Blonder Josip, Chan King C, Issaq Haleem J, Veenstra Timothy D
Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Cancer Institute at Frederick, P.O. Box B, Frederick, Maryland 21702, USA.
Nat Protoc. 2006;1(6):2784-90. doi: 10.1038/nprot.2006.359.
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.
对哺乳动物膜蛋白进行高效蛋白质组规模分析的核心前提条件是有效的分离、溶解、消化以及多维液相色谱-串联质谱（LC-MS/MS）。本方案用于分析哺乳动物膜蛋白质组，该方法依赖于在含有60%（体积/体积）甲醇的缓冲液中溶解膜蛋白并进行胰蛋白酶消化。胰蛋白酶消化后，进行强阳离子交换（SCX）色谱和反相（RP）色谱，并与MS/MS在线联用进行蛋白质鉴定。使用基于甲醇的缓冲液无需使用会干扰肽段色谱分辨率和离子化的试剂（如去污剂、离液剂、无机盐）。由于溶解和消化在同一管中进行，避免了这些步骤之间的任何样品转移或缓冲液交换，样品损失降至最低。本方案与蛋白质和肽水平的稳定同位素标记兼容，能够鉴定和定量整合膜蛋白。整个过程——从分离膜组分开始到获取MS数据结束——需要4至5天。
