Complementary peptide sequence coverage using alternative enzymes for on-line digestion with a triaxial electrospray probe

Abstract

Using alternative enzymes for on-line digestion with a triaxial electrospray probe extends sequence coverage. This is the first report of utilization of our triaxial probe for on-line analysis with enzymes other than pepsin, suggesting potential for broader application. The probe allows access to processes occurring on a timescale and/or involving substrate conformations complementary to those for conventional (off-line) digestion. Some of the features observed in application to A␤ fibrils are suggestive of unique reactive intermediates during dissolution. Data obtained with enzyme mixtures suggest synergistic effects. (J Am Soc Mass Spectrom 2009, 20, 1983-1987) © 2009 American Society for Mass Spectrometry H ydrolysis followed by tandem electrospray (ES) mass spectrometry is widely used for "bottom up" sequencing and localization of hydrogen/ deuterium exchange (HDX). For HDX studies, pepsin is a preferred enzyme because it tolerates the low pH that minimizes artifactual exchange during sample processing. There are cases where sequence coverage using pepsin is suboptimal, especially in the short hydrolysis times used to reduce HDX artifacts. This challenge was amplified in HDX studies of A␤ (1-40) fibrils [1,2] because of the need to accomplish both hydrolysis and rapid fibril dissolution with minimal scrambling and artifactual exchange. A triaxial probe [3] was designed and proven to accomplish this by on-line mixing of a fibril suspension with a solution that quenched HDX and initiated dissolution and hydrolysis, followed ϳ12 s later by addition of acetonitrile to provide a strong and stable ES signal. Stocks and Konermann [4] employed a similar mixing apparatus to probe denaturation via oxidative labeling, but coupled it with conventional LC/MS rather than attempting on-line hydrolysis. Our probe was designed for the specialized application requiring on-line hydrolysis; it was therefore of interest to determine whether it might be usable with other enzymes compatible with low pH constraints. We report here the efficacy of using alternative pepsin-like enzymes and their mixtures for improving sequence coverage with on-line hydrolysis.

Characterizing the Range of Extracellular Protein Post-Translational Modifications in a Cellulose-Degrading Bacteria Using a Multiple Proteolyic Digestion/Peptide Fragmentation Approach

“…To probe the PTM signature of a particular organism, a multifaceted approach must be utilized in order to increase the number of protein identifications, the number of modified peptide identifications, and confidence in these results. The value of using multiple proteases in proteomic experiments has been demonstrated in multiple studies. − In addition to multiple proteases, the introduction of alternate peptide fragmentation methods has also given rise to another dimension in PTM identification. For example, electron-transfer dissociation (ETD) , induces peptide fragmentation that is distinct compared to collisionally activated dissociation (CAD) .…”