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

Shajahan, Asif; Supekar, Nitin T.; Heiß, Christian; Ishihara, Mayumi; Azadi, Parastoo

doi:10.1021/acs.analchem.7b01730

Cited by 35 publications

(42 citation statements)

References 48 publications

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“…The reaction was quenched by adding water (1 ml) and the permethylated N-glycans were extracted by organic phase separation using dichloromethane (2 ml). The excess of dichloromethane was removed by stream of nitrogen and subsequently, prepared for MALDI-MS analysis (84).…”

Section: Methodsmentioning

confidence: 99%

“…Data were obtained from AB SCIEX MALDI TOF/TOF 5800 (Applied Biosystem MDS Analytical Technologies, Foster City, CA) mass spectrometer in reflector positive-ion mode. Data analysis was performed by using Data Explorer V4.5, and the assignment of glycan structure was based on the primary mass (m/z) coupled with MS/MS fragmentation profile using the Expasy database online and the glycowork bench software analysis (84, 85).…”

Section: Methodsmentioning

confidence: 99%

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CRISPR/Cas9 gene editing for the creation of an MGAT1 deficient CHO cell line to control HIV-1 vaccine glycosylation

Byrne

O’Rourke

Alexánder

et al. 2018

Preprint

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Over the last decade multiple broadly neutralizing monoclonal antibodies (bN-mAbs) to the HIV-1 envelope protein, gp120, have been described. Surprisingly many of these recognize epitopes consisting of both amino acid and glycan residues. Moreover, the glycans required for binding of these bN-mAbs are early intermediates in the N-linked glycosylation pathway. This type of glycosylation substantially alters the mass and net charge of HIV envelope (Env) proteins compared to molecules with the same amino acid sequence but possessing mature, complex (sialic acid containing) carbohydrates. Since cell lines suitable for biopharmaceutical production that limit N-linked glycosylation to mannose-5 (Man5) or earlier intermediates are not readily available, the production of vaccine immunogens displaying these glycan dependent epitopes has been challenging. Here we report the development of a stable suspension adapted CHO cell line that limits glycosylation to Man5 and earlier intermediates. This cell line was created using the CRISPR/Cas9 gene editing system and contains a mutation that inactivates the gene encoding Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase (MGAT1). Monomeric gp120s produced in the MGAT1- CHO cell line exhibit improved binding to prototypic glycan dependent bN-mAbs directed to the V1/V2 domain (e.g. PG9) and the V3 stem (e.g. PGT128 and 10–1074) while preserving the structure of the important glycan independent epitopes (e.g. VRC01). The ability of the MGAT1-CHO cell line to limit glycosylation to early intermediates in the N-linked glycosylation pathway, without impairing the doubling time or ability to grow at high cell densities, suggest that it will be a useful substrate for the biopharmaceutical production of HIV-1 vaccine immunogens.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

CRISPR/Cas9 gene editing for the creation of an MGAT1 deficient CHO cell line to control HIV-1 vaccine glycosylation

Byrne

O’Rourke

Alexánder

et al. 2018

Preprint

Self Cite

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“…Recent advances in multi-mode fragmentation allow the acquisition of complementary fragmentation spectra on the time-scale of eluting peptide species. (51) The utility of this has been the focus of several recent studies (50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67). We acquired the data using an HCD-triggered EThcD approach relying on detection of an N-acetylglucosamine oxonium ion (4,57,(68)(69)(70)(71)(72)(73)(74).…”

