Quantitative analytical method for determining the levels of gastric inhibitory polypeptides GIP_(1-42) and GIP_(3-42) in human plasma using LC-MS/MS/MS

摘要

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 GIP_(1-42) and GIP_(3-42) from human plasma. We selected the surrogate peptide to avoid oxidative modification, and the endoproteinase Asp-N was selected for the proteolysis of GIP_(1-42) and GIP_(3-42). The phenylalanine residue at position 6 in both GIP_(1-42) and GIP_(3-42) was substituted with ~(13)C_9,~(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 GIP_(1-42) and 10 pM for GIP_(3-42) by using 200 μL of plasma. Quantification of GIP_(1-42) and GIP _(3-42) 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 GIP_(1-8), and MRM for GIP _(3-8) to achieve accurate, precise, and quantitative analysis that can be validated to support large clinical trials.