Online electrospray ionization mass spectrometric monitoring of protease-catalyzed reactions in real time.
Although there are a lot of well established methods for monitoring enzyme-catalyzed reactions, most of them are based on changes in spectroscopic properties during the conversion of substrates to products. However, reactions without optical changes are common, which are inapplicable to these spectroscopic methods. As an alternative technique for enzymologic research, mass spectrometry (MS) is favored due to its specificity, sensitivity, and the ability to obtain stoichiometric information. In this work, probe electrospray ionization (PESI) source coupled with a time of flight mass spectrometer was employed to monitor some typical protease-catalyzed reactions, including pepsinolysis and trypsinolysis of cytochrome c in real time. Due to the high electrical conductivity of each reaction system, corona discharges are likely to occur, which would decrease intensities of mass spectrometric signals. An ultra-fine sampling probe and an auxiliary vapor spray were adopted to prevent corona discharges. Experimental results from peptic and tryptic digestions of cytochrome c showed different and characteristic catalytic pathways. With the data presented in this study, PESI-MS can be considered as a potential tool for real-time monitoring of enzymatic reactions because of its simplicity in instrumental configuration, wide applicability under harsh conditions, and flexibility in combination with other techniques.
Clean Energy Research Center, University of Yamanashi, 4-3-11 Takeda, Kofu Yamanashi, 400-8511, Japan.
Zhan
Z
Yu
Lee Chuin
LC
Chen
Mridul Kanti
MK
Mandal
Hiroshi
H
Nonami
Rosa
R
Erra-Balsells
Kenzo
K
Hiraoka
D000818:Animals; D019149:Bioreactors; D002417:Cattle; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D010434:Pepsin A / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D059748:Proteolysis; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation* / Q000379:methods*; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
D010446:Peptide Fragments; D045304:Cytochromes c; D014357:Trypsin; D010434:Pepsin A