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 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 alpha-amylase and its microbial inhibitor tendamistat using ESI-MS. Crude porcine pancreatic alpha-amylase was purified using a glycogen precipitation method. Noncovalent complexes between porcine pancreatic alpha-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 alpha-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.
Douglas
D J
DJ
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, B.C., V6T 1Z1, Canada.
Collings
B A
BA
Numao
S
S
Nesatyy
V J
VJ
D000818:Animals; D002853:Chromatography, Liquid; D004791:Enzyme Inhibitors / Q000032:analysis*; D008961:Models, Structural; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis*; D011485:Protein Binding; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013552:Swine; D000516:alpha-Amylases / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000302:isolation & purification
D004791:Enzyme Inhibitors; D010455:Peptides; D000516:alpha-Amylases; C030526:tendamistate