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

Abstract

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.

Simultaneous quantification of intracellular and secreted active and inactive glucagon-like peptide-1 from cultured cells

“…Unlike GIP peptides, GLP-1 peptides are subject to both N-and C-terminal processing. Therefore, enzymatic digestion cannot be used for these peptides, as described by Miyachi and coworkers [22]. In addition, intact GLP-1 peptides are hydrophilic, meaning that they are highly adsorptive at lower concentrations (pM levels) [33].…”

“…Wolf and coworkers [14,15] used immunoprecipitation and mass spectrometry to analyze incretin peptide hormones. Selected reaction monitoring (SRM) is now widely employed for peptide hormone quantification owing to its high sensitivity, specificity, and multiplicity [16][17][18][19][20][21][22]. For instance, Miyachi and coworkers developed a method to analyze GIP peptides [22].…”

“…Selected reaction monitoring (SRM) is now widely employed for peptide hormone quantification owing to its high sensitivity, specificity, and multiplicity [16][17][18][19][20][21][22]. For instance, Miyachi and coworkers developed a method to analyze GIP peptides [22]. GIP contains a methionine at position 14, and trypsin digestion produces methionine-containing GIP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] and GIP [3][4][5][6][7][8][9][10][11][12][13][14][15][16] peptides, which can be easily http://dx.doi.org/10.1016/j.ab.2014.…”

Microbial degradation of linear peptides by strain B-9 of Sphingosinicella and its application in peptide quantification using liquid chromatography-mass spectrometry

“…The calibration curve in plasma was assessed by the correlation coefficient obtained using the following equation: Y ¼ A Â X þ B (the weighting factor was 1/X 2 ), where Y is the ratio of the relative peak area of GLP-1 [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] to that of the IS GIP 1-8 , X is the concentration of GLP-1, and A and B are the values from least squares regression analysis. Acceptance criterion for the calibration curve and QC samples is that the back-calculated concentration for each calibration sample is within 100 AE 15% of the theoretical concentration (100 AE 20% at the lower limit of quantification: LLOQ).…”

“…To overcome undesirable properties, Siskos et al proposed the use of a surrogate peptide (4). We have also successfully developed a quantification method for GIPs using LC-MS via their surrogate peptides, in which we selected the endoproteinase Asp-N instead of trypsin because the surrogate peptide GIP 1-8 obtained with Asp-N was excellent in terms of oxidation resistance, compared to GIP 1-16 obtained with trypsin (7). This strongly indicates the importance of careful selection of both the targeted peptide and the protease for the quantification of peptides.…”