An investigation into fragmentation of hEGF in triple quadrupole mass spectrometry and its quantitative application to human plasma

Abstract

A growing number of peptides are being used today as biomarkers and therapeutic drugs. Liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS), in providing a highly specific and reliable technique, is likely to have a substantial impact on peptide quantification. However, most of the applications developed in research laboratories have relied on capillary liquid chromatography, nanospray interfaces, and ion trap or other types of mass spectrometers, which are not commonly found in preclinical and clinical laboratories. Triple quadrupole mass spectrometers are not often considered for quantitative analysis of peptides, despite the fact that they are currently an increasingly common tool in preclinical and clinical laboratories. However, sensitive peptide analyses have been reported, as in the demonstration of structurally distinctive immonium ions that were formed in abundance. Thus, we have recently proposed that use of immonium ions might provide a potential pathway for the quantitative analysis of oligo-peptides with intra-chain disulfide bonds. In the present report, the capability of these ions in LC/MS/MS toward peptide determination was further explored using a polypeptide, human epidermal growth factor (hEGF). hEGF is much larger and more complex than the oligo-peptides previously investigated in our lab. Both the experiments and theoretical calculations were carried out. The results indicate that polypeptides with intra-chain disulfide bonds can increase fragmentation efficiency and raise the number of immonium product ions produced, on the condition that at least one of their intra-loops is sensitive to abrupt bond cleavage. In addition, the utilization of immonium product ions in LC/MS/MS was demonstrated for the determination of hEGF in human plasma. Good linearity and accuracy were achieved.