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  • Resolving the molecular details of glyco proteome

    2022-07-16

    Resolving the molecular details of (glyco)proteome variation in different tissues and organs of the human body is critical for the understanding of human biology and disease [59]. Glycoproteomic analysis of disease-related tissues and cells has provided valuable information to identify promising targets for their diagnosis, prognosis and therapy. In this part, we summarized recent glycoproteomic studies in 12 different tissues (and related cell lines), involving some closely related diseases (Table 1).
    Glycoproteins in special cell types There are some special cell types in human that researchers are particularly interested in. Hereby, glycoproteomic studies in special cell types are summarized, including human blood platelets, T cells, clemastine fumarate receptor and spermatozoa (Table 1). As a critical component of human blood, more than 1000 N-glycosylation sites have been identified from blood platelets by MS. In 2006, Lewandrowski et al. reported the first de-glycopeptide dataset from human blood platelets by using both lectin and hydrazide chemistry enrichment methods, PNGase F treatment and mass spectrometry [98]. Using this approach, they identified 70 different glycosylation sites from 41 different platelet glycoproteins. One year later, they published the second paper reporting 148 N-linked glycosylation sites on 79 glycoproteins from the platelet membrane [21]. In 2008, the same group reported 125 glycosylation sites on 66 proteins from platelets by glycopeptides enrichment using electrostatic repulsion hydrophilic interaction chromatography (ERLIC) [99]. In total, more than 250 glycosylation sites annotated for platelets were identified using three different enrichment strategies (four different enrichment methods) [99]. In 2017, our group published another MS dataset related to platelet glycoproteins. By using iTRAQ labeling and SPEG (hydrazide chemistry) enrichment, we identified 799 unique N-linked glycosylation sites in platelets [100], which further extended the depth of the glycoprotein coverage of human platelets. Up to now, more than one thousand N-glycoproteins have been identified from human platelets by MS. Besides blood platelets, glycoproteomic analysis on T cells, B cells and spermatozoa have been reported as well. From T cell line ACH-2, Yang et al. identified 563 unique glycopeptides from 247 unique glycoproteins using hydrazide chemistry enrichment method [101]. In 2014, Deeb et al. identified 2383 unique glycosylation sites on 1321 glycoproteins from the cell surface of diffuse large B-cell lymphoma subtypes by glyco-FASP method and MS analysis [102]. Using glyco-FASP method coupled with LC-MS/MS, Wang et al. identified 554 N-glycosylation sites and 297 N-glycoproteins from human spermatozoa [103].
    In addition to the canonical N-linked glycosylation motif asparagine-X-serine/threonine (N-X-S/T, X is any amino acid except proline) [5], [104], many sites with atypical sequons have been identified by MS as N-linked glycosylation sites. These reported atypical glycosites include N-X-C [105], N-X-V [106], [107] and N-X-G [107]. Except for the N-X-C motif, which has been confirmed in several known glycoproteins [105], all other atypical motifs were only identified based on the deamidation of asparagine (N) residues in the peptides after PNGase F treatment (with/without 18O labeling) using mass spectrometry-based glycoproteomics [106], [107]. However, the atypical sites identified based on deamidation of N are potentially false positives as it could occur naturally or be induced during sample preparation [108], [109]. Recently, by using the NGAG method coupled with high resolution mass spectrometry, we identified two atypical N-glycosites with 146N#HV and 156N#SC motifs on Protein sel-1 homolog 1 (SEL1L) and Kunitz-type protease inhibitor 2 (SPINT2) from OVCAR-3 cell line, respectively [110]. These two atypical glycosites were further confirmed via the direct identification of their intact N-glycopeptide forms (with glycan still attached at the glycosylation site). The results showed that the glycosite 146N#HV was modified by the glycan Man9 (Fig. 2A) while the glycosite 156N#SC was modified by four different oligo-mannose glycans (HexNAc2Hex7 - HexNAc2Hex10, Fig. 2B). Using the same strategy, another two atypical N-glycosites with the N-X-V motif, which included 68N#EV on Albumin (ALB) and 62N#GV on Alpha-1B-glycoprotein (A1BG), were identified and verified from human serum [111]. Both glycosites were modified by complex glycans N4H5S1 and N4H5S2 (Fig. 2C, D).