Quantitative analytical method for determining the levels of gastric inhibitory polypeptides GIP1-42 and GIP3-42 in human plasma using LC-MS/MS/MS.
Quantitative analytical method for determining the levels of gastric inhibitory polypeptides GIP1-42 and GIP3-42 in human plasma using LC-MS/MS/MS.
复制标题
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：
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发表时间：
2013
影响因子：
4.4
通讯作者：
K. Harada
中科院分区：
文献类型：
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作者：
Atsushi Miyachi;T. Murase;Yuichiro Yamada;T. Osonoi;K. Harada
胃抑制多肽（GIP）是一种肠促胰岛素，是内分泌学的重要课题。已经提出了一些 LC-MS 检测方法；然而，它们的敏感性不足以研究内源性人肠促胰岛素。在这里，我们描述了一种纳流 LC 混合三重四极杆/线性离子阱 MS 测定，用于同时定量人血浆中的 GIP1-42 和 GIP3-42。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GIP1-42和GIP3-42的蛋白水解。 GIP1-42和GIP3-42中6位的苯丙氨酸残基均被(13)C9,(15)N-标记的苯丙氨酸取代，并且这些取代的GIP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。使用 200 μL 血浆，GIP1-42 的定量下限为 1 pM，GIP3-42 的定量下限为 10 pM。使用该方法可以对 2 型糖尿病患者血浆中的 GIP1-42 和 GIP3-42 进行定量，其中包括蛋白质沉淀、Asp-N 蛋白水解、固相萃取、纳流 LC、GIP1-8 的正离子多反应监测立方 (MRM(3)) 和 GIP3-8 的 MRM，以实现准确、精确的定量分析，并可进行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 GIP1-42 and GIP3-42 from human plasma. We selected the surrogate peptide to avoid oxidative modification, and the endoproteinase Asp-N was selected for the proteolysis of GIP1-42 and GIP3-42. The phenylalanine residue at position 6 in both GIP1-42 and GIP3-42 was substituted with (13)C9,(15)N-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 GIP1-42 and 10 pM for GIP3-42 by using 200 μL of plasma. Quantification of GIP1-42 and GIP3-42 in 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 (MRM(3)) for GIP1-8, and MRM for GIP3-8 to achieve accurate, precise, and quantitative analysis that can be validated to support large clinical trials.
影响因子：
7.4
作者：
Cao, Jin;Covarrubias, Vanessa M.;Straubinger, Robert M.;Wang, Hao;Duan, Xiaotao;Yu, Haoying;Qu, Jun;Blanco, Javier G.
通讯作者：
Blanco, Javier G.