Mass spectrometric characterisation of the circulating peptidome following oral glucose ingestion in control and gastrectomised patients.

Abstract

Meal ingestion triggers secretion of a variety of gut and endocrine peptides, several of which are routinely measured in research studies by commercial immunoassays. We developed an LC-MS/MS based assay for parallel monitoring of multiple peptides in small volumes of human plasma, providing the benefit of analysing exact peptide sequences rather than immuno-reactivity, and potential advantages of cost and sample volumes for measuring multiple peptide hormones. The method involves acetonitrile precipitation of larger proteins, followed by solid phase extraction and nano-LC-MS/MS using an untargeted approach on an orbitrap mass spectrometer. Analysis of plasma from control subjects and patients who have undergone gastrectomy with Roux-en-Y reconstruction, revealed elevated levels of a number of peptides following glucose ingestion. These included GLP-1(7-36), GLP-1(9-36), glicentin, oxyntomodulin, GIP(1-42), GIP(3-42), PYY(1-36), PYY(3-36), neurotensin, insulin and C-peptide, as well as motilin, which decreased following glucose ingestion. Results showed good correlation with those peptides measured previously by immunoassay in the same samples. The gastrectomy group had higher, but non-glucose-dependent, circulating levels of peptides from PIGR and DMBT1. Overall, the method is fast, generic, reproducible and inexpensive, and requires only small plasma samples, making it potentially adaptable for multiplexed measurement of a variety of peptides.