Abstract
In microdroplets, rates of chemical or biomolecular reactions can exceed those in the bulk phase by more than a million times. As electrospray ionization-based mass spectrometry (MS) involves the formation of charged microdroplets, reaction acceleration and online MS monitoring of reaction products can readily be performed at the same time. We investigated accelerated enzymatic reactions in microdroplets and focused on the proteolytic enzyme pepsin. Electrosonic spray ionization (ESSI) was utilized for developing the ultrarapid pepsin in-spray digestion of two different proteins, cytochrome c and RocC, at low pH values. The optimization of the protein digestion aimed at achieving maximum sequence coverage for the analyzed proteins. Furthermore, carefully designed control experiments allowed us to unambiguously prove that enzymatic protein cleavage almost exclusively occurs within the spray at a millisecond time scale and not prior to microdroplet generation.
Highlights
Rapid developments in the field of electrospray-based ambient MS1 have paved the way to a variety of novel applications of mass spectrometry.[2]
Journal of the American Society for Mass Spectrometry pubs.acs.org/jasms digestion in the context of protein hydrogen−deuterium exchange experiments (HDX), we developed a method for ultrarapid pepsin digestion of proteins in charged microdroplets at low pH levels
The obtained mass spectra matched those from the intact protein; that is, no spontaneous cleavage was induced by Electrosonic spray ionization (ESSI) at low pH values (Figures S1 and S2)
Summary
Rapid developments in the field of electrospray-based ambient MS1 have paved the way to a variety of novel applications of mass spectrometry.[2] In particular, the phenomenon of reaction acceleration in airborne charged microdroplets attracts researchers from various backgrounds.[3−26] Compared to chemical reactions in the bulk phase, rates of reactions in microdroplets were found to be increased by several orders of magnitude, often showing exceptionally high yields. (optimum value for pepsin digestion), the following input parameters were varied: total flow rate of the reaction mixture (1 to 5 μL·min−1), the distance between the sprayer and the MS inlet (3 to 7 cm), the concentrations of protein, protease, and buffer additives, and the nitrogen back pressure at the ESSI sprayer (60 to 120 psi).
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