Automated N-Glycopeptide Identification Using a Combination of Single- and Tandem-MS

Goldberg, David; Bern, Marshall; Parry, Simon; Sutton-Smith, Mark; Panico, Maria; Morris, Howard R.; Dell, Anne

doi:10.1021/pr070239f

Cited by 92 publications

(95 citation statements)

References 23 publications

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“…Software exists that can address some of these issues in the case of N-glycosylation (28,29). Site assignment for Nlinked glycopeptides is aided by the fact that these peptides typically will have a single asparagine residue that matches the NXS/T/C motif.…”

Section: Post-translational Modifications (Ptms)mentioning

confidence: 99%

“…Identification of Additional Glycoforms Using LC-MS DataIt has previously been observed that the individual glycoforms (of any particular glycopeptide bearing a range of carbohydrate structures) generally co-elute on standard RP C18 chromatography (29). Therefore, it is potentially useful to examine the distribution of precursor mass values in the corresponding MS survey scans to assess the extent of microheterogeneity for a given modified peptide sequence.…”

Section: Determining the Distribution Of Detectible Glycan Structures-mentioning

confidence: 99%

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N- and O-Glycosylation in the Murine Synaptosome

Trinidad

Schoepfer²,

Burlingame

et al. 2013

Molecular & Cellular Proteomics

158

223

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We present the first large scale study characterizing both N-and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N-and O-linked carbohydrate structures. Liquid chromatography-MS (LC/ MS) analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N-and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues. Molecular & Cellular

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Section: Post-translational Modifications (Ptms)mentioning

confidence: 99%

Section: Determining the Distribution Of Detectible Glycan Structures-mentioning

confidence: 99%

N- and O-Glycosylation in the Murine Synaptosome

Trinidad

Schoepfer²,

Burlingame

et al. 2013

Molecular & Cellular Proteomics

158

223

View full text Add to dashboard Cite

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“…Up to the 10 most abundant ions (95000 counts) with charge states of 9+2 were sequentially isolated and fragmented via HCD within the HCD collision cell with an activation q=0. 25, an activation time of 40 ms, normalized collision energies of 27%, 35%, and 42% and at a target value of 50,000 ions; fragment ions were mass analyzed in the Orbitrap without the use of a lock mass; m/z selected for MS/MS were dynamically excluded for 45 s. Peak lists were generated using Mascot Daemon/ extract_msn (ver. 2.3; Matrix Science, London, England) using the default parameters.…”

Section: Mass Spectrometrymentioning

confidence: 99%

“…Similar DDA/MS experiments were conducted using collision induced dissociation (CID) with an activation q=0. 25, an activation time of 30 ms, normalized collision energy of 30%, and at a target value of 10,000 ions; fragment ions were mass analyzed in the linear ion trap. Each nano-LC DDA/MS experiment was replicated three times.…”

Section: Mass Spectrometrymentioning

confidence: 99%

“…The use of pronase to achieve nonspecific enzymatic proteolysis can also contribute towards an exposition of oligosaccharide structural and attachment site heterogeneity [18][19][20][21][22][23][24]. To characterize intact glycopeptides on a proteome-wide scale, the steps associated with such analyses must ultimately be addressed using automated, high throughput data analysis workflows such as Peptoonist [25], GlycoMiner [26] and the automated workflows of Joenväärä et al [27,28] (summarized in Scheme 1). Analyses of this breadth dictate that these workflows must be capable of being applied to complex mixtures featuring glycopeptides of low abundance.…”

Section: Introductionmentioning

confidence: 99%

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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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Glycosylation and Disease

Dwek

Markiv

2018

Encyclopedia of Life Sciences

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Glycosylation is the process of attachment of carbohydrates (glycans) to proteins and lipids to form the glycoproteins and glycolipids found in eukaryotic organisms. Glycosylation reactions are amongst the most common posttranslational modifications that have been reported on proteins, and they function to alter protein size, stability, charge and antigenicity. Glycosylation plays an important role in cancer, inherited diseases, pathogen–host interactions and immune recognition. Key Concepts The analysis of glycosylation is more complex than DNA and RNA or protein analysis as glycosylation is a non‐template‐driven event. N‐ linked glycosylation commences in the endoplasmic reticulum and is completed in the Golgi apparatus. O‐ linked glycosylation commences and is completed in the Golgi apparatus. N‐ linked glycosylation occurs on asparagine residues in the consensus sequon Asn‐X‐Ser/Thr. O‐ linked glycosylation occurs on serine and threonine residues. Many enzymes are involved in both N‐ and O‐ linked glycosylation. Glycans play a role in sperm–egg interactions, the implantation process and embryonic development. Congenital disorders in glycosylation result from autosomally dominant mutations in enzymes involved in glycosylation pathways. Glycosylation of proteins influences their half‐life in the serum. N‐ and O‐ linked glycosylation have been reported to be altered in cancer and implicated in all of the steps in the metastatic cascade.

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Automated N-Glycopeptide Identification Using a Combination of Single- and Tandem-MS

Cited by 92 publications

References 23 publications

N- and O-Glycosylation in the Murine Synaptosome

N- and O-Glycosylation in the Murine Synaptosome

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

Glycosylation and Disease

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