Title : Defining glycan residues for Mascot database search
Abstract :
A series of Y type glycosidic fragment ions are required for confident characterization of glycan structures and both collision-induced dissociation (CID) and higher-energy collisional induced dissociation ( HCD ) fragmentation techniques provide such pattern at lower normalized collision energy (NCE) values
A typical example of an HCD (NCE = 15) MS2 spectrum of a glycopeptide derived from bovine alpha-1-acid glycoprotein is presented in Fig. 1A
Starting with the Y1 ion by following the mass differences between the most intense peaks and the mass difference between the Y10 ion and the precursor, the glycan sequence can be easily determined
The complete glycan structure contains four HexNAc , five Hex and two Neu5Acresidues , which represent a di-sialylated biantennary N-glycan (Fig. 1A)
Assuming the glycan residues similar to amino acids, deducing the glycan structure from glycopeptide MS2 spectrum is similar to peptide sequencing
In order to use the Mascot search engine for automated glycopeptide analysis, the basic requirements include that (i) the sugar residues must be defined with unique one-letter codes in a Mascot readable format, (ii) each glycan structure must be defined in a linear format and (iii) a customized database must be prepared which consists of combined protein and glycan sequences
Mascot uses the Latin alphabet as one-letter codes and 20 of them are assigned to the standard amino acid residues , and B, X and Z are hard-coded
Of the remaining three letters (O, J and U), O was assigned to N-acetylhexosamine (GlcNAc, GalNAc), J to hexoses (Galactose, mannose) and U to sialic acid (Table 1)
Fucose was defined as a variable modification on N-acetylhexoseamines (O)
For Mascot, the three letters O, J and U must be defined in the unimod.xml file (Supplementary Fig. 1)