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Glycosylation events play an invaluable role in regulating cellular processes including enzymatic activity, immune recognition, protein stability, and cell–cell interactions. However, researchers have yet to realize the full range of glycan mediated biological functions due to a lack of appropriate chemical tools. Fortunately, the past 25 years has...
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... as a barrier for cells from physical or chemical damage. 36 Mucin MCRs have primarily been designed to target the initial GalNAc residue present in all mucin O-linked glycans. The first example came from Hang et al. who synthesized a GalNAc analog functionalized with an azide tag on the N-acetyl position, N-azidoacetylgalactosamine (GalNAz, Fig. 5) and demonstrated that it can label cells. 37 CHO cells were treated ...
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... ppGalNAcT isozymes has remained a major challenge. Recently, a collaboration between the Schumann and Bertozzi groups has started to address this limitation by developing bump-hole sugar-protein pairs. 41-44 Their strategy involves synthesizing GalNAc analogs with extended carbon chains ending in a bioorthogonal group on the N-acetyl position (Fig. 5). The additionally carbons add a ''bump'' that prevents endogenous ppGalNAcTs from turning over the corresponding UDP sugardonor. These reporters were partnered with mutated ppGalNacT enzymes with a complementary ''hole'' in the active site. Initial characterization in vitro proved mutant ppGalNAcT enzymes selective modify peptides ...
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... cellular functions including cell-cell and cell-pathogen interactions as well as being elevated during development. 50 Researchers have exploited the fucose salvage pathway to incorporate chemically reactive groups on cell surface glycans. 4 The first example of a fucose MCR came from the Sawa et al. as an azide-bearing fucose analog, Ac 4 FucAz (Fig. 5). 51 Ac 4 FucAz enters the fucose salvage pathway and is converted to GDPFucAz. From here, GDP-FucAz is transferred onto cell surface fucosylated glycans. Ac 4 FucAz incorporation can be visualized using CuAAC labeling with an alkyne-based fluorescence tag. Concurrently, Rabuka et al. synthesized fucose analogs with azide substitutions ...
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... the fucose salvage pathway and is converted to GDPFucAz. From here, GDP-FucAz is transferred onto cell surface fucosylated glycans. Ac 4 FucAz incorporation can be visualized using CuAAC labeling with an alkyne-based fluorescence tag. Concurrently, Rabuka et al. synthesized fucose analogs with azide substitutions at the C2, C4, and C6 positions (Fig. 5). 52 2-and 4-Azido fucose are not tolerated by the fucose salvage pathway and therefore not metabolically incorporated onto glycans. 6-Azido-fucose was successfully incorporated but showed high levels of cytotoxicity. Hsu et al. addressed this limitation by switching the orientation of the probe and tag, generating FucAlk probes and ...
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... on these findings, Kizuka et al. generated a fucose MCR with an extension off of the C5 position to give 7-alkynylfucose, eliminating these inhibitory affects (Fig. 5). 56 Subsequent analysis showed that FucAlk is selectively incorporated into the core position of N-linked glycans. Recently, Kizuka and coworkers compared the labeling efficiency of FucAlk and 7-alkynyl-fucose (Fig. 5) to learn more about fucose metabolism. 57 A comparison of these reporters across different cell lines showed ...
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... al. generated a fucose MCR with an extension off of the C5 position to give 7-alkynylfucose, eliminating these inhibitory affects (Fig. 5). 56 Subsequent analysis showed that FucAlk is selectively incorporated into the core position of N-linked glycans. Recently, Kizuka and coworkers compared the labeling efficiency of FucAlk and 7-alkynyl-fucose (Fig. 5) to learn more about fucose metabolism. 57 A comparison of these reporters across different cell lines showed differential incorporation that was both cell and protein dependent. Subsequent in vitro assays demonstrated that tolerance of FucTs dictates the labeling efficiency of fucose analogs. Cell lines expressing FucTs with larger ...
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... have been made by our lab and others to develop a selective O-GlcNAc reporters. This is challenging because UDPGlcNAc is a substrate for other glycosyltransferases and is incorporated into both N-linked and O-linked glycoproteins. Hang and Vocadlo et al. developed the first O-GlcNAc reporters termed Ac 4 GlcNAz and Ac 4 GalNAz (Fig. 5). 37,39 These two molecules are C4 epimers that can be interconverted by the enzyme UDP-glucose 4-epimerase (GALE). They synthesized UDP-GlcNAz and all upstream metabolites of the GlcNAc salvage pathway in order to demonstrate that all relevant enzymes including OGT could tolerate the azide analogs in vitro. Additionally, they showed ...
