Comparison of HPLC/ESI-FTICR MS versus MALDI-TOF/TOF MS for glycopeptide analysis of a highly glycosylated HIV envelope glycoprotein

Irungu, Janet; Go, Eden P.; Zhang, Ying; Dalpathado, Dilusha S.; Liao, Hua‐Xin; Haynes, Barton F.; Desaire, Heather

doi:10.1016/j.jasms.2008.05.010

Cited by 67 publications

(73 citation statements)

References 35 publications

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“…[21][22][23][24][25][26][27][28][29][30][31][32][33] However, less attention has been paid to the study of glycosylation during and after treatment. [34][35][36] In this regard, our present work is aimed to MS investigation of N-glycans in human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells vs oligosaccharide profiles obtained from cells treated by ex-vivo assay with Herceptin alone or combined with Lipofectamine (LipA) and plasmid DNA.…”

Section: Introductionmentioning

confidence: 99%

N-Glycomic Changes in Human Breast Carcinoma MCF-7 and T-Lymphoblastoid Cells After Treatment with Herceptin and Herceptin/Lipoplex

et al. 2010

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The humanized monoclonal antibody IgG1 in combination with chemotherapy has been demonstrated to enhance survival benefit in cancer treatment. Despite positive outcomes, some cancer cells develop multidrug resistance. Numerous mechanisms in cancers can be involved in the process of treatment therapy and most of them are not still well understood. To address how the carbohydrate moieties of cells are affected during treatment, the glycan profiles from the two most common cancer cell lines s human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells s were studied here and compared with profiles after treatment with Herceptin alone or in combination with Lipofectamine mixed with plasmid DNA to form Lipoplex. N-Glycans were released from total cells by digestion with PNGaseF and analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In summary, both original cell lines showed a dominant occurrence of high-mannose glycans. After treatment, these structures were suppressed and biantennary core-fucosylated glycans originating from IgG1 were the major carbohydrate products identified in cells. The high incidence of additional fucosylated or nonfucosylated galactosylated oligosaccharides, which were not detected in original cells or Herceptin, varied with conditions and time of exposure of cells to the antibody. The results presented in this study provide strong evidence for a role of glycosylation during antibody treatment.

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Section: Introductionmentioning

confidence: 99%

N-Glycomic Changes in Human Breast Carcinoma MCF-7 and T-Lymphoblastoid Cells After Treatment with Herceptin and Herceptin/Lipoplex

et al. 2010

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“…A typical analysis employs a bottom-up proteomics approach wherein glycoproteins are enzymatically digested using a specific or nonspecific protease to generate the peptide/glycopeptide mixture that is subsequently analyzed by a mass spectrometry (MS) platform in tandem with the chromatographic separation of choice (2,11,14,18,29,48,51,66). Following data acquisition, all possible glycopeptides are identified by peaks that are separated by monosaccharide units and/or by peaks of characteristic glycopeptide-marker ions in the MS 1 and tandem-MS (MS/MS) data (9,21,66).…”

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confidence: 99%

Characterization of Glycosylation Profiles of HIV-1 Transmitted/Founder Envelopes by Mass Spectrometry

