Figure 4.

Two distinct tandem mass spectra of peptide sequence “VSTTTGQNNNSNFAWTAGTK” of the AAV8 capsid protein (in the common region of VPs). Characterized CID spectra of glycosylated peptide after ¹⁸O mediated digestion. (a) The position of de-N-glycosylated asparagine 499 (¹⁸O-incorporated aspartic acid) was attested by mass increment of 2.8547 Da (theoretical mass difference 2.98 Da) to the series of b and y ion (the precursor ion mass: m/z 2101.93193). (b) The position of Non-glycosylated asparagine 499 in the sequence was confirmed by y12/b8 ions (the precursor ion mass: m/z 2099.0766 Da). The highlighted ions (marked with a star) shows the mass differences from the non-glycosylated peptide mass spectrum. y11+ (highlighted) ions are same in both the spectra. (c) Capsid protein amino acid sequence and possible N-glycosylation sites predicted by NetNglyc software based on the consensus sequence (NXT/S, “X” can be any amino acid except proline). Different N-terminal sequence of VP1, VP2 and VP3 are marked in the sequence. The glycosite identified peptide is highlighted on the sequence.