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Mass spectrometric analysis of the glycosphingolipid‐enriched microdomains of rat natural killer cells
Author(s) -
Man Petr,
Novák Petr,
Cebecauer Marek,
Horváth Ondrej,
Fišerová Anna,
Havlíček Vladimír,
Bezouška Karel
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200400887
Subject(s) - glycosphingolipid , cd38 , glycoprotein , differential centrifugation , proteome , natural killer cell , chemistry , receptor , membrane protein , tandem mass spectrometry , membrane , microbiology and biotechnology , biology , biochemistry , in vitro , stem cell , cytotoxicity , mass spectrometry , chromatography , cd34
Glycosphingolipid‐enriched microdomains (GEM) are membrane entities that concentrate glycosylphosphatiolylinositol(GPI)‐anchored, acylated and membrane proteins important for immune receptor signaling. Using rat leukemic cell line RNK‐16 we have initiated proteomic studies of microdomains in natural killer (NK) cells. Isolated plasma membranes were treated with Brij 58, or Nonidet‐P40, or sodium carbonate. Extracts were separated by sucrose density gradient centrifugation into very light membrane, medium light membrane and heavy fractions, and a complete protein profile was analyzed by tandem mass spectrometry. Up to 250 proteins were unambiguously identified in each analyzed fraction. The first study of the proteome of NK cell GEM revealed several new aspects including identification of molecules not expected to be expressed in rat NK cells ( e.g. , NAP‐22) or associated with GEM ( e.g. , NKR‐P1, CD45, CD2). Moreover, it provided clear data consolidating controversial views concerning the occurrence of major histcompatibility complex glycoproteins and RT6.1/CD73/CD38 complex in NK cells. Our results also identified a large number of receptors as candidates for future functional studies.