Intact glycopeptide characterization using mass spectrometry

Cao, Li; Qu, Yi; Zhang, Zhaorui; Wang, Zhe; Prytkova, Iya; Wu, Si

doi:10.1586/14789450.2016.1172965

Cited by 61 publications

(55 citation statements)

References 100 publications

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“…Despite the improvement in MS technologies and software solutions for protein sequencing, identification of protein/peptide modifications remains challenging, with the main difficulties related to the complexity and high glycan heterogeneity . Both O‐glycosylation and N‐glycosylation have been implicated in various biological processes, including inflammatory responses, angiogenesis, autoimmunity, and cancer .…”

Section: Discussionmentioning

confidence: 99%

Urinary Glycopeptide Analysis for the Investigation of Novel Biomarkers

Belczacka

Pejchinovski

Krochmal

et al. 2018

Proteomics Clinical Apps

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Purpose: Urine is a rich source of potential biomarkers, including glycoproteins. Glycoproteomic analysis remains difficult due to the high heterogeneity of glycans. Nevertheless, recent advances in glycoproteomics software solutions facilitate glycopeptide identification and characterization. The aim is to investigate intact glycopeptides in the urinary peptide profiles of normal subjects using a novel PTM-centric software-Byonic. Experimental design: The urinary peptide profiles of 238 normal subjects, previously analyzed using CE-MS and CE-MS/MS and/or LC-MS/MS, are subjected to glycopeptide analysis. Additionally, glycopeptide distribution is assessed in a set of 969 patients with five different cancer types: bladder, prostate and pancreatic cancer, cholangiocarcinoma, and renal cell carcinoma. Results: A total of 37 intact O-glycopeptides and 23 intact N-glycopeptides are identified in the urinary profiles of 238 normal subjects. Among the most commonly identified O-glycoproteins are Apolipoprotein C-III and insulin-like growth factor II, while titin among the N-glycoproteins. Further statistical analysis reveals that three O-glycopeptides and five N-glycopeptides differed significantly in their abundance among the different cancer types, comparing to normal subjects. Conclusions and clinical relevance: Through the established glycoproteomics workflow, intact O-and N-glycopeptides in human urine are identified and characterized, providing novel insights for further exploration of the glycoproteome with respect to specific diseases.

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Section: Discussionmentioning

confidence: 99%

Urinary Glycopeptide Analysis for the Investigation of Novel Biomarkers

Belczacka

Pejchinovski

Krochmal

et al. 2018

Proteomics Clinical Apps

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“…With glycans remaining attached to the protein sites, this strategy can elucidate the glycopeptide amino acid sequence, the glycosylation site information, and the glycan composition simultaneously (Mayampurath et al, ; Stadlmann, Pabst, Kolarich, Kunert, & Altmann, ). Due to the inherent complexities of intact glycopeptides, this analytical method faces several complications: (a) the glycopeptides can be suppressed by nonglycopeptides in the mass spectrometry because of their low ionization efficiency (Cao et al, ). Therefore, a glycopeptide enrichment step may help to enhance the mass spectrometry performance.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, a glycopeptide enrichment step may help to enhance the mass spectrometry performance. (b) The commonly used collision‐induced dissociation fragmentation method was reported to have difficulty in generating a diverse collection of peptide backbone fragments, which can make peptide sequence identification problematic (Cao et al, ). As an alternative approach, higher‐energy C‐trap dissociation (HCD) fragmentation, often equipped with orbitrap detection, represents a potentially powerful tool for generating high resolution tandem mass spectrometry (MS/MS) spectra.…”

Section: Introductionmentioning

confidence: 99%

“…As an alternative approach, higher‐energy C‐trap dissociation (HCD) fragmentation, often equipped with orbitrap detection, represents a potentially powerful tool for generating high resolution tandem mass spectrometry (MS/MS) spectra. HCD can provide superior performance in representing low m / z glycan oxonium ions, which normally contain signal ions for glycopeptides (Cao et al, ). By supporting multiple cleavage events, HCD fragmentation can provide a wealth of peptide identifications (Cao et al, ; Frese et al, ; Jedrychowski et al, ).…”

Section: Introductionmentioning

confidence: 99%

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Characterization of intact glycopeptides reveals the impact of culture media on site‐specific glycosylation of EPO‐Fc fusion protein generated by CHO‐GS cells

