Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

Thaysen‐Andersen, Morten; Packer, Nicolle H.; Schulz, Benjamin L.

doi:10.1074/mcp.o115.057638

Cited by 177 publications

(202 citation statements)

References 184 publications

(167 reference statements)

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“…This ∆ost3/∆ost6 yeast shows severe underglycosylation at many sites, due to inefficient transfer of glycan to asparagine [17,38]. N-glycopeptides can be difficult to detect and quantify because the N-glycan structures are large and heterogeneous [39,40]. To more easily measure site-specific glycosylation occupancy, we therefore deglycosylated N-glycoproteins with Endo H, which cleaves all yeast N-glycan structures and leaves a single N-acetylglucosamine (GlcNAc) monosaccharide on previously glycosylated asparagines [38].…”

Section: Accepted Manuscriptmentioning

confidence: 99%

High-performance targeted mass spectrometry with precision data-independent acquisition reveals site-specific glycosylation macroheterogeneity

Yeo

Chrysanthopoulos

Nouwens

et al. 2016

Analytical Biochemistry

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Protein glycosylation is a critical post-translational modification that regulates the structure, stability, and function of many proteins. Mass spectrometry is currently the preferred method for qualitative and quantitative characterization of glycosylation. However, the inherent heterogeneity of glycosylation makes its analysis difficult. Quantification of glycosylation occupancy, or macroheterogeneity, has proven to be especially challenging. Here, we used a variation of high-resolution multiple reaction monitoring (MRM(HR)) or pseudo-MRM for targeted data-independent acquisition that we term SWAT (sequential window acquisition of targeted fragment ions). We compared the analytical performance of SWATH (sequential window acquisition of all theoretical fragment ions), SWAT, and SRM (selected reaction monitoring) using a suite of synthetic peptides spiked at various concentrations into a complex yeast tryptic digest sample. SWAT provided superior analytical performance to SWATH in a targeted approach. We then used SWAT to measure site-specific N-glycosylation occupancy in cell wall glycoproteins from yeast with defects in the glycosylation biosynthetic machinery. SWAT provided robust measurement of occupancy at more N-glycosylation sites and with higher precision than SWATH, allowing identification of novel glycosylation sites dependent on the Ost3p and Ost6p regulatory subunits of oligosaccharyltransferase.

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Section: Accepted Manuscriptmentioning

confidence: 99%

High-performance targeted mass spectrometry with precision data-independent acquisition reveals site-specific glycosylation macroheterogeneity

Yeo

Chrysanthopoulos

Nouwens

et al. 2016

Analytical Biochemistry

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“…Deciphering glycan structures on glycoproteins is an important goal for better understanding the roles of protein glycosylation in normal and disease biology 1 . For disease research, studies using clinical specimens are particularly valuable, as they enable a direct look at the location and amount of expression of glycans in the biological context.…”

Section: Introductionmentioning

confidence: 99%

Characterizing Protein Glycosylation through On-Chip Glycan Modification and Probing

et al. 2016

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Glycans are critical to protein biology and are useful as disease biomarkers. Many studies of glycans rely on clinical specimens, but the low amount of sample available for some specimens limits the experimental options. Here we present a method to obtain information about protein glycosylation using a minimal amount of protein. We treat proteins that were captured or directly spotted in small microarrays (2.2 × 2.2 mm) with exoglycosidases to successively expose underlying features, and then we probe the native or exposed features using a panel of lectins or glycan-binding reagents. We developed an algorithm to interpret the data and provide predictions about the glycan motifs that are present in the sample. We demonstrated the efficacy of the method to characterize differences between glycoproteins in their sialic acid linkages and N-linked glycan branching, and we validated the assignments by comparing results from mass spectrometry and chromatography. The amount of protein used on-chip was about 11 nanograms. The method also proved effective for analyzing the glycosylation of a cancer biomarker in human plasma, MUC5AC, using only 20 μL of the plasma. A glycan on MUC5AC that is associated with cancer had mostly 2,3 linked sialic acid, whereas other glycans on MUC5AC had a 2,6 linkage of sialic acid. The on-chip glycan modification and probing (on-chip GMAP) method provides a platform for analyzing protein glycosylation in clinical specimens and could complement the existing toolkit for studying glycosylation in disease.

