Ye Xiaoying, Luke Brian T, Johann Donald J, Chan King C, Prieto Darue A, Ono Akira, Veenstra Timothy D, Blonder Josip
Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD, USA.
Methods Mol Biol. 2012;893:223-40. doi: 10.1007/978-1-61779-885-6_15.
The role of membrane proteins is critical for regulation of physiologic and pathologic cellular processes. Hence it is not surpassing that membrane proteins make ∼70% of contemporary drug targets. Quantitative profiling of membrane proteins using mass spectrometry (MS)-based proteomics is critical in a quest for disease biomarkers and novel cancer drugs. Post-digestion (18)O exchange is a simple and efficient method for differential (18)O/(16)O stable isotope labeling of two biologically distinct specimens, allowing relative quantitation of proteins in complex mixtures when coupled with shotgun MS-based proteomics. Due to minimal sample consumption and unrestricted peptide tagging, (18)O/(16)O stable isotope labeling is particularly suitable for amount-limited protein specimens typically encountered in membrane and clinical proteomics. This chapter describes a protocol that relies on shotgun proteomics for quantitative profiling of the detergent-insoluble membrane proteins isolated from HeLa cells, differentially transfected with plasmids expressing HIV Gag protein and its myristylation-defective N-terminal mutant. Whilst this protocol depicts solubilization of detergent-insoluble membrane proteins coupled with post-digestion (18)O labeling, it is amenable to any complex membrane protein mixture. Described approach relies on solubilization and tryptic digestion of membrane proteins in a buffer containing 60% (v/v) methanol followed by differential (18)O/(16)O labeling of protein digests in 20% (v/v) methanol buffer. After mixing, the differentially labeled peptides are fractionated using off-line strong cation exchange (SCX) followed by on-line reversed phase nanoflow reversed-phase liquid chromatography (nanoRPLC)-MS identification/quantiation of peptides/proteins. The use of methanol-based buffers in the context of the post-digestion (18)O exchange/labeling eliminates the need for detergents or chaotropes that interfere with LC separations and peptide ionization. Sample losses are minimized because solubilization, digestion, and stable isotope labeling are carried out in a single tube, avoiding any sample transfer or buffer exchange between these steps.
膜蛋白在调节生理和病理细胞过程中起着关键作用。因此，膜蛋白占当代药物靶点的70%左右也就不足为奇了。使用基于质谱（MS）的蛋白质组学对膜蛋白进行定量分析，对于寻找疾病生物标志物和新型癌症药物至关重要。消化后（18）O交换是一种简单有效的方法，用于对两个生物学上不同的样本进行差异（18）O/（16）O稳定同位素标记，当与基于鸟枪法MS的蛋白质组学结合使用时，可对复杂混合物中的蛋白质进行相对定量。由于样品消耗极少且肽标记不受限制，（18）O/（16）O稳定同位素标记特别适用于膜和临床蛋白质组学中常见的量有限的蛋白质样本。本章描述了一种方案，该方案依赖于鸟枪法蛋白质组学对从HeLa细胞中分离出的去污剂不溶性膜蛋白进行定量分析，这些细胞分别用表达HIV Gag蛋白及其肉豆蔻酰化缺陷型N端突变体的质粒进行差异转染。虽然该方案描述了去污剂不溶性膜蛋白的溶解以及消化后（18）O标记，但它适用于任何复杂的膜蛋白混合物。所描述的方法依赖于在含有60%（v/v）甲醇的缓冲液中溶解和胰蛋白酶消化膜蛋白，然后在20%（v/v）甲醇缓冲液中对蛋白质消化物进行差异（18）O/（16）O标记。混合后，使用离线强阳离子交换（SCX）对差异标记的肽进行分级分离，然后通过在线反相纳流反相液相色谱（nanoRPLC）-MS对肽/蛋白质进行鉴定/定量。在消化后（18）O交换/标记过程中使用基于甲醇的缓冲液，无需使用会干扰液相色谱分离和肽电离的去污剂或离液剂。由于溶解、消化和稳定同位素标记在单个试管中进行，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。
