PMC:7108637 / 20337-23549 JSONTXT

{"project":"2_test","denotations":[{"id":"29579213-25284204-45167322","span":{"begin":480,"end":484},"obj":"25284204"},{"id":"29579213-22944675-45167323","span":{"begin":1460,"end":1464},"obj":"22944675"},{"id":"29579213-26202417-45167324","span":{"begin":1476,"end":1480},"obj":"26202417"},{"id":"29579213-22256963-45167325","span":{"begin":1688,"end":1692},"obj":"22256963"},{"id":"29579213-28348411-45167326","span":{"begin":1705,"end":1709},"obj":"28348411"},{"id":"29579213-27984693-45167327","span":{"begin":1900,"end":1904},"obj":"27984693"},{"id":"29579213-25284204-45167328","span":{"begin":2094,"end":2098},"obj":"25284204"},{"id":"29579213-19095697-45167329","span":{"begin":2350,"end":2354},"obj":"19095697"},{"id":"29579213-22944673-45167330","span":{"begin":2370,"end":2374},"obj":"22944673"},{"id":"29579213-23242014-45167331","span":{"begin":2495,"end":2499},"obj":"23242014"},{"id":"29579213-25915761-45167332","span":{"begin":2517,"end":2521},"obj":"25915761"},{"id":"29579213-23722953-45167333","span":{"begin":2851,"end":2855},"obj":"23722953"},{"id":"29579213-14760718-45167334","span":{"begin":3073,"end":3077},"obj":"14760718"},{"id":"29579213-20510933-45167335","span":{"begin":3096,"end":3100},"obj":"20510933"},{"id":"29579213-25153361-45167336","span":{"begin":3116,"end":3120},"obj":"25153361"},{"id":"29579213-25284204-45167337","span":{"begin":3136,"end":3140},"obj":"25284204"},{"id":"T68230","span":{"begin":480,"end":484},"obj":"25284204"},{"id":"T66672","span":{"begin":1460,"end":1464},"obj":"22944675"},{"id":"T30510","span":{"begin":1476,"end":1480},"obj":"26202417"},{"id":"T85525","span":{"begin":1688,"end":1692},"obj":"22256963"},{"id":"T87920","span":{"begin":1705,"end":1709},"obj":"28348411"},{"id":"T94890","span":{"begin":1900,"end":1904},"obj":"27984693"},{"id":"T60505","span":{"begin":2094,"end":2098},"obj":"25284204"},{"id":"T59671","span":{"begin":2350,"end":2354},"obj":"19095697"},{"id":"T27324","span":{"begin":2370,"end":2374},"obj":"22944673"},{"id":"T54114","span":{"begin":2495,"end":2499},"obj":"23242014"},{"id":"T41554","span":{"begin":2517,"end":2521},"obj":"25915761"},{"id":"T29714","span":{"begin":2851,"end":2855},"obj":"23722953"},{"id":"T84008","span":{"begin":3073,"end":3077},"obj":"14760718"},{"id":"T90102","span":{"begin":3096,"end":3100},"obj":"20510933"},{"id":"T734","span":{"begin":3116,"end":3120},"obj":"25153361"},{"id":"T17533","span":{"begin":3136,"end":3140},"obj":"25284204"}],"text":"While glycoprofiling studies provide important information on distribution and composition of different glycan structures present on a given protein, it lacks the information on individual glycosite location, occupancy and glycan structure heterogeneity. The recent development of instruments equipped with ECD or ETD MS2 fragmentation has allowed for simultaneous determination of the peptide sequence and the position to which the carbohydrate moiety is attached (Levery et al. 2015). When relevant reporter glycan oxonium ions are present and it is easy to predict the glycosylation position within peptides, HCD MS2 fragmentation can also be used (Wuhrer et al. 2007). Interfacing the mass spectrometer with capillary liquid chromatography enables separation of complex proteome-scale glycopeptide mixtures and allows identification of thousands of glycopeptides in a single run. At the single protein level, it is now possible to determine the exact position, structure heterogeneity, and site occupancy for each individual glycosite (Wuhrer et al. 2007). Both N- and O-glycosylation can be analyzed by tandem MS. Biosynthetic features of N-glycosylation enable relatively easy identification and quantification of deglycosylated sites by MS2 sequencing. Due to asparagine deamidation during enzymatic N-glycan removal, the site occupancy can be determined by calculating the intensity ratio of naked (Asn) and deglycosylated (Asp) peptides (Pabst et al. 2012; An et al. 2015). However, the more appropriate methodology includes carrying out the reaction using heavy-oxygen water (O18–H2O), which discriminates deglycosylated sites from spontaneous Asn deamidation (Palmisano et al. 2012; Cao et al. 2017). It is also possible to evaluate the overall distribution of N-glycosite occupancy on intact glycoproteins by metabolically simplifying the glycans to homogeneous structures (Struwe et al. 2017). For O-linked glycans, complete O-glycan removal does not result in chemical peptide modification and cannot be used for identification of glycosylated amino acid positions (Levery et al. 2015). ETD and ECD MS2 techniques allow analysis of intact O-glycopeptides by fragmentation of the peptide backbone without the loss of O-glycan modification, however, it is of limited use for determining site occupancy (Wuhrer et al. 2007; Sihlbom et al. 2009; Zauner et al. 2012). Approximations are yet often made by comparing intensities of nonmodified and glycosylated peptides (Brautigam et al. 2013; Stansell et al. 2015). The relative quantitation of both N- and O-glycosylated peptides is not as accurate due to different ionization efficiencies of nonglycosylated peptides and peptides carrying complex glycans. Dependent on the complexity of the glycans, glycopeptides generally exhibit poorer ionization compared to peptides (Stavenhagen et al. 2013). The analysis of complex proteins therefore often requires enrichment with hydrophilic interaction chromatography or specific lectins, which is particularly relevant for proteome-wide applications (Bunkenborg et al. 2004; Zielinska et al. 2010; Khatri et al. 2014; Levery et al. 2015). However, information regarding site occupancy needs to be sacrificed."}