Absolute quantification of the model biomarker prostate-specific antigen in serum by LC-MS/MS using protein cleavage and isotope dilution mass spectrometry
Absolute quantification of the model biomarker prostate-specific antigen in serum by LC-MS/MS using protein cleavage and isotope dilution mass spectrometry
复制标题
DOI：
10.1021/pr049963d
复制
发表时间：
2004-05-01
影响因子：
4.4
通讯作者：
Muddiman, DC
中科院分区：
文献类型：
--
作者：
Barnidge, DR;Goodmanson, MK;Muddiman, DC
蛋白质裂解-同位素稀释质谱法（PC-IDMS）可用于定量蛋白质，用同位素标记的肽片段类似物作为内标。在这里，我们研究了使用标准LC-MS/MS平台通过该技术直接从血清中定量模型生物标志物。我们合成了一个与PSA n端色氨酸片段相同的肽（IVGGWECEK），但每个甘氨酸含有两个C-13原子和一个N-15原子。用尿素使无PSA的人血清变性，然后引入PSA标准品和稳定同位素标记的内标肽。然后用胰蛋白酶水解样品，并在三重四极杆质谱仪上使用LC-MS/ MS进行定量。将5种不同浓度的PSA添加到血清中并消化（每配制3份，随机注射3次），得到线性最小二乘校准曲线，平均斜率为0.973 (SE = 0.023)，截距为-0.003 (SE = 0.022)， R-2为0.971。校准器的回收率为70 ~ 85%，运行间平均CV为13%，运行内平均CV为5.7%。PC-IDMS是一种很有前途的蛋白质定量技术，涵盖了广泛的应用，从标准化免疫测定到监测翻译后修饰，再到在免疫测定的开发和实施之前定量新发现的生物标志物，仅举几例。围绕PC-IDMS用于蛋白质绝对定量的应用问题包括选择可在宽动态范围内切割和可重复分离的蛋白质水解片段进行定量，稳定的同位素标记合成肽标准，提供一致的结果，LC-MS/MS方法提供足够的灵敏度和再现性，而不会产生不实际的分析时间。本研究结果表明，在LC-MS/MS分析时，可以对引入变性血清的模型生物标志物PSA进行绝对定量。然而，与现有的免疫测定法相比，PC-IDMS的敏感性以及在不同基质中进行的可重复性仍然存在问题。
Protein cleavage-isotope dilution mass spectrometry (PC-IDMS) can be used to quantify proteins, with an isotope-labeled analogue of the peptide fragment used as an internal standard. Here, we investigate use of a standard LC-MS/MS platform for quantifying a model biomarker directly from serum by this technique. We synthesized a peptide (IVGGWECEK) identical to the N-terminal tryptic fragment of PSA but with each glycine containing two C-13 atoms and one N-15 atom. PSA-free human serum was denatured with urea followed by the introduction of PSA standard and the stable isotope labeled internal standard peptide. The sample was then proteolyzed with trypsin and subjected to quantification using LC-MS/ MS on a triple quadrupole mass spectrometer. A linear least squares calibration curve made from five different concentrations of PSA added to serum and digested (each made in triplicate and randomly injected three times) had a mean slope of 0.973 (SE = 0.023), intercept of -0.003 (SE = 0.022), and R-2 of 0.971. Recovery of calibrators ranged from 70 to 85% with a mean run-to-run CV of 13% and a mean within-run CV of 5.7%. PC-IDMS is a promising technique for quantifying proteins covering a broad range of applications from standardizing immunoassays to monitoring post-translational modifications to quantifying newly discovered biomarkers prior to the development and implementation of an immunoassay, just to name a few. Issues surrounding the application of PC-IDMS for the absolute quantification of proteins include selection of a proteolytic fragment for quantification that can be cleaved and isolated reproducibly over a broad dynamic range, stable isotope labeled synthetic peptide standards that give consistent results, and LC-MS/MS methods that provide adequate sensitivity and reproducibility without creating impractical analysis times. The results presented here show that absolute quantification can be performed on the model biomarker PSA introduced into denatured serum when analyzed by LC-MS/MS. However, concerns still exist regarding sensitivity compared to existing immunoassays as well as the reproducibility of PC-IDMS performed in different matrixes.