Chemical-Mediated Digestion: An Alternative Realm for Middle-down Proteomics?
Authors: Mikhail V Gorshkov
Yury O Tsybin
Anna A Lobas
Konstantin O Zhurov
Gennady Nikitin
Luca Fornelli
Kristina Srzentić
Journal: Journal of Proteome Research (2018)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: FTMS; Fourier transform mass spectrometry; MD; MS; Orbitrap
Affiliations: Ecole Polytechnique Fédérale de Lausanne, Lausanne 1015, Switzerland, V. L. Talrose Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Leninsky Prospect 38, Moscow 119334, Russia, Moscow Institute of Physics and Technology (State University), 9 Institutskiy per., Dolgoprudny, Moscow 141707, Russia, Spectroswiss, EPFL Innovation Park, Lausanne 1015, Switzerland

Abstract:  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 (&gt;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. 

Retracted: No

PubPeer comments:
 Hello, Great paper with exciting implications for future developments in middle-down mass spectrometry. I noticed one issue however that I am hoping the authors can address here and issue a correction for. It looks like Figure 2 was duplicated and also placed into Figure 3 . Could the authors provide the actual Figure 3 here and correct the error in the article? Many thanks! , 
 #1 Hello and thank you! apparently the journal made a mistake during the production. The Proofs were all correct, but at the publication stage Figure 2 was published twice. Too bad. Thanks a lot for pointing to this error! We wrote to the journal and ACS proofs. Please write directly to the corresponding author of the paper if you have any questions. This website seems to be a strange place and is not working properly (I cannot even register with my e-mail as it is not accepted). cheers! ,