Wang

Yang

Wang

et al. 2019

Biotech & Bioengineering

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With the increasing demand to provide more detailed quality attributes, more sophisticated glycan analysis tools are highly desirable for biopharmaceutical manufacturing. Here, we performed an intact glycopeptide analysis method to simultaneously analyze the site-specific N-and O-glycan profiles of the recombinant erythropoietin Fc (EPO-Fc) protein secreted from a Chinese hamster ovary glutamine synthetase stable cell line and compared the effects of two commercial culture media, EX-CELL (EX) and immediate advantage (IA) media, on the glycosylation profile of the target protein. EPO-Fc, containing the Fc region of immunoglobulin G1 (IgG1) fused to EPO, was harvested at Day 5 and 8 of a batch cell culture process followed by purification and N-and O-glycopeptide profiling. A mixed anion exchange chromatographic column was implemented to capture and enrich N-linked glycopeptides. Using intact glycopeptide characterization, the EPO-Fc was observed to maintain their individual EPO and Fc N-glycan characteristics in which the EPO region presented bi-, tri-, and tetra-branched N-glycan structures, while the Fc Nglycan displayed mostly biantennary glycans. EPO-Fc protein generated in EX medium produced more complex tetra-antennary N-glycans at each of the three EPO N-sites while IA medium resulted in a greater fraction of bi-and tri-antennary N-glycans at these same sites. Interestingly, the sialylation content decreased from sites 1-4 in both media while the fucosylation progressively increased with a maximum at the final IgG Fc site. Moreover, we observed that low amounts of Neu5Gc were detected and the content increased at the later sampling time in both EX and IA media. For O-glycopeptides, both media produced predominantly three structures, N1F1F0SOG0, N1H1F0S1G0, and N1H1F0S2G0, with lesser amounts of other structures. This intact glycopeptide method can decipher site-specific glycosylation profile and provide a more detailed characterization of N-and O-glycans present for enhanced understanding of the key product quality attributes such as media on recombinant proteins of biotechnology interest. K E Y W O R D S CHO-GS cells, culture media, EPO-Fc protein, intact glycopeptide analysis, site-specific glycosylation SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section.

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“…Detection of multiple glycoforms of glycopeptides, which tend to be poorly ionized and are often present as minor fractions relative to peptides, is currently a challenging task in the glycoproteomic profiling of glycoproteins. 17,18 Moreover, glycopeptides of low charge state are poor candidates for the electron transfer dissociation (ETD) fragmentation methodology, which is presently state-of the art technology for accurate site mapping of post-translational modification on peptides. 19 Multifaceted work-flows for the enrichment of glycopeptides from among the peptides are often associated with profiling of glycoprotein from a complex proteome.…”

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confidence: 99%

Tool for Rapid Analysis of Glycopeptide by Permethylation via One-Pot Site Mapping and Glycan Analysis

et al. 2017

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To overcome the challenges in the analysis of protein glycosylation, we have developed a comprehensive and universal tool through permethylation of glycopeptides and their tandem mass spectrometric analysis. This method has the potential to simplify glycoprotein analysis by integrating glycan sequencing and glycopeptide analysis in a single experiment. Moreover, glycans with unique glycosidic linkages, particularly from prokaryotes, which are resistant to enzymatic or chemical release, could also be detected and analyzed by this methodology. Here we present a strategy for the permethylation of intact glycopeptides, obtained via controlled protease digest, and their characterization by using advanced mass spectrometry. We used bovine RNase B, human transferrin, and bovine fetuin as models to demonstrate the feasibility of the method. Remarkably, the glycan patterns, glycosylation site, and their occupancy by N-glycans are all detected and identified in a single experimental procedure. Acquisition on a high resolution tandem-MSn system with fragmentation methodologies such as high-energy collision dissociation (HCD) and collision induced dissociation (CID), provided the complete sequence of the glycan structures attached to the peptides. The behavior of 20 natural amino acids under the basic permethylation conditions was probed by permethylating a library of short synthetic peptides. Our studies indicate that the permethylation imparts simple, limited, and predictable chemical transformations on peptides and do not interfere with the interpretation of MS/MS data. In addition to this, permethylated O-glycans in unreduced form (released by β elimination) were also detected, allowing us to profile O-linked glycan structures simultaneously.

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Intact glycopeptide characterization using mass spectrometry

Cited by 61 publications

References 100 publications

Urinary Glycopeptide Analysis for the Investigation of Novel Biomarkers

Urinary Glycopeptide Analysis for the Investigation of Novel Biomarkers

Characterization of intact glycopeptides reveals the impact of culture media on site‐specific glycosylation of EPO‐Fc fusion protein generated by CHO‐GS cells

Tool for Rapid Analysis of Glycopeptide by Permethylation via One-Pot Site Mapping and Glycan Analysis

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