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... et al. improved labeling by generating Ac 4 GlcNAlk, an alkyne substituted reporter that can be labeled with CuAAC (Fig. 5). 38 GlcNAlk showed robust labeling with an improved signal-to-noise ratio compared to GlcNAz. This study further demonstrated that both GlcNAlk and GlcNAz were incorporated and removed as similar rates indicating that GlcNAlk does not affect modification dynamics. Unfortunately, both GlcNAz and GlcNAlk are not specifically incorporated ...
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... an effort to generate an MCR specific for O-GlcNAcylation, we sought to exploit OGT's promiscuity to make more exotic sugar analogs. Inspired by work demonstrating that UDP-6-azido-6-deoxy-GlcNAc was a substrate for OGT in vitro, we synthesized the reporter Ac 3 6AzGlcNAc (Fig. 5). 73,74 Treatment of cells with 6AzGlcNAc showed similar labeling patterns and intensity compared to cells treated with GlcNAz demonstrating that the GlcNAc salvage pathway tolerates modifications made on the C6 position. Enrichment of cell lysate treated with 6AzGlcNAc pulled-down known O-GlcNAc modified proteins NEDD4, pyruvate ...
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... success of Ac 3 6AzGlcNAc lead us to seek to improve the labeling efficiency by generating an alkyne derivative also modified at the C6 position: Ac 3 6AlkGlcNAc (Fig. 5). 75 Fluorescent labeling with 6AlkGlcNAc showed a similar banding pattern and intensity compared to 6AzGlcNAc but with a better signal-to-noise ratio. Cells treated with 6AlkGlcNAc and then submitted to a proteomic workflow identified caspase-3 and caspase-8 as O-GlcNAc modified proteins. Both were confirmed via enrichment and western ...
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... test the breadth of OGT's promiscuity, two additional reporters have been developed; Ac 4 2AzGlc and 4-deoxy-GlcNAz (Ac 3 4dGlcNAz) (Fig. 5). 76,77 Both are successfully turned over by OGT and appear to be selective for O-GlcNAcylation. However, the per-acetylated form of 2AzGlc, Ac 4 2AzGlc, is toxic in cells treated for longer periods of time (200 mM for 16 hours). This was consistent with work published by the Yarema lab showing that per-acetylated versus partially ...
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... that label a fully formed proteoglycan. However, a few reporters that are incorporated as modified core monosaccharides leading to premature truncation of the GAG polysaccharide chain but allowing for downstream tagging. One example comes from Linhardt's lab in the synthesis of UDP sugar-donors of the hexosamine subunits, 4AzGlcNAc and 4AzGalNAc (Fig. 5). 87 Linhardt was able to incorporate these probes onto HS and HA polysaccharide chains in vitro. Unfortunately, azide modifications on the C4 position of hexosamine sugars are not tolerated by salvage pathway enzymes, limiting cellular applications for these MCRs. 88 In a similar approach, the Bertozzi lab generated C4 analogs of ...
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... HA polysaccharide chains in vitro. Unfortunately, azide modifications on the C4 position of hexosamine sugars are not tolerated by salvage pathway enzymes, limiting cellular applications for these MCRs. 88 In a similar approach, the Bertozzi lab generated C4 analogs of xylose by synthesizing the UDP donor-sugar of 4-azido-4-deoxy-xylse (4AzXyl) (Fig. 5). 89 To bypass salvage pathway limitations, UDP-4-AzXyl was directly injected into zebrafish embryos resulting in its incorporation into the core position of CS/DS and HS proteoglycans preventing GAG extension but allowing for tagging and analysis of the abundance and distribution proteoglycans of zebrafish cells at different stages of ...
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... This is achieved by feeding a chemically modified building block (e.g., lipid, amino acid, nucleotide, carbohydrate) to a biological system such as cells or animals. [47][48][49] As many components are distinct in microbial pathogens from eukaryotic cells, substantial progress in host-pathogen interaction studies has been made using metabolic labelling. [50][51][52][53][54][55][56][57][58] In both approaches, chemical probes may directly be modified with a reporter group such as a fluorophore or an affinity handle (e.g., biotin) to enable the analysis of target proteins. ...
