrane proteins. The remaining species were largely involved in cellular processes and molecular functions that could be predicted to be transiently associated with membranes. The analytical approaches presented in this study offer robust generic methods for the identification and characterization of membrane proteins. These observations highlight the fact that the membrane is a dynamic entity that is composed of integral and stably associated proteins. Copyright © 2009 John Wiley & Sons, Ltd.