Design and synthesis of metabolic chemical reporters for the visualization and identification of glycoproteins

Pedowitz, Nichole J.; Pratt, Matthew R.

doi:10.1039/d1cb00010a

Cited by 35 publications

(38 citation statements)

References 124 publications

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“…4,22 Metabolic oligosaccharide engineering (MOE), a recent addition to the field of intact O-glycoproteomics, is emerging as an important strategy to profile mucin-type O-glycans. 23,24 In the MOE strategy, monosaccharides are chemically modified with tags that are stable towards the environment in the living cell, but reactive towards methods of bioorthogonal (or "click") chemistry such as copper-mediated azide-alkyne cycloaddition (CuAAC). 25 A collection of bioorthogonal GalNAc-based monosaccharides relevant to this work is shown in Figure 1A.…”

Section: Introductionmentioning

confidence: 99%

Benefits of Chemical Sugar Modifications Introduced by Click Chemistry for Glycoproteomic Analyses

Calle

Bineva-Todd

Marchesi

et al. 2021

J. Am. Soc. Mass Spectrom.

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Mucin-type O-glycosylation is among the most complex post-translational modifications. Despite mediating many physiological processes, O-glycosylation remains understudied compared to other modifications, simply because the right analytical tools are lacking. In particular, analysis of intact O-glycopeptides by mass spectrometry is challenging for several reasons; O-glycosylation lacks a consensus motif, glycopeptides have low charge density which impairs ETD fragmentation, and the glycan structures modifying the peptides are unpredictable. Recently, we introduced chemically modified monosaccharide analogs that allowed selective tracking and characterization of mucin-type O-glycans after bioorthogonal derivatization with biotin-based enrichment handles. In doing so, we realized that the chemical modifications used in these studies have additional benefits that allow for improved analysis by tandem mass spectrometry. In this work, we built on this discovery by generating a series of new GalNAc analog glycopeptides. We characterized the mass spectrometric signatures of these modified glycopeptides and their MOE signature residues left by bioorthogonal enrichment reagents. Our data indicate that chemical methods for glycopeptide profiling offer opportunities to optimize attributes such as increased charge state, higher charge density, and predictable fragmentation behavior.

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Section: Introductionmentioning

confidence: 99%

Benefits of Chemical Sugar Modifications Introduced by Click Chemistry for Glycoproteomic Analyses

Calle

Bineva-Todd

Marchesi

et al. 2021

J. Am. Soc. Mass Spectrom.

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“…To assess the utility of our optimized workflow for other PTMs, we used the previously described Ac34dGlcNAz probe (GlcNAz) for the pull-down of O-linked β-N-acetyl-glucosamine glycosylated (O-GlcNAcylation) proteins (Fig 5A) (Yang & Qian, 2017;Laughlin & Bertozzi, 2007;Li et al, 2016;Pedowitz & Pratt, 2021). Numerous metabolic labels have been developed for characterisation of O-GlcNAcylated proteins (Li et al, 2016).…”

Section: Application Of the Sp2e Workflow For Analysis Of Protein O-glcnacylationmentioning

confidence: 99%

Transforming chemical proteomics enrichment into high-throughput method using SP2E workflow

Becker

Wiest

Telek

et al. 2022

Preprint

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Protein post-translational modifications (PTMs) play a critical role in regulation of protein catalytic activity, localization and protein-protein interactions. An attachment of PTMs onto proteins significantly diversifies their structure and function resulting in so-called proteoforms. However, the sole identification of post-translationally modified proteins, which are often cell type and disease specific, is still a highly challenging task. Sub-stoichiometric amounts and modification of low abundant proteins necessitate purification or enrichment of the modified proteins. Although the introduction of the mass spectrometry-based chemical proteomic strategy has enabled to screen protein PTMs with increased throughput, sample preparation has remained highly time consuming and tedious. Here, we report an optimized workflow for enrichment of PTM proteins in 96-well plate format which can be possible extended to robotic automatization. This platform allows to significantly lower the input of total protein, which opens up the opportunity to screen specialized and difficult to culture cell lines in high-throughput manner. The presented SP2E protocol is robust, time- and cost-effective as well as suitable for large-scale screening of proteoforms. Application of the SP2E protocol will thus enable the characterization of proteoforms in various processes such as neurodevelopment, neurodegeneration and cancer and may contribute to an overall acceleration of the recently launched Human Proteoform Project.

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“…Following these initial reports, the scope of MOE has expanded to include derivatives of a wide variety of different monosaccharides and diverse types of bioorthogonal tags, which cannot all be covered here. Instead, for comprehensive overviews of the various chemical reporters and bioorthogonal chemistries that have been developed to date, we direct the readers to recent reviews [8,9,[15][16][17][18][19][20].…”

Section: Metabolic Oligosaccharide Engineeringmentioning

confidence: 99%

“…While these methods are still used today, they are limited to the labelling of sialic acid and galactose-or GalNAc-containing glycans and offer little flexibility in the choice of labelling chemistry. Over the past decades, various chemical reporters have been developed that enable the labelling of endogenous glycans through metabolic and chemoenzymatic glycan engineering strategies [14][15][16][17][18][19]. In these approaches, termed metabolic oligosaccharide engineering (MOE, Figure 1B) and chemoenzymatic glycan labelling (CeGL, Figure 1C), unnatural carbohydrate derivatives are incorporated into glycans by the cell's own metabolic machinery or by the action of recombinant glycosyltransferases, respectively.…”

Section: Introductionmentioning

confidence: 99%

Chemical reporters to study mammalian O-glycosylation

Huxley

Willems

2021

Biochemical Society Transactions

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Glycans play essential roles in a range of cellular processes and have been shown to contribute to various pathologies. The diversity and dynamic nature of glycan structures and the complexities of glycan biosynthetic pathways make it challenging to study the roles of specific glycans in normal cellular function and disease. Chemical reporters have emerged as powerful tools to characterise glycan structures and monitor dynamic changes in glycan levels in a native context. A variety of tags can be introduced onto specific monosaccharides via the chemical modification of endogenous glycan structures or by metabolic or enzymatic incorporation of unnatural monosaccharides into cellular glycans. These chemical reporter strategies offer unique opportunities to study and manipulate glycan functions in living cells or whole organisms. In this review, we discuss recent advances in metabolic oligosaccharide engineering and chemoenzymatic glycan labelling, focusing on their application to the study of mammalian O-linked glycans. We describe current barriers to achieving glycan labelling specificity and highlight innovations that have started to pave the way to overcome these challenges.

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Design and synthesis of metabolic chemical reporters for the visualization and identification of glycoproteins

Abstract: This review details a brief history of the synthesis and characterization of metabolic chemical reporters used to study glycosylation before describing recent applications and finishing with considerations and limitations of reporter molecules.

Cited by 35 publications

References 124 publications

Benefits of Chemical Sugar Modifications Introduced by Click Chemistry for Glycoproteomic Analyses

Benefits of Chemical Sugar Modifications Introduced by Click Chemistry for Glycoproteomic Analyses

Transforming chemical proteomics enrichment into high-throughput method using SP2E workflow

Chemical reporters to study mammalian O-glycosylation

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