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Identification of membrane proteins from mammalian cell/tissue using methanol-facilitated solubilization and tryptic digestion coupled with 2D-LC-MS/MS


The core prerequisites for an efficient proteome-scale analysis of mammalian membrane proteins are effective isolation, solubilization, digestion and multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). This protocol is for analysis of the mammalian membrane proteome that relies on solubilization and tryptic digestion of membrane proteins in a buffer containing 60% (vol/vol) methanol. Tryptic digestion is followed by strong cation exchange (SCX) chromatography and reversed phase (RP) chromatography coupled online with MS/MS for protein identification. The use of a methanol-based buffer eliminates the need for reagents that interfere with chromatographic resolution and ionization of the peptides (e.g., detergents, chaotropes, inorganic salts). Sample losses are minimized because solubilization and digestion are carried out in a single tube avoiding any sample transfer or buffer exchange between these steps. This protocol is compatible with stable isotope labeling at the protein and peptide level, enabling identification and quantitation of integral membrane proteins. The entire procedure—beginning with isolated membrane fraction and finishing with MS data acquisition—takes 4–5 d.

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Figure 1: Flow chart showing sample preparation protocol used for multidimensional proteomic analysis of mammalian membrane proteins.
Figure 2: Cell adhesion proteins identified from the keratinocyte plasma membrane isolated from human skin (epidermis) depicted in accordance to their junction affiliation.
Figure 3: Flow chart showing isolation of the human keratinocyte plasma membrane.
Figure 4: Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis comparing Halobacterium halobium purple membrane sample containing bacteriorhodopsin (e.g., hydrophobic multipass integral membrane protein) before (lane 1) and after (lane 2) tryptic digestion in the presence of 60% (vol/vol) methanol.
Figure 5: Multidimensional proteomic analysis of mammalian membrane proteome using two-dimensional LC coupled with MS/MS.

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This project has been funded in whole or in part by federal funds from the National Cancer Institute, National Institutes of Health, under Contract NO1-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the United States government.

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Correspondence to Josip Blonder.

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Blonder, J., Chan, K., Issaq, H. et al. Identification of membrane proteins from mammalian cell/tissue using methanol-facilitated solubilization and tryptic digestion coupled with 2D-LC-MS/MS. Nat Protoc 1, 2784–2790 (2006).

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