Peptidomic analysis of endogenous plasma peptides from patients with pancreatic neuroendocrine tumours

Abstract

RationaleDiagnosis of pancreatic neuroendocrine tumours requires the study of patient plasma with multiple immunoassays, using multiple aliquots of plasma. The application of mass spectrometry based techniques could reduce the cost and amount of plasma required for diagnosis.MethodsPlasma samples from two patients with pancreatic neuroendocrine tumours were extracted using an established acetonitrile‐based plasma peptide enrichment strategy. The circulating peptidome was characterised using nano and high flow rate liquid chromatography/mass spectrometry (LC/MS) analyses. To assess the diagnostic potential of the analytical approach, a large sample batch (68 plasmas) from control subjects, and aliquots from subjects harbouring two different types of pancreatic neuroendocrine tumour (insulinoma and glucagonoma), were analysed using a 10‐min LC/MS peptide screen.ResultsThe untargeted plasma peptidomics approach identified peptides derived from the glucagon prohormone, chromogranin A, chromogranin B and other peptide hormones and proteins related to control of peptide secretion. The glucagon prohormone derived peptides that were detected were compared against putative peptides that were identified using multiple antibody pairs against glucagon peptides. Comparison of the plasma samples for relative levels of selected peptides showed clear separation between the glucagonoma and the insulinoma and control samples.ConclusionsThe combination of the organic solvent extraction methodology with high flow rate analysis could potentially be used to aid diagnosis and monitor treatment of patients with functioning pancreatic neuroendocrine tumours. However, significant validation will be required before this approach can be clinically applied.

Quantitative mass spectrometry for human melanocortin peptides in vitro and in vivo suggests prominent roles for β-MSH and desacetyl α-MSH in energy homeostasis

“…In addition, we found that acetylated POMC-derived peptides, including α-MSH, were readily detectable from mouse plasma using the same peptide extraction and LC-MS/MS methods used to analyse human neuronal samples, ruling out any potential detection bias ( Fig. S4 ) [66] . To test whether the higher concentration of β-EP (1-31) relative to d-α-MSH(1-13) and β-MSH(1-18) could be explained by technical artefacts, such as degradation of the standard peptide, we acquired a fresh stock of synthetic β-EP (1-31) and found good agreement (within 16%) with our previous measurements, indicating that the substantially higher concentrations of β-EP (1-31) we observed were not likely due to degradation of the stock peptide.…”

“…In addition, the acetylated α-MSH peptide standard was efficiently recovered during solid phase extraction ( Fig. S3 ), and acetylated α-MSH is robustly detected from mouse plasma using very similar sample preparation methods [66] ( Fig. S4 ).…”

Small-protein Enrichment Assay Enables the Rapid, Unbiased Analysis of Over 100 Low Abundance Factors from Human Plasma

“…These extraction methods facilitate dissociation of small proteins from larger factors (13,14) and the bulk removal of large plasma proteins by precipitation. For example, these studies have successfully quantified several commonly studied small protein hormones such as insulin (8,15,16), hepcidin (9,16), and IGF-1 (7,10,11,16). For quantification of analytes, these studies generally use high flow rate liquid chromatography separations with online targeted mass spectrometry detection.…”

Assessment and Management of Anti-Insulin Autoantibodies in Varying Presentations of Insulin Autoimmune Syndrome

“…Then, 250 μL of each sample of known peptide concentration, available patient plasma, and unfortified pooled plasma was transferred to different wells of a 2-mL 96-well plate. Five patient and 34 control samples were extracted using a combination of acetonitrile precipitation and solid-phase extraction–liquid chromatography ( 23 ) along with quality control samples and analyzed with two separately extracted sets of calibration samples. MS data were acquired from m/z 700 to 1600, with a resolution of 70,000 and an automatic gain control target of 3e6 ions.…”