With the rapid emergence and the dissemination of microbial resistance to conventional chemotherapy, the shortage of novel antimicrobial drugs has raised a global health threat. As molecular interactions between microbial pathogens and their mammalian hosts are crucial to establish virulence, pathogenicity, and infectivity, a detailed understanding of these interactions has the potential to reveal novel therapeutic targets and treatment strategies. Bidirectional molecular communication between microbes and eukaryotes is essential for both pathogenic and commensal organisms to colonise their host. In particular, several devastating pathogens exploit host signalling to adjust the expression of energetically costly virulent behaviours. Chemical proteomics has emerged as a powerful tool to interrogate the protein interaction partners of small molecules and has been successfully applied to advance host–pathogen communication studies. Here, we present recent significant progress made by this approach and provide a perspective for future studies.
... This approach has been applied to label glycans with fluorescent dyes, which allows us to visualize and track glycans in cells. [9][10][11][12][13][14] Based on MGL methods, various strategies enabling multiple fluorescent labeling in cells have been developed using different sugar reporters and bioorthogonal chemistry. [15][16][17][18][19] Given the biological importance of protein glycosylation in physiological and pathological processes, the large-scale characterization of glycoproteins could expand our understanding of cellular glycosylation processes in both healthy and diseased states. ...
... Indeed, bioorthogonal chemistry in combination with MGL has emerged as a valuable approach for the analysis of glycans in live cells. [9][10][11][12][13][14]35] Typically, bioorthogonal functional groups are metabolically incorporated into target glycoconjugates, allowing covalent conjugation by corresponding bioorthogonal reactions with either fluorescent or affinity tags for subsequent visualization or enrichment, respectively. However, these existing methods remain challenging for simultaneous site-specific labeling of sialylated glycoproteins and direct visualization in live cells, followed by simple enrichment and site-specific identification of intact glycoproteins in the same experiment for spatial glycoproteomes. ...
Multicolor labeling for monitoring the intracellular localization of the same target type in the native environment using chemical fluorescent dyes is a challenging task. This approach requires both bioorthogonal and biocompatible ligations with an excellent fluorescence signal‐to‐noise ratio. Here, we present a metabolic glycan labeling technique that uses homemade fluorogenic dyes to investigate glycosylation patterns in live cells. These dyes allowed us to demonstrate rapid and efficient simultaneous multilabeling of glycoconjugates with minimum fluorescence noise. Our results demonstrate that this approach is capable of not only probing sialylation and GlcNAcylation in cells but also specifically labeling the cell‐surface and intracellular sialylated glycoconjugates in live cells. In particular, we performed site‐specific dual‐channel fluorescence imaging of extra and intracellular sialylated glycans in HeLa and PC9 cancer cells as well as identified fluorescently labeled sialylated glycoproteins and glycans by a direct enrichment approach combined with an MS‐based proteomic analysis in the same experiment. In conclusion, this study provides multilabeling tools in cellular systems for simultaneous site‐specific glycan imaging and glycoproteomic analysis to study potential cancer‐ and disease‐associated glycoconjugates.
... Generally, the released acetyl groups have no significant effect on the cells when using SAM concentrations up to 100 mM. 12 Note that non-acetylated SAM can be applied for metabolic engineering in some bacteria that express active sialic acid uptake mechanisms. 13 Note: Most SAMs have reversible effects that dilute with cell division and competing endogenous sialic acid biosynthesis. ...
Mammalian glycans show a diversity in sialic acid capping, constituting the sialome. Sialic acids can be extensively modified chemically, yielding sialic acid mimetics (SAMs). Here, we present a protocol for detecting and quantifying incorporative SAMs using microscopy and flow cytometry, respectively. We detail steps for linking SAMS to proteins with western blotting. Lastly, we detail procedures for incorporative or inhibitory SAMs and how SAMs can be used for the on-cell synthesis of high-affinity Siglec ligands.
