Defining the membrane proteome of NK cells

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.

A Cell-Surface Membrane Protein Signature for Glioblastoma

“…Captured cell-surface proteins were subjected to high-resolution mass spectrometry in triplicate (technical replicates) and the proteins were identified using the Global Proteome Machine [(the GPM) (http://www.theGPM.org)] with minimum log expectation scores of <10 −3 (Craig and Beavis, 2003, 2004; Fenyo et al, 2007; Ghosh et al, 2010). We identified a total of 868, 813, 541, and 564 non-redundant proteins from U87MG, T98, neural stem cell, and cancer stem cell populations, respectively (Figure 2A–D and Table S1).…”

High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis

“…The difference of separation selectivity between the low and high pH RPLC comes from the changes in the charge distribution within peptide chains upon altering pH of the eluent [22]. The 2D RPLC strategy has been employed in various proteomics applications to separate stable isotopic labeled [23–26] or non-labeled peptides [27,28]. The orthogonality of RPLC-RPLC can be further improved [5,11] by using different reversed phase packing materials, such as polyRP and octadecylsilanized particles that lead to different LC retention behaviors of peptides.…”

Human NK cell lytic granules and regulation of their exocytosis

“…FasL and TRAIL are pro-apoptotic molecules that have been found to localize to the lytic granules of NK cells (Montel et al, 1995; Bossi and Griffiths, 1999; Monleon et al, 2001; Schmidt et al, 2009; Ghosh et al, 2010). FasL has been shown to be sorted to lysosomes through recognition of a proline-rich domain in its cytoplasmic tail (Blott et al, 2001).…”