Cheung Crystal S F, Anderson Kyle W, Wang Meiyao, Turko Illarion V
Institute for Bioscience and Biotechnology Research , Rockville, Maryland 20850, United States.
Anal Chem. 2015 Jan 20;87(2):1097-102. doi: 10.1021/ac503697j. Epub 2014 Dec 31.
Quantification by targeted proteomics has largely depended on mass spectrometry and isotope-labeled internal standards. In addition to traditionally used recombinant proteins or synthetic peptides, concatenated peptides (QconCATs) were introduced as a conceptually new source of internal standard. In the present study, we focused on assessing the length of natural flanking sequences, which surround each peptide included in QconCAT and provide for identical rates of analyte and standard digestion by trypsin. We have expressed, purified, and characterized a set of seven (15)N-labeled QconCATs that cover seven tryptic peptides from human clusterin with a length of natural flanking sequences ranging from none (+0) to six amino acid residues (+6) for each tryptic peptide. Individual QconCATs were mixed with recombinant human clusterin at a 1:1 molar ratio and digested, and the actual ratios for each combination of peptide/flanking sequence were measured with a multiple reaction monitoring assay. Data analysis suggested that natural flanking sequences shorter than +6 residues can cause a quantitative error because the random appearance of other amino acid residues in close proximity to trypsin cleavage sites has unpredictable consequences for the digestion rates of QconCATs.
靶向蛋白质组学的定量分析很大程度上依赖于质谱和同位素标记的内标。除了传统使用的重组蛋白或合成肽外，串联肽（QconCATs）作为一种概念上新的内标来源被引入。在本研究中，我们着重评估天然侧翼序列的长度，这些序列围绕着QconCAT中包含的每个肽段，并确保胰蛋白酶对分析物和标准品的消化速率相同。我们表达、纯化并表征了一组七个用（15）N标记的QconCATs，它们覆盖了来自人簇集蛋白的七个胰蛋白酶肽段，每个胰蛋白酶肽段的天然侧翼序列长度从无（+0）到六个氨基酸残基（+6）不等。将单个QconCATs与重组人簇集蛋白以1:1的摩尔比混合并进行消化，然后用多反应监测分析法测量每种肽段/侧翼序列组合的实际比例。数据分析表明，短于+6个残基的天然侧翼序列可能会导致定量误差，因为在胰蛋白酶切割位点附近随机出现的其他氨基酸残基会对QconCATs的消化速率产生不可预测的影响。
