Neubauer G, Mann M
European Molecular Biology Laboratory, Heidelberg, Germany.
Anal Chem. 1999 Jan 1;71(1):235-42. doi: 10.1021/ac9804902.
Precursor ion scans have proven to be extremely useful for the characterization of unseparated peptide mixtures. In conjunction with the nanoelectrospray source, precursor ion scans provide a sensitive tool for the detection of posttranslationally modified peptides and have been used to determine phosphorylation sites of proteins digested in solution. In this report, we extend our previous work to the determination of protein phosphorylation sites of gel-isolated proteins. The in-gel digestion procedure developed in our laboratory for protein microsequencing was found to be suitable for phosphorylation mapping as well. The risk of losing hydrophilic peptides in the desalting step was decreased by using column packing material designed for the purification of oligonucleotides and by adjusting the pH conditions to the needs of phosphopeptide analysis. With this method, the tryptic phosphopeptides of beta-casein were detected after in-gel digestion at a sensitivity of 250 fmol of protein applied to the gel. The phosphorylation sites of two other proteins, Src-delta U and Op18, have similarly been mapped. Subpicomole to low-picomole amounts of protein starting material are needed in general, although we and others have reported attomole sensitivity for the detection of model phosphopeptides using precursor ion scans. This indicates that the success in determining phosphorylation sites depends crucially on the digestion, extraction, and detection efficiency for individual phosphopeptides.
前体离子扫描已被证明对于未分离的肽混合物的表征极为有用。与纳米电喷雾源相结合，前体离子扫描为检测翻译后修饰的肽提供了一种灵敏的工具，并已用于确定溶液中消化的蛋白质的磷酸化位点。在本报告中，我们将先前的工作扩展到凝胶分离蛋白质的蛋白质磷酸化位点的测定。我们实验室开发的用于蛋白质微量测序的凝胶内消化程序也被发现适用于磷酸化图谱分析。通过使用专为寡核苷酸纯化设计的柱填充材料并根据磷酸肽分析的需要调整pH条件，降低了脱盐步骤中丢失亲水性肽的风险。用这种方法，在凝胶内消化后检测到β-酪蛋白的胰蛋白酶磷酸肽，其灵敏度为施加到凝胶上的250飞摩尔蛋白质。另外两种蛋白质Src-δU和Op18的磷酸化位点也已类似地绘制出来。一般需要亚皮摩尔到低皮摩尔量的蛋白质起始材料，尽管我们和其他人已经报道了使用前体离子扫描检测模型磷酸肽的阿托摩尔灵敏度。这表明确定磷酸化位点的成功关键取决于单个磷酸肽的消化、提取和检测效率。
