Dual Proteolytic Enzyme Digestion with Trypsin and Chymotrypsin

Dual proteolytic digestion is an approach that involves the sequential use of two enzymes. Chymotrypsin and trypsin can be used to generate SUMO-isopeptides with shorter SUMO iso-chains in comparison to the full-length isochains generated from typical tryptic digestion [46]. First, trypsin is used to digest a poly-SUMO(2)ylated protein resulting in SUMO(2)-isopeptides with a SUMO(2) iso-chain containing 32 amino acids. Second, chymotrypsin is used to cleave C-terminal to the phenylalanine residue at position 87 resulting in the SUMO(2) iso-chains containing 6 amino acids, QQQTGG. Chymotrypsin has a C-terminal cleavage preference for a number of amino acid residues including phenylalanine, tyrosine, tryptophan, and leucine. QTOF ion mobility- based mass spectrometry with low-energy CID analysis was used to analyze SUMO(2)-isopeptides from dual digestion of poly-SUMO(2)ylated protein (data not shown). The use of ion mobility facilitated the successful separation of the predominantly higher charge state SUMO(2)-isopeptide ions under a narrow drift-time distribution from the predominantly lower charge state linear peptide ions generated under nESI conditions. Subsequent CID analysis of the SUMO(2)-isopeptide ions typically facilitated CID MS/MS spectra, which contained predominantly y-type ions from the backbone of the SUMO(2)-isopeptides as expected with additional b'-type-related product ions generated from the neutral loss of NH3 from the SUMO(2) iso-chain, which could be manually interpreted by matching theoretical ions generated in silico. This approach to the analysis of SUMO(2)-isopeptides was applied to a simple digestion mixture; it would benefit from demonstrating applicability in a more complex digestion mixture such as one enriched for more SUMO-isopeptides, where its robustness could be assessed. The QQQTGG iso-chains generated from this dual-enzyme digestion strategy may benefit from utility in LC-MS/MS CRA(K)-based approaches [39]. However, the length of this iso-chain is susceptible to multiple events of glutamine-related deamidation, which would thereby add additional complexity to bioinformatic analyses, in comparison to the QTGG and TGG iso-chains, which offer fewer permutations of deamidation and, therefore, a decrease in complexity.

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