Hewawasam

Liao

et al. 2011

J Virol

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116

150

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The analysis of HIV-1 envelope carbohydrates is critical to understanding their roles in HIV-1 transmission as well as in binding of envelope to HIV-1 antibodies. However, direct analysis of protein glycosylation by glycopeptide-based mass mapping approaches involves structural simplification of proteins with the use of a protease followed by an isolation and/or enrichment step before mass analysis. The successful completion of glycosylation analysis is still a major analytical challenge due to the complexity of samples, wide dynamic range of glycopeptide concentrations, and glycosylation heterogeneity. Here, we use a novel experimental workflow that includes an up-front complete or partial enzymatic deglycosylation step before trypsin digestion to characterize the glycosylation patterns and maximize the glycosylation coverage of two recombinant HIV-1 transmitted/founder envelope oligomers derived from clade B and C viruses isolated from acute infection and expressed in 293T cells. Our results show that both transmitted/founder Envs had similar degrees of glycosylation site occupancy as well as similar glycan profiles. Compared to 293T-derived recombinant Envs from viruses isolated from chronic HIV-1, transmitted/founder Envs displayed marked differences in their glycosylation site occupancies and in their amounts of complex glycans. Our analysis reveals that the glycosylation patterns of transmitted/founder Envs from two different clades (B and C) are more similar to each other than they are to the glycosylation patterns of chronic HIV-1 Envs derived from their own clades.The systematic mapping and characterization of protein glycosylation provide a wealth of molecular information that is crucial for understanding a wide variety of biochemical and cellular processes. However, comprehensive analysis of protein glycosylation has proven to be difficult due to the wide dynamic range of glycopeptide concentrations and immense structural diversity of glycans. Glycan modifications on proteins undergo a series of glycan processing steps from the endoplasmic reticulum (ER) to the Golgi apparatus with a diverse array of glycan processing enzymes that compete for available substrate, resulting in multiple glycosylation patterns for each glycosylation site for a given protein and variation in glycosylation site occupancy (35,52,53). Moreover, protein glycosylation varies significantly across different cell types, cell states, tissues, and organisms (3,12,16,34,35,53,56,62). Despite these challenges, recent advances in proteomics have accelerated the pace of the development of efficient methods and technologies that can be tailored for the analysis of protein glycosylation (6,51,66). To date, the analysis of protein glycosylation by mass spectrometry (MS) is underpinned by an array of sample preparation methods that include affinity/enrichment schemes (6, 18, 26), modern chromatographic methods (31,50,54), and remarkable improvements in mass spectrometry instrumentation (47,48,51). When used effectively, these methods provi...

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“…As glycoproteomics studies have not approached the scale of proteomics studies, several distinct studies were needed to generate a sufficient number of glycopeptides to test GRAEZ classifications. These studies examined a variety of different samples, including glycoprotein standards (19), fetal bovine serum (20), human urine (21), (24), hepatitis C glycoprotein (25), HIV envelope glycoprotein gp140 (26), and human IgG subclasses (27). In total, 624 nonredundant, intact tryptic glycopeptides were identified in these studies within the mass range of 1500 -5000 daltons.…”

Section: Resultsmentioning

confidence: 99%

A Classifier Based on Accurate Mass Measurements to Aid Large Scale, Unbiased Glycoproteomics

Froehlich

Dodds

Wilhelm

et al. 2013

Molecular & Cellular Proteomics

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Determining which glycan moieties occupy specific N-glycosylation sites is a highly challenging analytical task. Arguably, the most common approach involves LC-MS and LC-MS/MS analysis of glycopeptides generated by proteases with high cleavage site specificity; however, the depth achieved by this approach is modest. Nonglycosylated peptides are a major challenge to glycoproteomics, as they are preferentially selected for data-dependent MS/MS due to higher ionization efficiencies and higher stoichiometric levels in moderately complex samples. With the goal of improving glycopeptide coverage, a mass defect classifier was developed that discriminates between peptides and glycopeptides in complex mixtures based on accurate mass measurements of precursor peaks. By using the classifier, glycopeptides that were not fragmented in an initial data-dependent acquisition run may be targeted in a subsequent analysis without any prior knowledge of the glycan or protein species present in the mixture. Additionally, from probable glycopeptides that were poorly fragmented, tandem mass spectra may be reacquired using optimal glycopeptide settings. We demonstrate high sensitivity (0.892) and specificity (0.947) based on an in silico dataset spanning >100,000 tryptic entries. Comparable results were obtained using chymotryptic species. Further validation using published data and a fractionated tryptic digest of human urinary proteins was performed, yielding a sensitivity of 0.90 and a specificity of 0.93. Lists of glycopeptides may be generated from an initial proteomics experiment, and we show they may be efficiently targeted using the classifier. Considering the growing availability of high accuracy mass analyzers, this approach represents a simple and broadly applicable means of increasing the depth of MS/MS-based glycoproteomic analyses. Molecular &

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Comparison of HPLC/ESI-FTICR MS versus MALDI-TOF/TOF MS for glycopeptide analysis of a highly glycosylated HIV envelope glycoprotein

Cited by 67 publications

References 35 publications

N-Glycomic Changes in Human Breast Carcinoma MCF-7 and T-Lymphoblastoid Cells After Treatment with Herceptin and Herceptin/Lipoplex

N-Glycomic Changes in Human Breast Carcinoma MCF-7 and T-Lymphoblastoid Cells After Treatment with Herceptin and Herceptin/Lipoplex

Characterization of Glycosylation Profiles of HIV-1 Transmitted/Founder Envelopes by Mass Spectrometry

A Classifier Based on Accurate Mass Measurements to Aid Large Scale, Unbiased Glycoproteomics

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