Identification of Chondroitin Sulfate Linkage Region Glycopeptides Reveals Prohormones as a Novel Class of Proteoglycans

Abstract

Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far, only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites, and identities of core proteins. CSPGs were enriched from human urine and cerebrospinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CSlyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1, and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may also influence the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules. Molecular & Cellular

Structural characterisation of glycosylation of paramyxovirus attachment and fusion proteins by mass spectrometry

“…As described in Chapter 4 for NDV HN, this enabled the distinction between Sulf and Phos to be made based on the accuracy of the mass differences between sulfated and non-sulfated oxonium ions (238,267). Typically Sulf substituents on glycans have been identified in negative ion mode after the glycans have been released from a protein and analysed with or without derivatisation (343).…”

“…In positive ion mode, ion-pairing reagents can be used to stabilise labile Sulf substituents on glycopeptides before direct infusion into the mass spectrometer (344). The use of lower dissociation energies in positive ion mode has been used for the characterisation of enriched O-linked glycopeptides containing sulfated glycosaminoglycan chains (238,267). The present study complements this research by showing that stepped HCD and EThcD can be used to produce sulfated oxonium ions from N-linked glycopeptides in positive ion mode under typical LC-MS conditions.…”

“…Enrichment is particularly relevant for complex samples and, depending on the approach, can facilitate the isolation of a broad range of glycopeptides (206,213,227,230) or those carrying certain monosaccharides or substituents (235)(236)(237)(238)(239). Hydrophilic interaction liquid chromatography is commonly reported in the literature (234) with several different types of resins and methods available (240)(241)(242).…”

“…Glycans that contained Sulf or Phos, as determined by precursors mass, were confidently assigned as sulfated if oxonium ions HexNAc 1 Sulf 1 or HexNAc 1 Hex 1 Sulf 1 were present. The distinction between sulfated and phosphorylated glycopeptides can be made using mass shifts between glycan oxonium ions in a single spectrum (238,267). For all confidently assigned sulfated compositions the mass shifts between ions matching the m/z for HexNAc 1 …”

“…The list of the oxonium ions is stored in an Excel spreadsheet within the data folder of OxoExtract which can be modified by the user. The oxonium ions used in this work were observed in samples, theoretical or were defined in the literature (219,220,238,251,(267)(268)(269) and have been represented in Table 3 …”