Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies (SISCAPA)
Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies (SISCAPA)
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DOI：
10.1021/pr034086h
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发表时间：
2004-03-01
影响因子：
4.4
通讯作者：
Pearson, TW
中科院分区：
文献类型：
--
作者：
Anderson, NL;Anderson, NG;Pearson, TW
描述了一种用于对复杂消化中肽进行定量的方法(记为SISCAPA)。在该方法中，用固定在100纳升纳米亲和色谱柱上的抗肽抗体与相同序列的添加稳定同位素标记的内标一起富集特定的多肽。当从抗肽抗体载体上洗脱后，使用电喷雾质谱仪对多肽(天然的和标记的)进行定量。在一系列的初步实验中，从10203个候选胰蛋白酶多肽中选择了四种人血浆蛋白(血凝素、α1抗糜蛋白酶、白细胞介素6和肿瘤坏死因子-α)的胰酶测试肽，这些多肽代表了237种已知的血浆成分。针对所选择的多肽序列制备的兔多克隆抗体被亲和纯化并共价固定在POOS载体上。通过选择离子监测(SIM)或选择反应监测(SRM)电喷雾质谱检测，结合和洗脱这些载体提供的抗原肽的平均浓缩倍数是其他载体的120倍。这些柱可以循环使用，结合能力几乎没有损失，并产生了多肽离子电流测量，循环间变异系数接近5%。抗肽抗体的浓缩将有助于提高MS分析的灵敏度，特别是对血浆中丰度较低的蛋白质，并可能最终允许快速结合/洗脱过程取代目前用作在线MS多肽分析前奏的耗时的反相分离过程。该方法适合于快速生成已定义蛋白质的分析，并应在大样本集的诊断蛋白质板的验证中得到应用。
A method (denoted SISCAPA) for quantitation of peptides in complex digests is described. In the method, anti-peptide antibodies immobilized on 100 nanoliter nanoaffinity columns are used to enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence. Upon elution from the anti-peptide antibody supports, electrospray mass spectrometry is used to quantitate the peptides (natural and labeled). In a series of pilot experiments, tryptic test peptides were chosen for four proteins of human plasma (hemopexin, alpha1 antichymotrypsin, interleukin-6, and tumor necrosis factor-alpha) from a pool of 10203 in silico tryptic peptide candidates representing 237 known plasma components. Rabbit polyclonal antibodies raised against the chosen peptide sequences were affinity purified and covalently immobilized on POROS supports. Binding and elution from these supports was shown to provide an average 120-fold enrichment of the antigen peptide relative to others, as measured by selected ion monitoring (SIM) or selected reaction monitoring (SRM) electrospray mass spectrometry. The columns could be recycled with little loss in binding capacity, and generated peptide ion current measurements with cycle-to-cycle coefficients of variation near 5%. Anti-peptide antibody enrichment will contribute to increased sensitivity of MS-based assays, particularly for lower abundance proteins in plasma, and may ultimately allow substitution of a rapid bind/elute process for the time-consuming reverse phase separation now used as a prelude to online MS peptide assays. The method appears suitable for rapid generation of assays for defined proteins, and should find application in the validation of diagnostic protein panels in large sample sets.