Identification of components of trans‐Golgi network‐derived transport vesicles and detergent‐insoluble complexes by nanoelectrospray tandem mass spectrometry

Abstract

Epithelial cells have to deliver newly synthesized proteins to the apical and the basolateral plasma membrane domains of the polarized cell surface. Sorting takes place in the trans-Golgi network and at least two vesicular carriers exist for apical and basolateral delivery. After immuno-isolation, the composition of these vesicle preparations was analyzed by two-dimensional (2-D) gel electrophoresis and detergent extraction. In this paper we compare the constituents of detergent-insoluble complexes in different cell lines of polarized or nonpolarized origin and present the identification of five previously uncharacterized proteins. We show that our protein identification strategy can be successfully applied to the problem of small hydrophobic proteins from organisms that have not been substantially sequenced. The high sensitivity of nanoelectrospray tandem mass spectrometry allowed us to identify two proteins that belong to the p23/p24 family of putative cargo receptors for vesicular trafficking. Furthermore we have mapped CD9 and CD81, two members of a large family of proteins consisting of highly hydrophobic four transmembrane proteins. In addition we have identified caveolin-2 as a constituent of basolateral transport vesicles. We have also extended our analysis of immuno-isolated vesicles to a more basic pI range and show that this region on 2-D gels is devoid of proteins. With these approaches and with the previously published data we have now identified most of the major low molecular weight proteins recovered in detergent-insoluble glycolipid-enriched complexes.

Visualization and identification of hepatitis c viral particles by atomic force microscopy combined with ms/ms analysis

“…Для этого к 5 мкл 50 мM NH 4 HCO 3 буферного раствора (рН 7,4) добавляли 1 мкл раствора трипсина (10 -9 М). Трипсинолиз проводили по стандартной схеме, аналогично описанной в [32]. После этого трипсинолитическая смесь смывалась с АСМ-наночипа для масс-спектрометрического анализа.…”

Mass-spectrometric identification of interaction sites for cytochrome P450 2b4/nadph cytochrome P450 reductase

“…Полосы, соответствующие d-2B4, d-Fp и их комплексам вырезали из геля, обесцвечивали раствором феррицианида калия (0,2 г/л в 0,2 М NH 4 HCO 3 ) и дальнейший трипсинолиз белков проводили согласно [34] с небольшими модификациями: полосы из геля промывали дважды 50 мМ гидрокарбонатом аммония в 50% ацетонитриле, затем их дегидратировали ацетонитрилом и высушили на воздухе. После чего к ним был добавлен раствор трипсина (25 мкг/мл) в 50 мМ гидрокарбонате аммония, рН 8,2.…”

Intact‐protein based sample preparation strategies for proteome analysis in combination with mass spectrometry

“…Shevchenko et al (1997) used this approach to isolate trans-Golgi network (TGN)-derived apical and basolateral transport vesicles, followed by 2D PAGE and Bio-MS. Two proteins that belong to the p23/p24 family of putative cargo receptors for vesicular trafficking were identified, and caveolin-2 was also characterized as a constituent of basolateral transport vesicles. This approach can also be used in combination with gradient centrifugation to further reduce protein cross-contamination from different organelles, such as endoplasmic reticulum, Golgi membrane, and plasma-membrane proteins.…”

“…Nano-electrospray techniques (Wilm & Mann, 1996;Shevchenko et al, 1997) combined with a hybrid quadrupole time-of-flight mass spectrometer tandem mass analyzer (ESI Q-TOF MSMS) enable extensive fragmentations to produce collision-induced dissociation (CID) spectra that allow unambiguous protein identification by peptide sequence tags through protein sequence database searches. High-throughput proteomic analysis may also be performed with a MALDI Q-TOF MSMS tandem instrument Shevchenko et al, 2000) and MALDI-TOF-TOF MSMS tandem MS (Medzihradszky et al, 2000).…”