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“…In addition to increasing the relative amounts of c-and z-ions these combined fragmentation methods also produce ions that are characteristic of CID/HCD fragmentation of glycopeptides (19). This proves beneficial as it provides informative spectra where both peptide and monosaccharide constituents can be elucidated along with the site of glycan attachment.…”

Section: Mass Spectrometry For Protein Glycosylationmentioning

confidence: 99%

“…For disordered glycoproteins, small-angle X-ray scattering may be used to provide molecular weight (MW) determination or low resolution characterisation of protein conformation (143). Another tool that is widely used for the structural characterisation of protein glycosylation is MS [reviewed in (19,(144)(145)(146)(147)(148)(149)(150)(151)(152)(153)(154)]. Depending on the type of analysis implemented, a range of qualitative, quantitative, structural and site-specific information can be obtained.…”

Section: Methods To Analyse Protein Glycosylationmentioning

confidence: 99%

“…More recently, an unconventional glycan processing pathway has been described, prompted by the growing observation of truncated N-linked structures in mammals (19)(20)(21)(22)(23). These glycans stem from trimming of hybrid or complex glycans and the truncated N-glycans containing one to four mannose residues (Man 1-4 ) are described as paucimannose and those without Man residues are described as chitobiose core type glycans (19). Remodelling of the Glc 3 Man 9 GlcNAc 2 structure takes place after it is transferred to a nascent polypeptide chain in the ER (top left).…”

Section: N-linked Glycosylationmentioning

confidence: 99%

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

Pegg¹

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The family Paramyxoviridae (paramyxovirus) contains several significant human and animal pathogens.Represented within this family are human respiratory syncytial virus (hRSV) and Newcastle disease virus (NDV). The former contributes significantly to severe respiratory tract disease in infants, children and immunocompromised individuals. At present, highly efficacious therapeutics or safe and effective vaccines are not available for hRSV. NDV is the causative agent of Newcastle disease, afflicting a wide range of avian species. The desire to study NDV is due not only to the significant economic impact it has on the poultry industry worldwide, but also its potential use as an oncolytic agent and vaccine vector in humans and animals. Additionally, findings on NDV may be translated to closely related viruses that cause disease in humans, such as parainfluenza viruses.As outlined in Chapter 1 of this thesis, the infectious processes of all members of this family are driven by two major membrane glycoproteins whose ectodomains project from the viral envelope: the fusion (F) and attachment glycoproteins. The F glycoprotein is responsible for viral entry by means of fusion with host cell membranes while the variable attachment glycoproteins, haemagglutinin, haemagglutininneuraminidase (HN) and major surface glycoprotein (G), are involved in viral attachment to host cells.Previous studies have shown that altering the glycosylation profile of these proteins can modulate the ability of the virus to infect host cells and stimulate the host immune system. As yet, site-specific glycan heterogeneity of hRSV and NDV surface glycoproteins has not been defined at a chemical level. As described in Chapter 2, reverse-phase liquid chromatography tandem mass spectrometry (MS) strategies were implemented to characterise intact glycopeptides from the attachment and F glycoproteins of hRSV and NDV. Site-occupancy and monosaccharide compositions at a given site were determined using collision-induced dissociation, higher-energy collision dissociation, electron-transfer dissociation and electron-transfer dissociation combined with collision dissociation. As described in Chapter 3, a spectral processing program was developed called OxoExtract to aid identification of intact glycopeptides that were fragmented with higher-energy collision dissociation.The F and HN proteins of NDV were derived from virions propagated in embryonic eggs. Analyses of HN in Chapter 4 revealed high mannose N-linked glycans and complex or hybrid N-linked glycans that were variably fucosylated, sialylated and sulfated or phosphorylated. In total 63, 58, and 37 glycans were identified at sites N341, N433 and N481, respectively. In addition, a previously undocumented Olinked glycosylation site was identified in the stalk domain of the protein. Observed glycans from NDV F described in Chapter 5 were mainly high mannose, containing variations of five to nine mannose ii residues across four sites, N85, N191, N366 and N471. There was also evidence of fucosylated c...

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Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

Cited by 177 publications

References 184 publications

High-performance targeted mass spectrometry with precision data-independent acquisition reveals site-specific glycosylation macroheterogeneity

High-performance targeted mass spectrometry with precision data-independent acquisition reveals site-specific glycosylation macroheterogeneity

Characterizing Protein Glycosylation through On-Chip Glycan Modification and Probing

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

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