Department of Chemistry, University of California, Berkeley, California 94720, United States.
ACS Chem Biol. 2011 Aug 19;6(8):829-36. doi: 10.1021/cb100338x. Epub 2011 Jun 16.
Directed proteomics applies mass spectrometry analysis to a subset of information-rich proteins. Here we describe a method for targeting select proteins by chemical modification with a tag that imparts a distinct isotopic signature detectable in a full-scan mass spectrum. Termed isotopic signature transfer and mass pattern prediction (IsoStamp), the technique exploits the perturbing effects of a dibrominated chemical tag on a peptide's mass envelope, which can be detected with high sensitivity and fidelity using a computational method. Applying IsoStamp, we were able to detect femtomole quantities of a single tagged protein from total mammalian cell lysates at signal-to-noise ratios as low as 2.5:1. To identify a tagged-peptide's sequence, we performed an inclusion list-driven shotgun proteomics experiment where peptides bearing a recoded mass envelope were targeted for fragmentation, allowing for direct site mapping. Using this approach, femtomole quantities of several targeted peptides were identified in total mammalian cell lysate, while traditional data-dependent methods were unable to identify as many peptides. Additionally, the isotopic signature imparted by the dibromide tag was detectable on a 12-kDa protein, suggesting applications in identifying large peptide fragments, such as those containing multiple or large posttranslational modifications (e.g., glycosylation). IsoStamp has the potential to enhance any proteomics platform that employs chemical labeling for targeted protein identification, including isotope coded affinity tagging, isobaric tagging for relative and absolute quantitation, and chemical tagging strategies for posttranslational modification.
定向蛋白质组学将质谱分析应用于信息丰富的蛋白质子集。在这里，我们描述了一种通过用赋予独特同位素特征的标签化学修饰来靶向选择蛋白质的方法，该特征在全扫描质谱中可检测到。该技术称为同位素特征转移和质量模式预测（IsoStamp），它利用了二溴化化学标签对肽质量包络的干扰效应，可使用计算方法以高灵敏度和保真度进行检测。应用 IsoStamp，我们能够从总哺乳动物细胞裂解物中检测到低至 2.5:1 的信噪比的单个标记蛋白的飞摩尔量。为了鉴定标记肽的序列，我们进行了包含列表驱动的鸟枪法蛋白质组学实验，其中携带重编码质量包络的肽被靶向进行碎片化，从而可以直接进行位点映射。使用这种方法，在总哺乳动物细胞裂解物中鉴定了几个靶向肽的飞摩尔量，而传统的基于数据的方法则无法鉴定那么多的肽。此外，二溴化物标签赋予的同位素特征在 12kDa 蛋白质上可检测到，这表明该方法可用于鉴定大的肽片段，例如含有多个或大的翻译后修饰（例如糖基化）的肽片段。IsoStamp 有可能增强任何使用化学标记进行靶向蛋白质鉴定的蛋白质组学平台，包括同位素编码亲和标记、相对和绝对定量的等重标记、以及翻译后修饰的化学标记策略。
