Improving sensitivity for the targeted LC-MS/MS analysis of the peptide bradykinin using a design of experiments approach

For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays. Show more

Kinins, which are produced by the action of kallikrein enzymes, are blood-derived local-acting peptides that have broad effects mediated by two related G-protein-coupled receptors termed the bradykinin receptors. The endogenous kallikrein-kinin system controls blood circulation and kidney function, and promotes inflammation and pain in pathological conditions, which has led to interest in developing modulators of bradykinin receptors as potential therapeutics. This review discusses recent progress in our understanding of the genetics, molecular biology and pathophysiology of kinins and their receptors, as well as developments in medicinal chemistry, which have brought us closer to therapeutic applications of kinin receptor ligands in various indications. The potential of kinin receptor antagonists as novel analgesic agents that do not result in tolerance or have a liability for abuse has attracted particular interest. Show more

The unpredictable nature of peptide binding to surfaces requires optimization of experimental containers to be used. To demonstrate the variable recoveries of peptides from multiple surfaces commonly employed in peptide research, we tested the recovery of radiolabeled (125)I endocrine peptides under different conditions and provide guidelines for determining the surfaces to use for other peptides. (125)I-labeled peptides (ghrelin, sulfated cholecystokinin-8, corticotropin-releasing factor, glucagon-like peptide-1 [GLP-1], insulin, leptin, nesfatin-1, and peptide YY), representing a wide spectrum in net charge, size, end group, and modification, were incubated for 48 h in glass and plastic tubes untreated or coated with siliconizing fluid. Best surfaces were chosen and peptides were incubated with bovine serum albumin (BSA, 1%) with or without subsequent lyophilization. Recovery of (125)I-labeled peptides was determined by gamma counting. Important differences in (125)I-labeled peptide binding capacities to various types of surfaces exist. Siliconization decreased, whereas the addition of BSA improved recovery from surfaces tested. Lyophilizing solutions containing (125)I-labeled peptides and BSA in the tubes best suited for individual peptides rendered more than 89% recovery for all peptides. Ghrelin specifically displaced (125)I-ghrelin from borosilicate glass, whereas GLP-1 and Fmoc-arginine did not. Choosing the appropriate experimental container avoids unpredictable peptide loss that results in inaccurate measurements and false conclusions. Show more