Detection of noncovalent complex between α‐amylase and its microbial inhibitor tendamistat by electrospray ionization mass spectrometry

Abstract

Electrospray ionization mass spectrometry (ESI-MS) is now routinely used for detection of noncovalent complexes. However, detection of noncovalent protein-protein complexes is not a widespread practice and still produces some challenges for mass spectrometrists. Here we demonstrate the detection of a noncovalent protein-protein complex between α-amylase and its microbial inhibitor tendamistat using ESI-MS. Crude porcine pancreatic α-amylase was purified using a glycogen precipitation method. Noncovalent complexes between porcine pancreatic α-amylase and its microbial inhibitor tendamistat are probed and detected using ESI-MS. The atmosphere-vacuum ESI conditions along with solution conditions and the ratio of inhibitor over enzyme strongly affect the detection of noncovalent complexes in the gas phase. ESI mass spectra of α-amylase at pH 7 exhibited charge states significantly lower than that reported previously, which is indicative of a native protein conformation necessary to produce a noncovalent complex. Detection of noncovalent complexes in the gas phase suggests that further use of conventional biochemical approaches to provide a qualitative, and in some cases even quantitative, characterization of equilibria of noncovalent complexes in solution is possible. Copyright © 2001 John Wiley & Sons, Ltd.