Sequential Digestion with Trypsin and Elastase in Cross-Linking Mass Spectrometry

Abstract

Cross-linking mass spectrometry has become an important approach for studying protein structures and protein–protein interactions. The amino acid compositions of some protein regions impede the detection of cross-linked residues, although it would yield invaluable information for protein modeling. Here, we report on a sequential-digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin-cleavage sites. We exploited intrinsic substrate-recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4–12 complex allowed us to identify cross-links in previously inaccessible regions.

Proteomics Using Protease Alternatives to Trypsin Benefits from Sequential Digestion with Trypsin

“…In this way trypsin leads to a concentration increase of peptides by reducing sample complexity. At least when trypsin is used first an additional mechanism must be considered that was previously described for sequential digestion 12,14 . The second enzyme does not cleave shorter peptides with high efficiency effectively leading to short tryptic peptides being protected from proteinase K. In either case, the complexity that is normally introduced through proteinase K is reduced by the tryptic treatment.…”

“…The data on trypsin, elastase, trypsin-elastase and elastasetrypsin were taken from our previous work 14 and retrieved from PRIDE with the data set identifier PXD011459.…”

“…Proteinase K for example, was used, to "shave" surface exposed loops from proteins in membrane vesicles 10,11 Our group has previously shown that the number of identified peptides, when using alternatives to trypsin, could largely be improved by a sequential combination with trypsin. This includes AspN, GluC, chymotrypsin and elastase for the detection of crosslink sites [12][13][14] and elastase applied to S. pombe whole cell lysates 14 . The sequential digestion increased the number of identified crosslinks up to 19-fold for the Taf4-12 complex compared to digesting with elastase alone 14 .…”

“…This includes AspN, GluC, chymotrypsin and elastase for the detection of crosslink sites [12][13][14] and elastase applied to S. pombe whole cell lysates 14 . The sequential digestion increased the number of identified crosslinks up to 19-fold for the Taf4-12 complex compared to digesting with elastase alone 14 . Introducing positively charged C-termini through trypsin improves the detection of previously non-tryptic peptides.…”

“…Introducing positively charged C-termini through trypsin improves the detection of previously non-tryptic peptides. Importantly, smaller peptides are protected from the second protease 12,14 . Thus, use of two proteases does not lead to the very small peptides that in silico digestion would predict.…”