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.
Highlights
The application of mass spectrometry to protein analysis is common place, and routine in academic proteomics core facilities
The liquid chromatography/mass spectrometry (LC/MS) analysis identified a total of nine peptides from the pro‐peptide, three below the 1% false discovery rate (FDR) value (Table 2), including two previously characterised peptides from Chromogranin A (CHGA) known as LF‐19 and GR‐44
This plasma peptidomics methodology has demonstrated the ability to detect peptide hormones produced by Pancreatic neuroendocrine tumours (PNETs) as well as peptides from other regulatory prohormones such as Glucose‐dependent insulinotropic polypeptide (GIP) and insulin
Summary
The application of mass spectrometry to protein analysis is common place, and routine in academic proteomics core facilities. Other sample handling procedures (for both human and rodent plasma) have used size‐exclusion‐based extraction techniques, such as ultrafiltration spin devices.[6,7] Some plasma peptidomic studies have employed solid‐ phase extraction (SPE), whereby plasma is extracted by SPE followed by high‐performance liquid chromatography (HPLC) fractionation and subsequent enzymatic digestion prior to LC/MS analysis.[8] These previous methodologies have shown effectiveness at extracting peptides from plasma, they are not considered high throughput, and in the case of immunoaffinity extraction, have to be performed consecutively. Pancreatic islet α‐cell tumours that overexpress proglucagon are typically associated with the glucagonoma syndrome, a rare disease characterised by necrolytic migratory erythema, impaired glucose tolerance, thromboembolic complications and psychiatric disturbance.[15] Currently, diagnosis of a PNET is made through a combination of clinical examination and history as well as radiological, histological and biochemical investigations The latter are based, primarily, on immunoassays directed towards a limited number of peptide hormone biomarkers. Samples were analysed by low‐ and high‐throughput LC/MS methodologies to assess the feasibility of developing these techniques for use in a clinical setting
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