The majority of large-scale glycoproteomic studies features trypsin for the generation of (glyco) peptides
Trypsin is the proteolytic gold standard in LC-MS/MS based peptide identification and quantification, as it reproducibly generates predictable peptides that can be readily retained on reversed-phase column and that give enough fragment ions for an unambiguous peptide identification, in most cases
In terms of glycoproteomics, though, the cleavage specificity of trypsin can be a limiting factor for the identification and the localization of certain glycosylation sites , in particular for densely clustered O-glycosylation sites
Hence, the use of broad- and nonspecific proteases, like Pronase E or Proteinase K was proposed, to reduce the number of nonglycosylated peptides and to make certain glycosylation sites analytically amenable (34)
Proteinase K , for instance, has been successfully used in a number of publications that are centered on the O-glycoproteomic analysis of single proteins ; though, the use of Proteinase K in large-scale glycoproteomic studies on complex samples has not been described so far
In the present study we could show that Proteinase K generates (glyco) peptides from a complex sample, like human blood plasma, in a reproducible and nonrandom manner, which is in agreement with a report from Hua et al. (34)
We could show that, most of the time, Proteinase K generates shorter peptides compared with trypsin (supplemental Fig
S7), and that Proteinase K cleaves effectively in-between densely O-glycosylated regions—thus, rendering the determination of the occupied O-glycosylation site (s) less difficult
In fact we could show that Proteinase K can generate O-glycopeptides that exhibit only one potential O-glycosylation site , thus allowing for an unambiguous localization of the occupied site
We could clearly show that some O-glycosylation sites could only be identified and pinpointed by the use of Proteinase K , because tryptic peptides would have been too long and would have harbored too many potential O-glycosylation sites