The process of annotation of the resulting MS2 spectra is made quicker due to the development of UniCarb-DB, an LC/MS2 database of annotated N- and O-glycan structures [16]. The database provides mass spectrometric structural assignment of structures, which is based on LC/MS2 fragmentation. The database contains extensive information about glycan analysis including their HPLC details such

as column types, solvents, gradients, flow rates and MS details such Inhibitors,research,lifescience,medical as modification, mode of detection, data acquisition and the type of devices used during analysis. In addition, the database provides MS2 spectra and annotated MS2 peak list of the identified structures. This allows a parent mass to be searched for and the comparison of the MS2 spectra of these known spectra to be compared to experimental data, therefore, reducing the necessity of manual annotation of glycan data analysis. A previous study has shown a successful Inhibitors,research,lifescience,medical strategy of combined exoglycosidase digestion and MS2 spectral

matching of N-linked oligosaccharides [17]. In the present Inhibitors,research,lifescience,medical study, O-linked oligosaccharides from human synovial lubricin, mucin from porcine gastric stomach and salivary glycoproteins (MUC5B and MUC7) was spectral matched with spectra from UniCarb-DB. The lack of confirmative matches in the database triggered within the sample an exoglycosidase treatment, wherein the structure of the generated product could again

be subjected to spectral matching. The specificity of the exoglycosidase used allowed the identification of the oligosaccharide Inhibitors,research,lifescience,medical sequence of the substrate. It was also investigated how MSn could be used to identify non-reducing monosaccharide units, where Inhibitors,research,lifescience,medical the lack of specific exoglycosidases prevented them to be removed. 2. Results The schematic workflow in Figure 1 shows how the MS2 peak list (m/z and relative intensity) of the isolated chromatographic peaks were compared with the MS2 peak list of the structures reported in the MS2 glycomic database UniCarb-DB. The isothipendyl structures, in particular sialylated structures, which did not give a good match, were exoglycosidase digested (in particular de-sialylated). The MS2 peak list of the exoglycosidase products generated were again compared with the MS2 peak list of the structures reported in the MS2 database UniCarb-DB. For structures wherein a specific exoglycosidase was lacking, an MS3 approach was used. The MS3 peak lists of Dactolisib unknown structures were compared with the MS2 peak lists from the UniCarb-DB database (if fragments were Y-ions), or compared MS3 spectra of fragments generated from standards. Figure 1 Schematic workflow for structural assignment of O-glycans using MSn spectral match.