Tandem Mass Spectrometry and Glycoproteins

Dolashka, Pavlina

doi:10.5772/30829

Cited by 5 publications

(4 citation statements)

References 36 publications

(29 reference statements)

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“…Mass spectrometry coupled with liquid chromatography is one of the most powerful tools that can be used to acquire high-throughput and accurate informatics for mapping protein glycosylation patterns [ 12 , 13 ]. Since the protein glycosylation pattern of antibody therapeutics is considered a critical quality attribute affecting a wide spectrum of the biological processes of the therapeutics, precise mapping of glycosylation patterns is very important to evaluate safety and efficacy of the drug [ 14 , 15 ].…”

Section: Introductionmentioning

confidence: 99%

Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping

Lee

Choi²,

Hwang³

et al. 2022

IJMS

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The evaluation of the protein glycosylation states of samples of aflibercept obtained from three different regions was conducted by site-specific N-linked glycan microheterogeneity profiling. Glycopeptide-based nano-LC MSMS mapping of tryptic-digested samples of each aflibercept lot provided site-specific information about glycan microheterogeneity on each of the five N-glycosylation sites (two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region). Next, the glycopeptide-mapping results obtained from the three different aflibercept lots were compared to evaluate the similarity between the samples. Three aflibercept lots showed a high degree of similarity in glycan composition, fucosylation level, sialylation level, and branching, when all five N-glycosylation sites were assessed together as a group. On the other hand, noticeable variations between lots in the glycan types and sialylation levels on the two sites of the VEGFR-2 domain were observed when each of the five N-glycosylation sites were assessed using the glycopeptide-based method. The presence of N-glycolylneuraminic acid (NeuGc) glycans, which may mediate adverse immune reactions in antibody therapeutics, were also detected on the sites of VEGFR1 and VEGFR2 domains, but not on the IgG Fc domain site. These results imply that analyses of the glycosylation profiles of fusion proteins containing multiple N-glycosylation sites, such as aflibercept, being done as a part of quality control for the therapeutics manufacturing process or for biosimilar development, can be done with a more applicable outcome by assessing each site separately.

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Section: Introductionmentioning

confidence: 99%

Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping

Lee

Choi²,

Hwang³

et al. 2022

IJMS

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“…In the first approach, functional units (FUs) of the two subunits RvH1 and RvH2 are treated with trypsin and the obtained glycopeptides are identified through LC/ESI-MS, LC-Q-trap-MS/MS or nano-ESI-MS [ 28 , 29 , 30 , 31 , 32 , 33 ]. The second approach includes isolation of glycans after hydrolysis of the subunits of RvH with specific glycosidases (PNGase F) and analysis of the glycan structures with MALDI-TOF-MS, СЕ-MS, and Q-trap instrumental systems [ 28 , 29 , 34 ].…”

Section: Carbohydrate Structure Of Hemocyanins From the Marine Snamentioning

confidence: 99%

De Novo Structural Determination of the Oligosaccharide Structure of Hemocyanins from Molluscs

et al. 2020

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A number of studies have shown that glycosylation of proteins plays diverse functions in the lives of organisms, has crucial biological and physiological roles in pathogen–host interactions, and is involved in a large number of biological events in the immune system, and in virus and bacteria recognition. The large amount of scientific interest in glycoproteins of molluscan hemocyanins is due not only to their complex quaternary structures, but also to the great diversity of their oligosaccharide structures with a high carbohydrate content (2–9%). This great variety is due to their specific monosaccharide composition and different side chain composition. The determination of glycans and glycopeptides was performed with the most commonly used methods for the analysis of biomolecules, including peptides and proteins, including Matrix Assisted Laser Desorption/Ionisation–Time of Flight (MALDI-TOF-TOF), Liquid Chromatography - Electrospray Ionization-Mass Spectrometry (LC/ESI-MS), Liquid Chromatography (LC-Q-trap-MS/MS) or Nano- Electrospray Ionization-Mass Spectrometry (nano-ESI-MS) and others. The molluscan hemocyanins have complex carbohydrate structures with predominant N-linked glycans. Of interest are identified structures with methylated hexoses and xyloses arranged at different positions in the carbohydrate moieties of molluscan hemocyanins. Novel acidic glycan structures with specific glycosylation positions, e.g., hemocyanins that enable a deeper insight into the glycosylation process, were observed in Rapana venosa, Helix lucorum, and Haliotis tuberculata. Recent studies demonstrate that glycosylation plays a crucial physiological role in the immunostimulatory and therapeutic effect of glycoproteins. The remarkable diversity of hemocyanin glycan content is an important feature of their immune function and provides a new concept in the antibody–antigen interaction through clustered carbohydrate epitopes.

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“…Recent advances in mass spectrometry have allowed for high-throughput methods and accurate digitalized informatics for mapping glycosylation patterns [7,8,9]. A widely used analytical method for glycans released in IgG monoclonal antibodies is 2-AB labeling, in which the glycans are labeled with 2-aminobenzamide (2-AB) and analyzed by liquid chromatography equipped with fluorescent detection or by mass spectrometer [9,10].…”

Section: Introductionmentioning

confidence: 99%

Investigation of Site-Specific Differences in Glycan Microheterogeneity by N-Glycopeptide Mapping of VEGFR-IgG Fusion Protein

Hahm¹,

Lee

Ahn

2019

Molecules

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A biosimilar fusion protein VEGFR-IgG consisting of vascular endothelial growth factor receptors 1 and 2 (VEGFR-1, VEGFR-2) and the Fc portion of human IgG1 was prepared for this study. The prepared fusion protein was expected to possess a total of five N-linked glycosylation sites: two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region. For site-specific glycan analysis, the fusion protein was hydrolyzed with trypsin, and the resulting tryptic digests were analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). The expected N-linked glycosylation sites were successfully identified and site-specific glycopeptide mapping was completed by Integrated GlycoProteome Analyzer (I-GPA) for the resulting raw tandem mass data. Finally, it was clearly confirmed that N-linked glycans for each glycosylation site showed significantly different patterns in microheterogeneity, which may indicate certain functions for each glycosylation site in the protein. Based on the mapping results, the unique features in glycan microheterogeneity for the five glycosylation sites of VEGFR-IgG fusion protein were compared site-specifically and further discussed to understand the functional meaning of each glycosylation pattern.

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Tandem Mass Spectrometry and Glycoproteins

Cited by 5 publications

References 36 publications

Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping

Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping

De Novo Structural Determination of the Oligosaccharide Structure of Hemocyanins from Molluscs

Investigation of Site-Specific Differences in Glycan Microheterogeneity by N-Glycopeptide Mapping of VEGFR-IgG Fusion Protein

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