Parallel Determination of Polypeptide and Oligosaccharide Connectivities by Energy-Resolved Collison-Induced Dissociation of Protonated O-Glycopeptides Derived from Nonspecific Proteolysis

Kelly, Maia I.; Dodds, Eric D.

doi:10.1021/jasms.9b00065

Cited by 12 publications

(15 citation statements)

References 59 publications

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“…Tandem mass spectrometry (MS) is the premier method for O-glycosite mapping, but O-glycopeptides are largely intractable using standard glycoproteomic approaches, which have focused almost entirely on N-glycans. To this end, recent efforts have focused on improving O-glycopeptide analyses, [7][8][9][10] ranging from developments in sample preparation, [11][12][13][14][15] data acquisition, [16][17][18][19][20][21] and post-acquisition data analysis. [22][23][24][25][26][27] One promising avenue to emerge from these investigations is the description of O-glycoproteases that can generate O-glycopeptides more amenable to MS characterization.…”

mentioning

confidence: 99%

Electron-Based Dissociation Is Needed for O-Glycopeptides Derived from OpeRATOR Proteolysis

2020

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The recently described O-glycoprotease OpeRATOR presents exciting opportunities for O-glycoproteomics. This bacterial enzyme purified from Akkermansia muciniphila cleaves N-terminally to serine and threonine residues that are modified with (preferably asialylated) O-glycans. This provides orthogonal cleavage relative to canonical proteases (e.g., trypsin) for improved O-glycopeptide characterization with tandem mass spectrometry (MS/MS). O-glycopeptides with a modified N-terminal residue, such as those generated by OpeRATOR, present several potential benefits, perhaps the most notable being de facto O-glycosite localization without the need of glycan-retaining fragments in MS/MS spectra. Indeed, O-glycopeptides modified exclusively at the N-terminus would enable O-glycoproteomic methods to rely solely on collision-based fragmentation rather than electron-driven dissociation because glycan-retaining peptide fragments would not be required for localization. The caveat is that modified peptides would need to reliably contain only a single O-glycosite.Here we use methods that combine collision-and electron-based fragmentation to characterize the number of O-glycosites that are present in O-glycopeptides derived from OpeRATOR digestion of four known O-glycoproteins. Our data show that over 50% of O-glycopeptides generated from combined digestion using OpeRATOR and trypsin contain multiple O-glycosites, indicating that collision-based fragmentation alone is not sufficient. Electron-based dissociation methods are necessary to capture the O-glycopeptide diversity present in OpeRATOR digestions.

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confidence: 99%

Electron-Based Dissociation Is Needed for O-Glycopeptides Derived from OpeRATOR Proteolysis

2020

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“…Numerous alternative strategies enrich glycopeptides from whole cell or tissue lysate, but such approaches do not provide evidence for subcellular localization akin to that provided by the cell surface glycoprotein focused methods [ 55 , 66 , 108 , 110 , 113 , 118 , 120 ]. Methods for intact glycopeptide analysis by MS enable determination of monosaccharide compositions present at specific sites on a protein, but do not typically generate sufficient information to fully characterize glycan structures or structural isomers [ 36 , 43 , 44 , 50 , 86 , 94 , 97 ].…”

Section: Expanding the Assessment Of Protein Glycosylation In Hpsc-cmmentioning

confidence: 99%

Importance of evaluating protein glycosylation in pluripotent stem cell-derived cardiomyocytes for research and clinical applications

Kelly

Albahrani

Castro

et al. 2021

Pflugers Arch - Eur J Physiol

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Proper protein glycosylation is critical to normal cardiomyocyte physiology. Aberrant glycosylation can alter protein localization, structure, drug interactions, and cellular function. The in vitro differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CM) has become increasingly important to the study of protein function and to the fields of cardiac disease modeling, drug testing, drug discovery, and regenerative medicine. Here, we offer our perspective on the importance of protein glycosylation in hPSC-CM. Protein glycosylation is dynamic in hPSC-CM, but the timing and extent of glycosylation are still poorly defined. We provide new data highlighting how observed changes in hPSC-CM glycosylation may be caused by underlying differences in the protein or transcript abundance of enzymes involved in building and trimming the glycan structures or glycoprotein gene products. We also provide evidence that alternative splicing results in altered sites of glycosylation within the protein sequence. Our findings suggest the need to precisely define protein glycosylation events that may have a critical impact on the function and maturation state of hPSC-CM. Finally, we provide an overview of analytical strategies available for studying protein glycosylation and identify opportunities for the development of new bioinformatic approaches to integrate diverse protein glycosylation data types. We predict that these tools will promote the accurate assessment of protein glycosylation in future studies of hPSC-CM that will ultimately be of significant experimental and clinical benefit.

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“…This supports a recent report by Kelly and Dodds, where they found that O-glycopeptides require lower collision energies for precursor depletion for a small pool of O-glycopeptides. 88 Although some increase in Figures S14-S16 provide distributions of precursor peptide lengths, m/z values, and charge state distributions of identified N-and O-glycoPSMs for each method. As expected, EThcD methods extend the m/z range of ETD for successfully identified glycopeptides, making their distributions more similar to HCD and EThcD methods.…”

Section: Comparisons Between N-and O-glycopeptide Datamentioning

confidence: 99%

“…In 2017, Liu et al used sceHCD methods for identification of ~10,000 N-glycosites from five mouse tissues, 78 which contributed to its popularity in recent N-glycopeptide analysis. [79][80][81][82][83][84][85][86] A few studies have looked to extend the application of sceHCD to O-glycopeptides, 75,76,79,87,88 but this has not been as widespread. Limited comparisons between sceHCD and ETD methods have been performed for N-glycopeptides, 78 as have comparisons of HCD and EThcD for Oglycopeptides; 64,69 however, a comprehensive head-to-head comparison of standard HCD, sceHCD, ETD, and EThcD has not been reported.…”

Section: Introductionmentioning

confidence: 99%

Optimal Dissociation Methods Differ for N- and O-glycopeptides

Riley

Malaker²,

Drießen³

et al. 2020

Preprint

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<p><a>Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods, indicating that ETD-based methods are not required for routine N-glycoproteomics. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.</a></p>

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Parallel Determination of Polypeptide and Oligosaccharide Connectivities by Energy-Resolved Collison-Induced Dissociation of Protonated O-Glycopeptides Derived from Nonspecific Proteolysis

Cited by 12 publications

References 59 publications

Electron-Based Dissociation Is Needed for O-Glycopeptides Derived from OpeRATOR Proteolysis

Electron-Based Dissociation Is Needed for O-Glycopeptides Derived from OpeRATOR Proteolysis

Importance of evaluating protein glycosylation in pluripotent stem cell-derived cardiomyocytes for research and clinical applications

Optimal Dissociation Methods Differ for N- and O-glycopeptides

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