A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea
A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea
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DOI：
10.1021/pr0603396
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发表时间：
2006-12-01
影响因子：
4.4
通讯作者：
Zhao, Yingming
中科院分区：
文献类型：
--
作者：
Kim, Sung Chan;Chen, Yue;Zhao, Yingming
来自细胞器或全细胞裂解物的复杂蛋白质混合物的蛋白水解消化通常在变性缓冲液（例如 1-2 M 尿素）的稀溶液中进行。随后必须通过 C18 珠去除尿素，然后进行下游分析（例如 HPLC/MS/MS）或完全甲基化，然后进行 IMAC 分离磷酸肽。在这里，我们描述了在没有变性剂的情况下消化复杂蛋白质混合物的程序。混合物中的蛋白质用三氯乙酸/丙酮沉淀，进行变性和除盐，然后重悬于 NH4HCO3 缓冲液中。胰蛋白酶分解后，所得肽不会被尿素或其他非挥发性盐污染，并且可以在 SpeedVac 中干燥以去除 NH4HCO3。当将该方案应用于 A431 细胞提取物时，96.8% 的胰蛋白酶肽被完全消化（即，没有遗漏的裂解位点），而在尿素缓冲液中消化产生的肽为 87.3%。我们成功应用这种消化方法来分析 HeLa 细胞脂肪体的磷酸化蛋白质组，鉴定出 28 种不同蛋白质中的 33 个磷酸化位点。我们的消化方法无需在 HPLC/MS/MS 分析或甲基化和 IMAC 之前去除尿素，从而提高通量，同时减少样品处理中的样品损失和污染。我们相信这种方法对于蛋白质组学研究应该有价值。
Proteolytic digestion of a complicated protein mixture from an organelle or whole-cell lysate is usually carried out in a dilute solution of a denaturing buffer, such as 1-2 M urea. Urea must be subsequently removed by C18 beads before downstream analysis such as HPLC/MS/MS or complete methylation followed by IMAC isolation of phosphopeptides. Here we describe a procedure for digesting a complicated protein mixture in the absence of denaturants. Proteins in the mixture are precipitated with trichloroacetic acid/acetone for denaturation and salt removal and resuspended in NH4HCO3 buffer. After trypsinolysis, the resulting peptides are not contaminated by urea or other nonvolatile salts and can be dried in a SpeedVac to remove NH4HCO3. When this protocol was applied to an extract of A431 cells, 96.8% of the tryptic peptides were completely digested (i.e., had no missed cleavage sites), in contrast to 87.3% of those produced by digestion in urea buffer. We successfully applied this digestion method to analysis of the phosphoproteome of adiposomes from HeLa cells, identifying 33 phosphorylation sites in 28 different proteins. Our digestion method avoids the need to remove urea before HPLC/MS/MS analysis or methylation and IMAC, increasing throughput while reducing sample loss and contamination from sample handling. We believe that this method should be valuable for proteomics studies.