Positive Mode LC-MS/MS Analysis of Chondroitin Sulfate Modified Glycopeptides Derived from Light and Heavy Chains of The Human Inter-α-Trypsin Inhibitor Complex*

Toledo, Alejandro Gómez; Nilsson, Jonas; Noborn, Fredrik; Sihlbom, Carina; Larson, Göran

doi:10.1074/mcp.m115.051136

Cited by 39 publications

(80 citation statements)

References 46 publications

(46 reference statements)

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“…Strong anion exchange (SAX) chromatography was used to enrich GAG-peptides from trypsin-digested human urine, plasma, and cerebrospinal fluid (CSF) samples. Several CS linkage region-substituted glycopeptides (CS-glycopeptides) having a 6-mer saccharide structure were formed after chondroitinase ABC degradation (Figure 1) [21, 22]. High-energy collision dissociation (HCD)-based LC-MS/MS analysis enabled the simultaneous fragmentation and identification of glycan and peptide backbone in an integrated glycopeptide characterization.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Glycan Structures of Chondroitin Sulfate-Glycopeptides Facilitated by Sodium Ion-Pairing and Positive Mode LC-MS/MS

Nilsson

Noborn

Toledo

et al. 2016

J. Am. Soc. Mass Spectrom.

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Purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of glycopeptides, originating from protease digests of glycoproteins, enables site-specific analysis of protein N- and O-glycosylations. We have described a protocol to enrich, hydrolyze by chondroitinase ABC, and characterize chondroitin sulfate-containing glycopeptides (CS-glycopeptides) using positive mode LC-MS/MS. The CS-glycopeptides, originating from the Bikunin proteoglycan of human urine samples, had ΔHexAGalNAcGlcAGalGalXyl-O-Ser hexasaccharide structure and were further substituted with 0-3 sulfate and 0-1 phosphate groups. However, it was not possible to exactly pinpoint sulfate attachment residues, for protonated precursors, due to extensive fragmentation of sulfate groups using high-energy collision induced dissociation (HCD). To circumvent the well-recognized sulfate instability, we now introduced Na+ ions to form sodiated precursors, which protected sulfate groups from decomposition and facilitated the assignment of sulfate modifications. Sulfate groups were pinpointed to both Gal residues and to the GalNAc of the hexasaccharide structure. The intensities of protonated and sodiated saccharide oxonium ions were very prominent in the HCD-MS2 spectra, which provided complementary structural analysis of sulfate substituents of CS-glycopeptides. We have demonstrated a considerable heterogeneity of the bikunin CS linkage region. The realization of these structural variants should be beneficial in studies aimed at investigating the importance of the CS linkage region with regards to the biosynthesis of CS and potential interactions to CS binding proteins. Also, the combined use of protonated and sodiated precursors for positive mode HCD fragmentation analysis will likely become useful for additional classes of sulfated glycopeptides. Graphical Abstractᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-016-1539-1) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Characterization of Glycan Structures of Chondroitin Sulfate-Glycopeptides Facilitated by Sodium Ion-Pairing and Positive Mode LC-MS/MS

Nilsson

Noborn

Toledo

et al. 2016

J. Am. Soc. Mass Spectrom.

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“…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).…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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 …”

Section: Figure 3-1 Graphical User Interface Of Oxoextractmentioning

confidence: 99%

“…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 …”

Section: Amino Acid Sequence Changes In V4-var Hnmentioning

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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Comparative proteomic profiling of the serum differentiates pancreatic cancer from chronic pancreatitis

et al. 2017

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Finland ranks sixth among the countries having highest incidence rate of pancreatic cancer with mortality roughly equaling incidence. The average age of diagnosis for pancreatic cancer is 69 years in Nordic males, whereas the average age of diagnosis of chronic pancreatitis is 40–50 years, however, many cases overlap in age. By radiology, the evaluation of a pancreatic mass, that is, the differential diagnosis between chronic pancreatitis and pancreatic cancer is often difficult. Preoperative needle biopsies are difficult to obtain and are demanding to interpret. New blood based biomarkers are needed. The accuracy of the only established biomarker for pancreatic cancer, CA 19‐9 is rather poor in differentiating between benign and malignant mass of the pancreas. In this study, we have performed mass spectrometry analysis (High Definition MSE) of serum samples from patients with chronic pancreatitis (13) and pancreatic cancer (22). We have quantified 291 proteins and performed detailed statistical analysis such as principal component analysis, orthogonal partial least square discriminant analysis and receiver operating curve analysis. The proteomic signature of chronic pancreatitis versus pancreatic cancer samples was able to separate the two groups by multiple statistical techniques. Some of the enriched pathways in the proteomic dataset were LXR/RXR activation, complement and coagulation systems and inflammatory response. We propose that multiple high‐confidence biomarker candidates in our pilot study including Inter‐alpha‐trypsin inhibitor heavy chain H2 (Area under the curve, AUC: 0.947), protein AMBP (AUC: 0.951) and prothrombin (AUC: 0.917), which should be further evaluated in larger patient series as potential new biomarkers for differential diagnosis.

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Positive Mode LC-MS/MS Analysis of Chondroitin Sulfate Modified Glycopeptides Derived from Light and Heavy Chains of The Human Inter-α-Trypsin Inhibitor Complex*

Cited by 39 publications

References 46 publications

Characterization of Glycan Structures of Chondroitin Sulfate-Glycopeptides Facilitated by Sodium Ion-Pairing and Positive Mode LC-MS/MS

Characterization of Glycan Structures of Chondroitin Sulfate-Glycopeptides Facilitated by Sodium Ion-Pairing and Positive Mode LC-MS/MS

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

Comparative proteomic profiling of the serum differentiates pancreatic cancer from chronic pancreatitis

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