Section: Glycopeptide Mass Spectrometry Based Acquisitionmentioning

confidence: 99%

Glycoproteome Analysis of Human Serum and Brain Tissue

Brown

et al. 2019

Preprint

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Abbreviations: ammonium bicarbonate: ABC; electron-transfer/higher-energy collisional dissociation: EThcD; higher-energy collisional dissociation: HCD; hydrophilic interaction chromatography: HILIC; multi-lectin weak affinity chromatography: M-LWAC; posttranslational modification: PTM; reverse phase: RP Abstract Protein glycosylation represents one of the most common and heterogeneous posttranslational modifications (PTMs) in human biology. Herein, an approach for the enrichment of glycopeptides using multi-lectin weak affinity chromatography (M-LWAC), followed by fractionation of the enriched material, and multi-mode fragmentation LC/MS is described. Two fragmentation methods, high-energy collision induced dissociation (HCD) and electron transfer dissociation (EThcD), were independently analyzed. While each fragmentation method provided similar glycopeptide coverage, there was some dependence on the glycoform identity. From these data a total of 7,503 unique glycopeptides belonging to 666 glycoproteins from the combined tissue types, human serum and brain, were identified. Of these, 617 glycopeptides (192 proteins) were found in both tissues; 2,006 glycopeptides (48 proteins) were unique to serum, and 4,880 glycopeptides (426 proteins) were unique to brain tissue. From 379 unique glycoforms, 1,420 unique sites of glycosylation were identified, with an average of four glycans 2 per site. Glycan occurrences were significantly different between tissue types: serum showed greater glycan diversity whereas brain tissue showed a greater abundance of the high mannose family. Glycosylation co-occurrence rates were determined, which enabled us to infer differences in underlying biosynthetic pathways. Experimental procedures Brain Lysate Sample PreparationBrain specimens from all subjects were obtained during autopsies conducted at the Allegheny County Office of the Medical Examiner after receiving consent from the next-of-kin.Procedures were approved by the University of Pittsburgh Institutional Review Board and Committee for Oversight of Research Involving the Dead. Grey matter was harvested from the auditory cortex as previously described (40,41): Tissue slabs containing the superior temporal gyrus with Heschl's Gyrus located medial to the planum temporale were identified, and the superior temporal gyrus removed as single block. Grey matter (100 mg) was collected from HG by taking 100 μm frozen sections (40). Grey matter was homogenized in 1 mL of 8M urea with a FastPrep-24 benchtop homogenizer. Samples were stored at -20 ˚C. Protein PreparationSera (Sigma-Aldrich, St. Louis, MO) was denatured using 8 M urea (Sigma-Aldrich, St.Louis MO) in 100 mM ammonium bicarbonate (pH 7.4, Sigma-Aldrich, St. Louis MO).Disulfide bonds were reduced using 1 mM tris(2-carboxyethyl)phosphine (Sigma-Aldrich, St. Louis, MO) for 1 hour at 65 ˚C. Reduced cysteine residues were modified using 4.4 mM iodoacetamide (Sigma-Aldrich, St. Louis, MO) for 1 hour at room temperature. Following this, the sample was digested overnight at 37 ˚C with tryps...

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“…Recently, the mass spectrometric analysis of intact glycopeptides has emerged as a promising strategy to analyze glycoproteins for glycan modifications at site-and structure-specific levels (Sun et al, 2016;Yang et al, 2018;Xiao and Tian, 2019). However, the major limitation for this approach is the low detectability of intact glycopeptides in a complex mixture, which are suppressed by non-glycosylated peptides (Shajahan et al, 2017). Therefore, a capture method for intact glycopeptides before mass spectrometry analysis is highly desirable.…”

Section: Introductionmentioning

confidence: 99%

One-Step Enrichment of Intact Glycopeptides From Glycoengineered Chinese Hamster Ovary Cells

et al. 2020

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Recently, the glycoproteomic analysis of intact glycopeptides has emerged as an effective approach to decipher the glycan modifications of glycoproteins at the site-specific level. A rapid method to enrich intact glycopeptides is essential for the analysis of glycoproteins, especially for biopharmaceutical proteins. In this study, we established a one-step method for the rapid capture of intact glycopeptides for analysis by mass spectrometry. Compared to the conventional sequential enrichment method, the one-step intact glycopeptide enrichment method reduced the sample preparation time and improved the detection of intact glycopeptides with long sequences or non-polar amino acids. Moreover, an increased number of glycosite-containing peptides was identified by the one-step method compared with the sequential method. When we applied this method to the glycoproteomic analysis of glycoengineered Chinese hamster ovary (CHO)-K1 cells with α1,6-fucosyltransferase (FUT8) knockout, the results showed that the knockout of FUT8 altered the overall glycosylation profile of CHO-K1 cells with the elimination of core fucosylation and together with increases in high-mannose and sialylated N-glycans. Interestingly, the knockout of the FUT8 also appeared to regulate the expression of glycoproteins involved in several functions and pathways in CHO-K1 cells, such as the down-regulation of an intracellular lectin LMAN2 showing cellular adaptation to the alterations in FUT8 knockout cells. These findings indicate that the site-specific characterization of glycoproteins from glycoengineered CHO-K1 cells can be achieved rapidly using the one-step intact glycopeptide enrichment method, which could provide insights for bio-analysts and biotechnologists to better tailor therapeutic drugs.

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

Cited by 35 publications

References 48 publications

CRISPR/Cas9 gene editing for the creation of an MGAT1 deficient CHO cell line to control HIV-1 vaccine glycosylation

CRISPR/Cas9 gene editing for the creation of an MGAT1 deficient CHO cell line to control HIV-1 vaccine glycosylation

Glycoproteome Analysis of Human Serum and Brain Tissue

One-Step Enrichment of Intact Glycopeptides From Glycoengineered Chinese Hamster Ovary Cells

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