Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis

Woo, Christina M.; Iavarone, Anthony T.; Spiciarich, David R.; Palaniappan, Krishnan K.; Bertozzi, Carolyn R.

doi:10.1038/nmeth.3366

Cited by 232 publications

(330 citation statements)

References 48 publications

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“…We believe that this constitutes the first broad proteomic analysis of O-GlcNAc-modified proteins in primary human T cells. Many of the proteins that we identified appeared in prior proteomics studies (55,(76)(77)(78), supporting the validity of our results. However, we also identified novel O-GlcNAc substrates, including ARNT, HCLS1, ZAP-70, SHIP1, LCK, and PI3K.…”

Section: Discussionsupporting

confidence: 87%

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

Lund

Elias

Davis

2016

The Journal of Immunology

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T cell activation in response to Ag is largely regulated by protein posttranslational modifications. Although phosphorylation has been extensively characterized in T cells, much less is known about the glycosylation of serine/threonine residues by O-linked N-acetylglucosamine (O-GlcNAc). Given that O-GlcNAc appears to regulate cell signaling pathways and protein activity similarly to phosphorylation, we performed a comprehensive analysis of O-GlcNAc during T cell activation to address the functional importance of this modification and to identify the modified proteins. Activation of T cells through the TCR resulted in a global elevation of O-GlcNAc levels and in the absence of O-GlcNAc, IL-2 production and proliferation were compromised. T cell activation also led to changes in the relative expression of O-GlcNAc transferase (OGT) isoforms and accumulation of OGT at the immunological synapse of murine T cells. Using a glycoproteomics approach, we identified >200 O-GlcNAc proteins in human T cells. Many of the identified proteins had a functional relationship to RNA metabolism, and consistent with a connection between O-GlcNAc and RNA, inhibition of OGT impaired nascent RNA synthesis upon T cell activation. Overall, our studies provide a global analysis of O-GlcNAc dynamics during T cell activation and the first characterization, to our knowledge, of the O-GlcNAc glycoproteome in human T cells.

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Section: Discussionsupporting

confidence: 87%

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

Lund

Elias

Davis

2016

The Journal of Immunology

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“…Although the majority of these are nuclear and cytoplasmic proteins (Figure 2), 23% were associated with the secretory pathway. GlcNAz is the primary endpoint for Ac 4 GalNAz labeling in cell culture (95% of incorporated label (43)), but identified glycoproteins falling in the secretory category may bear glycans other than O-GlcNAz as Ac 4 GalNAz has the potential to label multiple types of glycans at O-glycosites (e.g., O-GalNAz) (42). While it is rare to observe the Tn antigen in healthy human T cells, it is possible for activated human T cells to display the Tn antigen (47) or for an extracellular glycoprotein to become intracellular through the ERAD pathway.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, for each glycopeptide sample an HCDpdETD scouting run was collected, followed by an inclusion list-driven run with an HCD/ETD/CID duty cycle ( Figure 1C). In previous work on an LTQ-Orbitrap XL, a 4-fold improvement in glycopeptide selection using the inclusion list was found (42). Using an LTQOrbitrap Elite, a HCDpdETD method yielded the most glycopeptide assignments, with the majority of glycopeptide assignments derived from HCD alone (50% for trypsin digests, 70% for chymotrypsin digests).…”

Section: Identification Of Over 2000 O-glcnaz Containing Peptides Frmentioning

confidence: 96%

“…Thus, specialized fragmentation methods to characterize glycopeptides have emerged, including electron transfer dissociation (ETD), higher-energy collision dissociation product dependent ETD (HCDpdETD), and electron transfer/higher-energy collision dissociation (EThcD) (40,41). Recently, a method to enrich metabolically labeled glycopeptides and confidently characterize the intact glycopeptides by MS was developed, termed Isotope Targeted Glycoproteomics (IsoTaG) (42). IsoTaG uses mass-independent MS to improve selection and identification of low abundance glycopeptides.…”

Section: Introductionmentioning

confidence: 99%

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Mapping and Quantification of Over 2000 O-linked Glycopeptides in Activated Human T Cells with Isotope-Targeted Glycoproteomics (Isotag)

Woo

Lund²,

Huang³

et al. 2018

Molecular & Cellular Proteomics

140

172

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Post-translational modifications (PTMs) on proteins often function to regulate signaling cascades, with the activation of T cells during an adaptive immune response being a classic example. Mounting evidence indicates that the modification of proteins by O-linked Nacetylglucosamine (O-GlcNAc), the only mammalian glycan found on nuclear and cytoplasmic proteins, helps regulate T cell activation. Yet, a mechanistic understanding of how O-GlcNAc functions in T cell activation remains elusive, partly because of the difficulties in mapping and quantifying O-GlcNAc sites. Thus, to advance insight into the role of O-GlcNAc in T cell activation, we performed glycosite mapping studies via direct glycopeptide measurement on resting and activated primary human T cells with a technique termed Isotope Targeted Glycoproteomics. This approach led to the identification of 2,219 intact O-linked glycopeptides across 1,045 glycoproteins. A significant proportion (>45%) of the identified O-GlcNAc sites lie in close proximity to or coincide with a known phosphorylation site, supporting the potential for PTM crosstalk. Consistent with other studies, we find that O-GlcNAc sites in T cells lack a strict consensus sequence. To validate our results, we employed gel shift assays based on conjugating mass tags to O-GlcNAc groups. Notably, we observed that the transcription factors c-JUN and JUNB show higher levels of O-GlcNAc glycosylation and higher levels of expression in activated T cells. Overall, our findings provide a quantitative characterization of O-GlcNAc glycoproteins and their corresponding modification sites in primary human T cells, which will facilitate mechanistic studies into the function of O-GlcNAc in T cell activation.

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“…Solid phase hydrazide-based glycopeptide capture has also seen developments (48) and applications (see Table I), but this approach is most commonly used in conjunction with peptide N-glycosidase F-catalyzed release and analysis of formerly N-glycosylated peptides and does not satisfy our definition of glycoproteomics. Other new enrichment methods of interest include the metabolic incorporation of N-azido sugars into N-glycopeptides to facilitate their specific enrichment and detection (84,85). The selective precipitation of glycopeptides by acetone (86), use of size exclusion chromatography (87,88) and the combined use of porous graphitized carbon (PGC) and reversed phase (RP) (89) and titanium dioxide (90) solid phase extraction (SPE) for efficient enrichment of glycopeptides and sialoglycopeptides, respectively, are also promising developments.…”

mentioning

confidence: 99%