Chemical-Mediated Digestion: An Alternative Realm for Middle-down Proteomics?

Protein digestion in mass spectrometry
(MS)-based bottom-up proteomics
targets mainly lysine and arginine residues, yielding primarily 0.6–3
kDa peptides for the proteomes of organisms of all major kingdoms.
Recent advances in MS technology enable analysis of complex mixtures
of increasingly longer (>3 kDa) peptides in a high-throughput manner
supporting the development of a middle-down proteomics (MDP) approach.
Generating longer peptides is a paramount step in launching an MDP
pipeline, but the quest for the selection of a cleaving agent that
would provide the desired 3–15 kDa peptides remains open. Recent
bioinformatics studies have shown that cleavage at the rarely occurring
amino acid residues such as methionine (Met), tryptophan (Trp), or
cysteine (Cys) would be suitable for MDP approach. Interestingly,
chemical-mediated proteolytic cleavages uniquely allow targeting these
rare amino acids, for which no specific proteolytic enzymes are known.
Herein, as potential candidates for MDP-grade proteolysis, we have
investigated the performance of chemical agents previously reported
to target primarily Met, Trp, and Cys residues: CNBr, BNPS-Skatole
(3-bromo-3-methyl-2-(2-nitrophenyl)­sulfanylindole), and NTCB
(2-nitro-5-thiobenzoic acid), respectively. Figures of merit such
as digestion reproducibility, peptide size distribution, and occurrence
of side reactions are discussed. The NTCB-based MDP workflow has demonstrated
particularly attractive performance, and NTCB is put forward here
as a potential cleaving agent for further MDP development.