Use of multiple enzymes in sequential in-gel digestions improves sequence coverage of botulinum neurotoxins for confident subtyping by mass spectrometry

s Toxins 2011 / Toxicon 68 (2013) 60–123 102 Use of multiple enzymes in sequential in-gel digestions improves sequence coverage of botulinum neurotoxins for confident subtyping by mass spectrometry D. Wang, J. Baudys, J. Rees, S.R. Kalb, J.R. Barr Division of Laboratory Science, National Center for Environmental Science, Centers for Disease Control and Prevention, Atlanta, GA, USA E-mail address: dov2@cdc.gov (D. Wang). Purpose of study: To develop a method for subtyping of botulinum neurotoxin (BoNT) by mass spectrometry with high sequence coverage. Methods used: Samples were separated by gel electrophoresis. The bands of interest were sliced, reduced, and alkylated followed by in-gel digestion with individual enzymes of various specificities. Sequential digestions were performed by re-digesting each of the gel bands with a select number of enzymes. The peptide mixtures were analyzed by LC-MS/MS on a FT-LTQ instrument. Summary of results: A SDS-PAGE purified protein, BoNT/A1, was initially examined with six commonly used enzymes. Four of these enzymes, including trypsin, chymotrypsin, Asp-N, and Lys-C resulted in substantial sequence coverage individually and yielded unique peptides that contributed to extended sequence coverage after a combination of the results. The data demonstrate that sequential digestion of up to four times on the same gel piece with different enzymes led to increased sequence coverage. A systematic study assessed the effect of different combinations of enzymes, sequential order and reaction conditions. As a result, 1285 of 1295 total amino acid residues of the BoNT/A1 protein were covered by a pool of detected peptides under one specific combination using a few micrograms of the protein. This nearcomplete sequence coverage should enable highly confident determination of known or unknown subtypes of BoNT. The strategy was applied to all serotypes of botulinum neurotoxins and significant improvements were observed in comparison with conventional single digestion methods. In addition, the primary sequence of a botulinum neurotoxin present in a carrot juice sample was almost completely determined (99% sequence coverage) and thus allowed the identification of its subtype and specific strain. Conclusions: Our study demonstrates in-gel digestion with the combination of different enzymes and various sequential orders improves the subtyping of botulinum neurotoxins by mass spectrometry. http://dx.doi.org/10.1016/j.toxicon.2012.07.118 Effects of botulinum-neurotoxin-complexing proteins on lymphatic cells L. Wang , Y. Sun , B.R. Singh a,b a BBTech Inc. Dartmouth, MA, USA Botulinum Research Center, University of Massachusetts Dartmouth,