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The glycoprotein-focused bottom-up glycoproteomics to top-down glycopeptidomic approach using “synthetic glycopeptides”. Glycopeptidic biomarkers pre-determined from purified human clusterin by the Iliopoulos group²⁴ were synthesized and employed for calibration standards in the SRM-based absolute quantitation of the targeted glycopeptides generated by the tryptic digestion of whole serum glycoproteins, namely the top-down glycopeptidomic approach. A specific fragment ion generated from the target analyte (selected glycopeptide, Q1) under the optimized collision energy (Q2) is selected in Q3 and guided to the detector. The number of target fragment ions such as mono- and oligosaccharide oxonium ions is counted over time, resulting in an SRM trace for each target glycopeptide. Notably, the SRM assay facilitates the accurate quantitation of the targeted glycopeptide without the influence of the large excess of other tryptic digests by using the designated SRM channel (Q1, Q2, and Q3, as well as LC retention time) optimized for each targeted glycopeptide. LC-ESI, liquid chromatography-electrospray ionization; XICs, extracted ion chromatograms
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Clusterin is a heavily glycosylated protein that is upregulated in various cancer and neurological diseases. The findings by the Hancock and Iliopoulos group that levels of the tryptic glycopeptide derived from plasma clusterin, 372Leu-Ala-Asn-Leu-Thr-Gln-Gly-Glu-Asp-Gln-Tyr-Tyr-Leu-Arg385 with a biantennary disialyl N-glycan (A2G2S2 or FA2G2S2) at...
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Citations
... CLU is a heterodimeric, evolutionarily conserved glycoprotein with an apparent molecular mass of approximately 80 kDa, of which approximately 30% comprises covalently bound glycans (Shiratori et al., 2022). The major isoform of CLU is secreted and described as an extracellular molecular chaperone. ...
... The major isoform of CLU is secreted and described as an extracellular molecular chaperone. CLU plays a role in BPs such as programmed cell death, complement regulation, and barrier cell protection in humans through signaling pathways, such as ERK, AKT, and NF-κB (Shiratori et al., 2022). In the present study, 22 and 9 intact N-glycopeptides were identified in CLU from BC and BM, respectively (Table S6). ...
N ‐glycosylation has important implications for the physicochemical properties and biochemical activity of breast milk proteins. However, an in‐depth characterization of site‐specific N ‐glycans in breast milk serum proteins is lacking. In this study, 875 site‐specific N ‐glycans attached to 114 serum glycoproteins from breast colostrum (BC) and 427 site‐specific N ‐glycans attached to 60 serum glycoproteins from breast mature milk (BM) were identified and quantified using label‐free site‐specific glycoproteomics. Among them, 24 site‐specific N ‐glycans mapping to 11 N ‐glycosites on 9 glycoproteins were significantly increased, and 76 site‐specific N ‐glycans mapping to 19 N ‐glycosites on 13 glycoproteins were significantly decreased. Gene ontology annotation analysis showed that BC and BM serum N ‐glycoproteins were mainly enriched in complement activation, classical pathway, extracellular exosome part, and immunoglobulin receptor binding. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that BC and BM serum N ‐glycoproteins were mainly involved in the PI3K‐Akt signaling pathway. Additionally, breast milk serum N ‐glycoproteins carried fucosylated core structures as well as Lewis and sialylated branch structures that may contribute to neonatal retinal homeostasis. These results provide a molecular basis for understanding the unique biological activities of breast milk serum N ‐glycoproteins.
... The use of pure standards is important to confirm the glycan structure and validation. In fact, it was demonstrated that although fragmentation patterns may be very similar between isomeric glycans, the detailed study of product ions' relative abundance can help shed light on the glycosidic bond isomerism and determine which is the correct structure of a biomarker initially detected by shotgun proteomics [45]. Such resolution of isomeric glycoconjugates is not always possible by MS/MS analysis alone, especially when the number of coexisting isomeric glycans is large. ...
This trends article provides an overview of the state of the art in the analysis of intact glycopeptides by proteomics technologies based on LC–MS analysis. A brief description of the main techniques used at the different steps of the analytical workflow is provided, giving special attention to the most recent developments. The topics discussed include the need for dedicated sample preparation for intact glycopeptide purification from complex biological matrices. This section covers the common approaches with a special description of new materials and innovative reversible chemical derivatization strategies, specifically devised for intact glycopeptide analysis or dual enrichment of glycosylation and other post-translational modifications. The approaches are described for the characterization of intact glycopeptide structures by LC–MS and data analysis by bioinformatics for spectra annotation. The last section covers the open challenges in the field of intact glycopeptide analysis. These challenges include the need of a detailed description of the glycopeptide isomerism, the issues with quantitative analysis, and the lack of analytical methods for the large-scale characterization of glycosylation types that remain poorly characterized, such as C-mannosylation and tyrosine O-glycosylation. This bird’s-eye view article provides both a state of the art in the field of intact glycopeptide analysis and open challenges to prompt future research on the topic.
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