For complete details on the use and execution of this protocol, please refer to Büll et al.¹ and Moons et al.²
... Such compounds are converted into glycoenzyme substrates within the cell, and metabolically incorporated into newlysynthesized glycoconjugates. Subsequent ligation on the reporter groups via the Nobel Prize-winning bioorthogonal reaction enables the dynamic and versatile glycan visualization, as well as glycoconjugate isolation and -omic analysis (Fig. 1a) 3,4 . So far, derivatizations on N-acetylmannosamine (ManNAc), sialic acid (Sia), Nacetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and fucose (Fuc), with or without acetylation on hydroxyl groups for better plasma membrane diffusion, has been realized as the MOE probes 5 . ...
Metabolic oligosaccharide engineering (MOE) is a classical chemical approach to perturb, profile and perceive glycans in physiological systems, but probes upon bioorthogonal reaction require accessibility and background signal readout makes it challenging to achieve absolute glycan quantification. Here we develop SeMOE, a selenosugar-based metabolic oligosaccharide engineering strategy that combines elemental analysis and MOE to enable the absolute quantification and mass spectrometric imaging of glycome in a concise procedure. We demonstrate that SeMOE probes allow for perception, absolute quantification and visualization of glycans in diverse biological contexts. We demonstrate that chemical reporters on conventional MOE can be integrated into a bifunctional SeMOE probe to provide multimodality signal readouts. We further show the anti-cancer potentiality of SeMOE probes. SeMOE thus provides a convenient and simplified method to "see more" of the glyco-world.
... However, due to the use of specialized equipment and complex decoupling of signals, real-time detection of glycans remains an attractive yet still unmet need in bioprocessing. Here, we point to the opportunity to resolve this gap using a modification of existing lectin-detection strategies [43] as an alternative for online sensing or the implementation of metabolically labeled substrates [59] as possible paths toward in situ biosensing. ...
Proteins continue to represent a large fraction of the therapeutics market, reaching over a hundred billion dollars in market size globally. One key feature of protein modification that can affect both structure and function is the addition of glycosylation following protein folding, leading to regulatory requirements for the accurate assessment of protein attributes, including glycan structures. The non-template-driven, innately heterogeneous N-glycosylation process thus requires accurate detection to robustly generate protein therapies. A challenge exists in the timely detection of protein glycosylation without labor-intensive manipulation. In this article, we discuss progress toward N-glycoprotein control, focusing on novel control strategies and the advancement of rapid, high-throughput analysis methods.
... [29] This metabolic labeling approach results in the installation of azido-or alkynyl O-GlcNAc analogs onto native substrates. [30] Then, O-GlcNAc substrates can be covalently labeled with a useful probe (e. g., an affinity handle for enrichment or a fluorophore for imaging) by a bioorthogonal "click" reaction, such as copper-catalyzed [3 + 2] azide-alkyne cycloaddition [31] or strain-promoted azide-cyclooctyne cycloaddition (Figure 2A). ...
O‐linked β‐N‐acetylglucosamine (O−GlcNAc) is a ubiquitous post‐translational modification in mammals, decorating thousands of intracellular proteins. O−GlcNAc cycling is an essential regulator of myriad aspects of cell physiology and is dysregulated in numerous human diseases. Notably, O−GlcNAcylation is abundant in the brain and numerous studies have linked aberrant O−GlcNAc signaling to various neurological conditions. However, the complexity of the nervous system and the dynamic nature of protein O−GlcNAcylation have presented challenges for studying of neuronal O−GlcNAcylation. In this context, chemical approaches have been a particularly valuable complement to conventional cellular, biochemical, and genetic methods to understand O−GlcNAc signaling and to develop future therapeutics. Here we review selected recent examples of how chemical tools have empowered efforts to understand and rationally manipulate O−GlcNAcylation in mammalian neurobiology.
... 4−10 Frequently, an azide is substituted in place of a hydroxyl group as in the commonly employed 9-azido-9-deoxy sialic acid (1, or its azido-ManNAc precursor) and as in the 6-azido-2-fluoro-2,6dideoxy-β-galactosyl fluoride (2) used to tag β-galactosidases in proteomes. 7,9,11 In other cases, it may replace a hydrogen atom, as in 6-azidofucose derivatives (3) and 5-azidoacetamido-sialic acid (4). 12 Studies of such types have provided useful insights in many cases, but care must be taken in the choice of position of substitution and in the interpretation of resultant data since the substitution is far from isosteric or isoelectronic. ...
