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

Biochimie. 2021 Mar 17:S0300-9084(21)00075-4. doi: 10.1016/j.biochi.2021.03.005. Online ahead of print.ABSTRACTOne 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.PMID:33744341 | DOI:10.1016/j.biochi.2021.03.005

Source: Biochimie - March 21, 2021 Category: Biochemistry Authors: Aleksandr S Taraskin Konstantin K Semenov Aleksandr V Protasov Alexey A Lozhkov Alexandr A Tyulin Aram A Shaldzhyan Edward S Ramsay Olga A Mirgorodskaya Sergey A Klotchenko Yana A Zabrodskaya Source Type: research