Quench me if you can: Alpha-2-macroglobulin trypsin complexes enable serum biomarker analysis by MALDI mass spectrometry

Abstract

One of the main functions of alpha-2-macroglobulin (A2M) in human blood serum is the binding of all classes of protease. It is known that trypsin, after such interaction, possesses modified proteolytic activity. Trypsin first hydrolyzes two bonds in A2M’s ‘bait region’, and the peptide 705VGFYESDVMGR715 is released from A2M. In this work, specifics of the A2M-trypsin interaction were used to determine A2M concentration directly in human blood serum using MALDI mass-spectrometry. Following exogenous addition of trypsin to human blood serum in vitro, the concentration of the VGFYESDVMGR peptide was measured, using its isotopically-labeled analogue (18O), and A2M concentration was calculated. The optimized mass spectrometric approach was verified using a standard method for A2M concentration determination (ELISA) and the relevant statistical analysis methods. It was also shown that trypsin’s modified proteolytic activity in the presence of serum A2M can be used to analyze other serum proteins, including potential biomarkers of pathological processes. Thus, this work describes a promising approach to serum biomarker analysis that can be technically extended in several useful directions.

A single protein to multiple peptides: Investigation of protein-peptide relationship using targeted alpha-2-macroglobulin analysis

“…A2MG a clinically important protein associated with liver 54 , diabetes 55 , neurological diseases [56][57][58] , and prostate cancer 55 , was selected as a reference protein. Recent structural investigations of the A2MG protein in the literature have mostly focused on the protein's conformational changes and complex formation [59][60][61] , binding interaction with drugs/small substrates [62][63][64][65][66][67][68][69] , and PTMs 70 . The position of the unique peptide in the protein structure and commonly observed mis-cleavages were examined in this study 71,72 The dynamic protein-unique peptide interaction is yet unknown, despite the widespread belief that all unique peptides that reflect the protein of interest behave in the same manner.…”