posted on 2020-03-20, 19:39authored byJohn P. Wilson, Jonathan J. Ipsaro, Samantha N. Del Giudice, Nikita Saha Turna, Carla M. Gauss, Katharine H. Dusenbury, Krisann Marquart, Keith D. Rivera, Darryl J. Pappin
Bottom-up proteomics is a mainstay
in protein identification and
analysis. These studies typically employ proteolytic treatment of
biological samples to generate suitably sized peptides for tandem
mass spectrometric (MS) analysis. In MS, fragmentation of peptides
is largely driven by charge localization. Consequently, peptides with
basic centers exclusively on their N-termini produce mainly b-ions.
Thus, it was long ago realized that proteases that yield such peptides
would be valuable proteomic tools for achieving simplified peptide
fragmentation patterns and peptide assignment. Work by several groups
has identified such proteases, however, structural analysis of these
suggested that enzymatic optimization was possible. We therefore endeavored
to find enzymes that could provide enhanced activity and versatility
while maintaining specificity. Using these previously described proteases
as informatic search templates, we discovered and then characterized
a thermophilic metalloprotease with N-terminal specificity for arginine
and lysine. This enzyme, dubbed Tryp-N, affords many advantages including
improved thermostability, solvent and detergent tolerance, and rapid
digestion time.