From individual proteins to proteomic samples: characterization of O-glycosylation sites in human chorionic gonadotropin and human-plasma proteins

Bai, Xue; Li, Daoyuan; Zhu, Jing; Guan, Yudong; Zhang, Qunye

doi:10.1007/s00216-014-8439-7

Cited by 20 publications

(19 citation statements)

References 40 publications

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“…S6 ), 54 EEAP S LRPAPPPIS 67 , m / z 706.27 3+ ) indicates O -glycosylation at the site Ser 58 . This contrasts recent findings by Bai et al who reported the site Ser 67 to be O -glycosylated, but not the site Ser 58 ( 76 ). In agreement with previous findings, both fibrinogen O -glycopeptides ( 524 ST GK T FPG 531 , m / z 725.78 2+ , 54 EEAP S LRPAPPPIS 67 , m / z 706.27 3+ ), detected in the present study, were found to be decorated with monosialylated T-antigens.…”

Section: Resultscontrasting

confidence: 99%

Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins

Hoffmann

Marx

Reichl

et al. 2016

Molecular & Cellular Proteomics

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Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1 mucin-type O-glycans (T-antigen). The developed workflow allows the identification and characterization of the major population of the human blood plasma O-glycoproteome and our results provide new insights, which can help to unravel structure-function relationships. The data were deposited to ProteomeXchange PXD003270.

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Section: Resultscontrasting

confidence: 99%

Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins

Hoffmann

Marx

Reichl

et al. 2016

Molecular & Cellular Proteomics

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“…A human serum-specific N-linked peptide database that was generated using solid phase extraction of glycosite-containing peptides (SPEG)1137 and solid phase extraction of N-linked glycans and glycosite-containing peptides (NGAG)18 in previous studies was used. The O-linked peptide database was also from previous studies3839. A minimum of two oxonium ions in top 10% of peaks in the spectra was required in an MS/MS spectrum to qualify for glycopeptide search.…”

Section: Methodsmentioning

confidence: 99%

Classification of Tandem Mass Spectra for Identification of N- and O-linked Glycopeptides

Eshghi

Yang

et al. 2016

Sci Rep

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Analysis of intact glycopeptides by mass spectrometry is essential to determining the microheterogeneity of protein glycosylation. Higher-energy collisional dissociation (HCD) fragmentation of glycopeptides generates mono- or disaccharide ions called oxonium ions that carry information about the structure of the fragmented glycans. Here, we investigated the link between glycan structures and the intensity of oxonium ions in the spectra of glycopeptides and utilized this information to improve the identification of glycopeptides in biological samples. Tandem spectra of glycopeptides from fetuin, glycophorin A, ovalbumin and gp120 tryptic digests were used to build a spectral database of N- and O-linked glycopeptides. Logistic regression was applied to this database to develop model to distinguish between the spectra of N- and O-linked glycopeptides. Remarkably, the developed model was found to reliably distinguish between the N- and O-linked glycopeptides using the spectral features of the oxonium ions using verification spectral set. Finally, the performance of the developed predictive model was evaluated in HILIC enriched glycopeptides extracted from human serum. The results showed that pre-classification of tandem spectra based on their glycosylation type improved the identification of N-linked glycopeptides. The developed model facilitates interpretation of tandem mass spectrometry data for assignment of glycopeptides.

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“…Numerous O-glycosylation studies rely on lectin affinity isolation. Jacalin has been used for O-glycosylation characterization of fetal calf serum (25,92), human plasma (140) and serum (79). Peanut agglutinin has been used for characterization of protein O-glycosylation of human plasma, platelets and endothelial cells (26) yielding the largest data set for protein O-glycosylation to date.…”

Section: O-glycosylation Studiesmentioning

confidence: 99%

Analysis of Mammalian O-Glycopeptides—We Have Made a Good Start, but There is a Long Way to Go

Darula

Medzihradszky

2018

Molecular & Cellular Proteomics

101

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Glycosylation is perhaps the most common post-translational modification. Recently there has been growing interest in cataloging the glycan structures, glycoproteins, and specific sites modified and deciphering the biological functions of glycosylation. Although the results are piling up for N-glycosylation, O-glycosylation is seriously trailing behind. In our review we reiterate the difficulties researchers have to overcome in order to characterize O-glycosylation. We describe how an ingenious cell engineering method delivered exciting results, and what could we gain from "wild-type" samples. Although we refer to the biological role(s) of O-glycosylation, we do not provide a complete inventory on this topic.

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From individual proteins to proteomic samples: characterization of O-glycosylation sites in human chorionic gonadotropin and human-plasma proteins

Cited by 20 publications

References 40 publications

Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins

Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins

Classification of Tandem Mass Spectra for Identification of N- and O-linked Glycopeptides

Analysis of Mammalian O-Glycopeptides—We Have Made a Good Start, but There is a Long Way to Go

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