Azido sugars have found frequent use as probes of biological systems in approaches ranging from cell surface metabolic labeling to activity-based proteomic profiling of glycosidases. However, little attention is typically paid to how well azide-substituted sugars represent the parent molecule, despite the substantial difference in size and structure of an azide compared to a hydroxyl. To quantitatively assess how well azides are accommodated, we have used glycosidases as tractable model enzyme systems reflecting what would also be expected for glycosyltransferases and other sugar binding/modifying proteins. In this vein, specificity constants have been measured for the hydrolysis of a series of azidodeoxy glucosides and N-acetylhexosaminides by a large number of glycosidases produced from expressed synthetic gene and metagenomic libraries. Azides at secondary carbons are not significantly accommodated, and thus, associated substrates are not processed, while those at primary carbons are productively recognized by only a small subset of the enzymes and often then only very poorly. Accordingly, in the absence of careful controls, results obtained with azide-modified sugars may not be representative of the situation with the natural sugar and should be interpreted with considerable caution. Azide incorporation can indeed provide a useful tool to monitor and detect glycosylation, but careful consideration should go into the selection of sites of azide substitution; such studies should not be used to quantitate glycosylation or to infer the absence of glycosylation activity.
... 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). ...
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.
... Metabolic chemical reporters (MCRs) of protein glycosylation are powerful chemical tools that have been used for over a decade to identify and characterize different types of glycans ( Figure 1a). 1,2 MCRs are typically analogues of naturally occurring monosaccharides that contain bio-orthogonal functionalities at different positions of the sugar ring. If these chemical modifications are relatively small, MCRs can take advantage of carbohydrate salvage pathway enzymes with different levels of substrate tolerance to yield the corresponding nucleotide sugar donors for subsequent transfer onto proteins by glycosyltransferases. ...
Bio-orthogonal chemistries have revolutionized many fields. For example, metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain a bio-orthogonal functionality, such as azides or alkynes. MCRs are metabolically incorporated into glycoproteins by living systems, and bio-orthogonal reactions can be subsequently employed to install visualization and enrichment tags. Unfortunately, most MCRs are not selective for one class of glycosylation (e.g., N-linked vs O-linked), complicating the types of information that can be gleaned. We and others have successfully created MCRs that are selective for intracellular O-GlcNAc modification by altering the structure of the MCR and thus biasing it to certain metabolic pathways and/or O-GlcNAc transferase (OGT). Here, we attempt to do the same for the core GalNAc residue of mucin O-linked glycosylation. The most widely applied MCR for mucin O-linked glycosylation, GalNAz, can be enzymatically epimerized at the 4-hydroxyl to give GlcNAz. This results in a mixture of cell-surface and O-GlcNAc labeling. We reasoned that replacing the 4-hydroxyl of GalNAz with a fluorine would lock the stereochemistry of this position in place, causing the MCR to be more selective. After synthesis, we found that 4FGalNAz labels a variety of proteins in mammalian cells and does not perturb endogenous glycosylation pathways unlike 4FGalNAc. However, through subsequent proteomic and biochemical characterization, we found that 4FGalNAz does not widely label cell-surface glycoproteins but instead is primarily a substrate for OGT. Although these results are somewhat unexpected, they once again highlight the large substrate flexibility of OGT, with interesting and important implications for intracellular protein modification by a potential range of abiotic and native monosaccharides.
... 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 toward the environment in the living cell but reactive toward 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. ...
... Tagged monosaccharides are rendered cell-permeable by derivatizing polar groups such as hydrophobic esters and thioesters. After cell entry and removal of esters by cytosolic esterases, tagged monosaccharides are metabolically activated into nucleotide-sugars that can be used by glycosyltransferases. 23,24,26,49 This process incorporates chemical tags into the glycoproteome of a living cell, allowing for the introduction of enrichment reagents such as biotin by CuAAC. 27 The ensuing enrichment, tryptic digest, and glycoproteomic analysis have enabled the assignment of glycosylation sites and glycan structures but are still hampered by ion suppression in the presence of nonglycosylated peptides. ...
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 analogues 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 analogue glycopeptides. We characterized the mass spectrometric signatures of these modified glycopeptides and their signature residues left by bioorthogonal resporter 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.
