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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

  1. Author:
    Blonder, J.
    Chan, K. C.
    Issaq, H. J.
    Veenstra, T. D.

  2. Author Address

    Natl Canc Inst Frederick, SAIC Frederick Inc, Lab Proteom & Anal Technol, Frederick, MD 21702 USA.;Blonder, J, Natl Canc Inst Frederick, SAIC Frederick Inc, Lab Proteom & Anal Technol, POB B, Frederick, MD 21702 USA.;blonder@ncifcrf.gov
    1. Year: 2006
  2. Journal: Nature Protocols
    1. 1
    2. 6
    3. Pages: 2784-2790
  4. Type of Article: Article
  5. ISSN: 1745-2473
  1. Abstract:

    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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External Sources

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  2. DOI: 10.1038/nprot.2006.359
  3. View record in Web of Science®
  4. WOS: 000251155700033

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