Online porous graphic carbon chromatography coupled with tandem mass spectrometry for post‐translational modification analysis

Chen, Rui; Stupak, Jacek; Williamson, Sam; Twine, Susan M.; Li, Jianjun

doi:10.1002/rcm.8459

Cited by 8 publications

(6 citation statements)

References 30 publications

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“…The relative distribution is shown in more detail in Figure S5A. This is consistent with ref , which showed separation of sialic acid isomers using PGC-LC–MS.…”

Section: Resultssupporting

confidence: 89%

Counterion Optimization Dramatically Improves Selectivity for Phosphopeptides and Glycopeptides in Electrostatic Repulsion-Hydrophilic Interaction Chromatography

et al. 2021

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A well-hydrated counterion can selectively and dramatically increase retention of a charged analyte in hydrophilic interaction chromatography. The effect is enhanced if the column is charged, as in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). This combination was exploited in proteomics for the isolation of peptides with certain post-translational modifications (PTMs). The best salt additive examined was magnesium trifluoroacetate. The well-hydrated Mg +2 ion promoted retention of peptides with functional groups that retained negative charge at low pH, while the poorly hydrated trifluoroacetate counterion tuned down the retention due to the basic residues. The result was an enhancement in selectivity ranging from 6-to 66-fold. These conditions were applied to a tryptic digest of mouse cortex. Gradient elution produced fractions enriched in peptides with phosphate, mannose-6-phosphate, and N-and O-linked glycans. The numbers of such peptides identified either equaled or exceeded the numbers afforded by the best alternative methods. This method is a productive and convenient way to isolate peptides simultaneously that contain a number of different PTMs, facilitating study of proteins with "crosstalk" modifications. The fractions from the ERLIC column were desalted prior to C-18-reversed phase liquid chromatography−tandem mass spectrometry analysis. Between 47−100% of the peptides with more than one phosphate or sialyl residue or with a mannose-6 phosphate group were not retained by a C-18 cartridge but were retained by a cartridge of porous graphitic carbon. This finding implies that the abundance of such peptides may have been significantly underestimated in some past studies.

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“…The relative distribution is shown in more detail in Figure S5A. This is consistent with ref , which showed separation of sialic acid isomers using PGC-LC–MS.…”

Section: Resultssupporting

confidence: 89%

Counterion Optimization Dramatically Improves Selectivity for Phosphopeptides and Glycopeptides in Electrostatic Repulsion-Hydrophilic Interaction Chromatography

et al. 2021

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“…The gel was sectioned into 8 slices according to molecular weight and analyzed by LC–MS/MS. Peptides were identified using a combination of Mascot, Sequest and X!Tandem UniProt bovine databases (Chen, Stupak, Williamson, Twine, & Li, ; Han, Maycock, Murray, & Boesch, ; Xie et al, ).…”

Section: Methodsmentioning

confidence: 99%

Safety of bovine milk derived extracellular vesicles used for delivery of RNA therapeutics in zebrafish and mice

Matsuda

Moirangthem

Angom

et al. 2019

J of Applied Toxicology

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Extracellular vesicles are endogenous biological nanoparticles that have potential for use as therapeutic nanoparticles or as delivery vehicles for therapeutic agents.Milk nanovesicles (MNV) are extracellular vesicles isolated from bovine milk that have been explored for use as delivery vehicles for RNA therapeutics such as small interfering RNA (siRNA). We performed in vivo toxicological studies of MNV or therapeutic MNV (tMNV) loaded with siRNA as a prelude to their clinical use.Development toxicity was assessed in zebrafish embryos. Acute toxicity was assessed in both mice and zebrafish whereas safety, biochemical, histological and immune effects after multiple dosing were assessed in mice. Zebrafish embryo hatching was accelerated with MNV and tMNV. While acute toxicity or effects on mortality were not observed in zebrafish, developmental effects were observed at high concentrations of MNV. There was a lack of discernable toxicity, mortality and systemic inflammatory or immunological responses in mice following administration of either MNVs or tMNVs. The tolerability and lack of discernable developmental or systemic in vivo toxicity support their use as biological nanotherapeutics. Adoption of a standardized protocol for systematic analysis of in vivo safety and toxicity will facilitate preclinical assessment of EV based formulations for therapeutic use.

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“…PGC-SPE formats have yet to be widely adopted for enrichment ( 271 , 272 ), likely because complicated retention mechanisms make method optimization challenging and less intuitive. That said, PGC has the attractive potential of chromatographically separating isomeric glycopeptides based on their glycan structures, and several studies have utilized online PGC separations to characterize intact glycopeptides ( 233 , 273 , 274 , 275 , 276 , 277 , 278 , 279 , 280 ). One challenge is that hydrophobic glycopeptides are difficult to elute from PGC, so most studies using online PGC separations have relied on nonspecific proteases to generate small peptide backbones for glycopeptides with limited hydrophobicity.…”

Section: Porous Graphitic Carbonmentioning

confidence: 99%

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

Riley

Bertozzi

Pitteri

2021

Molecular & Cellular Proteomics

144

158

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Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry (MS)-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography (HILIC) and its derivatives, porous graphitic carbon (PGC), reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as MS instrumentation and software improve, so this review aims to help equip researchers with necessary information to choose appropriate enrichment strategies that best complement these efforts.

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Online porous graphic carbon chromatography coupled with tandem mass spectrometry for post‐translational modification analysis

Cited by 8 publications

References 30 publications

Counterion Optimization Dramatically Improves Selectivity for Phosphopeptides and Glycopeptides in Electrostatic Repulsion-Hydrophilic Interaction Chromatography

Counterion Optimization Dramatically Improves Selectivity for Phosphopeptides and Glycopeptides in Electrostatic Repulsion-Hydrophilic Interaction Chromatography

Safety of bovine milk derived extracellular vesicles used for delivery of RNA therapeutics in zebrafish and mice

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

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