When can glycopeptides be assigned based solely on high-resolution mass spectrometry data?

Desaire, Heather; Hua, David

doi:10.1016/j.ijms.2008.12.001

Cited by 37 publications

(49 citation statements)

References 29 publications

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“…Mass Spectrometric Fragmentation Methods-Although choosing a separation strategy that maximizes the MS detection of glycopeptides is a key step in the success of glycopeptide analysis, detecting the m/z values of the glycoforms is typically not sufficient for high confidence analysis (34). Fragmentation data on the glycoforms should be used to support the assignments.…”

Section: Emerging Methods Supporting Glycopeptide Analysismentioning

confidence: 99%

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Glycopeptide Analysis, Recent Developments and Applications

Desaire

2013

Molecular & Cellular Proteomics

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Glycopeptide-based analysis is used to inform researchers about the glycans on one or more proteins. The method's key attractive feature is its ability to link glycosylation information to exact locations (glycosylation sites) on proteins. Numerous applications for glycopeptide analysis are known, and several examples are described herein. The techniques used to characterize glycopeptides are still emerging, and recently, research focused on facilitating aspects of glycopeptide analysis has advanced significantly in the areas of sample preparation, MS fragmentation, and automation of data analysis. These recent developments, described herein, provide the foundation for the growth of glycopeptide analysis as a blossoming field. Molecular & Cellular

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Section: Emerging Methods Supporting Glycopeptide Analysismentioning

confidence: 99%

“…Generally, both MS and MS/MS data are required to provide a high confidence assignment for any glycopeptide composition (34). Unfortunately, only a few tools are currently available to the public that make use of both types of data in one package.…”

Section: Emerging Methods Supporting Glycopeptide Analysismentioning

confidence: 99%

Glycopeptide Analysis, Recent Developments and Applications

Desaire

2013

Molecular & Cellular Proteomics

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“…In undertaking these steps, one approach-utilized by the GlycoMod tool [37] -involves matching theoretical glycopeptide masses, generated for base peptides associated with proteins identified in the sample, against those obtained experimentally. The simplicity of this technique is offset by its potential for high false discovery rates, even when high mass accuracy data is utilized [38]; MS/MS product ion scans are therefore often required to achieve adequate confirmation of glycopeptide matches. During step 2a this can be achieved using specific instrumentation [32,33,39,40] or fragmentation methods [5,[41][42][43][44][45][46]; of particular note is the use of the often intensive peptide+HexNAc (Y1) ion in CID derived MS/MS spectra [47], which can support the identification of base peptide masses [48].…”

Section: Introductionmentioning

confidence: 99%

Detection and Characterization of Low Abundance Glycopeptides Via Higher-Energy C-Trap Dissociation and Orbitrap Mass Analysis

Hart‐Smith

Raftery

2011

J. Am. Soc. Mass Spectrom.

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Broad-scale mass spectrometric analyses of glycopeptides are constrained by the considerable complexity inherent to glycoproteomics, and techniques are still being actively developed to address the associated analytical difficulties. Here we apply Orbitrap mass analysis and higherenergy C-trap dissociation (HCD) to facilitate detailed insights into the compositions and heterogeneity of complex mixtures of low abundance glycopeptides. By generating diagnostic oxonium product ions at mass measurement errors of G5 ppm, highly selective glycopeptide precursor ion detections are made at sub-fmol limits of detection: analyses of proteolytic digests of a hen egg glycoprotein mixture detect 88 previously uncharacterized glycopeptides from 666 precursor ions selected for MS/MS, with only one false positive due to co-fragmentation of a non-glycosylated peptide with a glycopeptide. We also demonstrate that by (1) identifying multiple series of glycoforms using high mass accuracy single stage MS spectra, and (2) performing product ion scans at optimized HCD collision energies, the identification of peptide+ N-acetylhexosamine (HexNAc) ions (Y1 ions) can be readily achieved at G5 ppm mass measurement errors. These data allow base peptide sequences and glycan compositional information to be attained with high confidence, even for glycopeptides that produce weak precursor ion signals and/or low quality MS/MS spectra. The glycopeptides characterized from low fmol abundances using these methods allow two previously unreported glycosylation sites on the Gallus gallus protein ovoglycoprotein (amino acids 82 and 90) to be confirmed; considerable glycan heterogeneities at amino acid 90 of ovoglycoprotein, and amino acids 34 and 77 of Gallus gallus ovomucoid are also revealed.

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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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When can glycopeptides be assigned based solely on high-resolution mass spectrometry data?

Cited by 37 publications

References 29 publications

Glycopeptide Analysis, Recent Developments and Applications

Glycopeptide Analysis, Recent Developments and Applications

Detection and Characterization of Low Abundance Glycopeptides Via Higher-Energy C-Trap Dissociation and Orbitrap Mass Analysis

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

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