A quantitative analytical method for determining the levels of gastric inhibitory polypeptides GIP1-42 and GIP3-42 in human plasma using LC-MS/MS/MS.
A quantitative analytical method for determining the levels of gastric inhibitory polypeptides GIP1-42 and GIP3-42 in human plasma using LC-MS/MS/MS.
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一种使用 LC-MS/MS/MS 测定人血浆中胃抑制多肽 GIP1-42 和 GIP3-42 水平的定量分析方法。
DOI：
10.1021/pr400069f
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Harada KI
中科院分区：
文献类型：
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作者：
Miyachi A;Murase T;Yamada Y;Osonoi T;Harada KI
胃抑制多肽（GIP）是一种肠促胰岛素，是内分泌学的重要研究对象。已经提出了一些LC-MS测定法;然而，它们的灵敏度不足以研究内源性人肠促胰岛素。在这里，我们描述了一种用于同时定量人血浆中GIP 1 - 42和GIP 3 - 42的纳米流LC混合三重四极杆/线性离子阱MS分析。我们选择替代肽以避免氧化修饰，并选择内切蛋白酶Asp-N用于GIP 1 - 42和GIP 3 -42的蛋白水解。用13 C9，15 N标记的苯丙氨酸取代GIP 1 - 42和GIP 3 - 42中第6位的苯丙氨酸残基，并将这些取代的GIPs用作内标。这有助于准确和精确的定量，因为在样品预处理和电离效率的所有步骤中都可以进行大的校正。采用200 μL血浆，GIP 1 - 42的定量下限为1 pM，GIP 3 - 42的定量下限为10 pM。使用该方法可以定量2型糖尿病患者血浆中的GIP 1 - 42和GIP 3 - 42，该方法包括蛋白质沉淀、Asp-N蛋白水解、固相萃取、纳米流LC和GIP 1 -8的正离子多反应监测立方体（MRM 3），以及GIP 3 - 8的MRM，以实现准确、精确和定量分析，可以验证以支持大型临床试验。
Gastric inhibitory polypeptide (GIP), an incretin, is an important subject in endocrinology. Some LC–MS assays have been proposed; however, their sensitivities are insufficient for the study of endogenous human incretin. Here, we describe a nanoflow LC hybrid triple quadrupole/linear ion trap MS assay for the simultaneous quantification of GIP1–42and GIP3–42from human plasma. We selected the surrogate peptide to avoid oxidative modification, and the endoproteinase Asp-N was selected for the proteolysis of GIP1–42and GIP3–42. The phenylalanine residue at position 6 in both GIP1–42and GIP3–42was substituted with13C9,15N-labeled phenylalanine, and these substituted GIPs were used as the internal standards. This facilitated accurate and precise quantification because large corrections are possible at all steps of sample pretreatment and ionization efficiency. The lower limit of quantification was 1 pM for GIP1–42and 10 pM for GIP3–42by using 200 μL of plasma. Quantification of GIP1–42and GIP3–42in plasma from patients with type 2 diabetes was possible using this method, which included protein precipitation, Asp-N proteolysis, solid-phase extraction, nanoflow LC, and positive-ion multiple reaction monitoring cubed (MRM3) for GIP1–8, and MRM for GIP3–8to achieve accurate, precise, and quantitative analysis that can be validated to support large clinical trials.