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To determine all potential N-glycosylation sites of a glycoprotein, one central aspect of every bottom-up N-glycoproteomic strategy is to generate suitable N-glycopeptides that can be detected and analyzed by mass spectrometry. Specific proteases, such as trypsin, bear the potential of generating N-glycopeptides that either carry more than one N-glycosylation site or are too long to be readily analyzed by mass spectrometry– both due to the lack of tryptic cleavage sites near the N-glycosylation site. Here, we present a newly identified cleavage specificity of flavastacin, a protease from  Flavobacterium menigosepticum , which - up to now - was only reported to cleave peptide bonds N-terminal to aspartic acid residues. In contrast to literature, we could not confirm this N-terminal specificity of flavastacin for aspartic acid. However, for the first time, we show a unique cleavage specificity of flavastacin towards the C-terminus of N-glycosylated asparagine residues. Implemented in an N-glycoproteomic workflow the use of flavastacin can thus not only render data analysis much easier, it can also significantly increase the confidence of MS-based N-glycoproteomic analyses. We demonstrate this newly discovered specificity of flavastacin by in-depth LC-MS(/MS) analysis of complex-type glycosylated human lactotransferrin and bovine serum albumin peptides and N-glycopeptides that were generated by trypsin and flavastacin digestion. Following to this work, further elucidation of the efficiency, specificity and mode of action of flavastacin is needed, but we believe that our discovery has great potential to facilitate and improve the characterization of N-glycoproteomes.

Improvement of the glycoproteomic toolbox with the discovery of a unique C-terminal cleavage specificity of flavastacin for N-glycosylated asparagine