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

Electrospray ionization mass spectrometry (ESI-MS) is now routinely used fo r detection of noncovalent complexes. However, detection of noncovalent pro tein-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 alpha -amylase and its microb ial inhibitor tendamistat using ESI-MS. Crude porcine pancreatic alpha -amy lase was purified using a glycogen precipitation method. Noncovalent comple xes between porcine pancreatic alpha -amylase and its microbial inhibitor t endamistat are probed and detected using ESI-MS. The atmosphere-vacuum ESI conditions along with solution conditions and the ratio of inhibitor over e nzyme strongly affect the detection of noncovalent complexes in the gas pha se. ESI mass spectra of alpha -amylase at pH 7 exhibited charge states sign ificantly lower than that reported previously, which is indicative of a nat ive protein conformation necessary to produce a noncovalent complex. Detect ion of noncovalent complexes in the gas phase suggests that further use of conventional biochemical approaches to provide a qualitative, and in some c ases even quantitative, characterization of equilibria of noncovalent compl exes in solution is possible. Copyright (C) 2001 John Wiley & Sons, Ltd.