Isotope Targeted Glycoproteomics (IsoTaG) to Characterize Intact, Metabolically Labeled Glycopeptides from Complex Proteomes

Woo, Christina M.; Bertozzi, Carolyn R.

doi:10.1002/9780470559277.ch150185

Cited by 15 publications

(29 citation statements)

References 33 publications

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“…The conditioned media were collected and reacted with azide probe 2 by CuAAC. For comparison, PC-3 cells were labeled with 100 µM Ac 4 ManNAz to produce the corresponding azido sialic acid (SiaNAz), and the conditioned media was reacted with alkyne biotin probe 1 [45,50]. Western blot analysis showed similar tagging and enrichment of conditioned media from cells treated with Ac 4 ManNAl or Ac 4 ManNAz, with little off-target tagging from cells treated with the DMSO vehicle (Figure 2A and ESM Figure S3).…”

Section: Resultsmentioning

confidence: 99%

Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars

et al. 2016

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Protein glycosylation is a post-translational modification (PTM) responsible for many aspects of proteomic diversity and biological regulation. Assignment of intact glycan structures to specific protein attachment sites is a critical step towards elucidating the function encoded in the glycome. Previously, we developed isotope targeted glycoproteomics (IsoTaG) as a mass-independent mass spectrometry method to characterize azide-labeled intact glycopeptides from complex proteomes. Here we extend the IsoTaG approach with the use of alkynyl sugars as metabolic labels, and employ new probes in analysis of the sialylated glycoproteome from PC-3 cells. Using an Orbitrap Fusion Tribrid mass spectrometer, we identified 699 intact glycopeptides from 192 glycoproteins. These intact glycopeptides represent a total of eight sialylated glycan structures across 126 N- and 576 O-glycopeptides. IsoTaG is therefore an effective platform for identification of intact glycopeptides labeled by alkynyl or azido-sugars and will facilitate further studies of the glycoproteome.

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Section: Resultsmentioning

confidence: 99%

Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars

et al. 2016

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“…The cell culture dishes were incubated for an additional 48 h at 37 °C in a humidified 5% CO 2 incubator. Media and cell lysates were harvested and concentrated as described previously [19,20]. Briefly, to obtain the “conditioned media fraction”, media from all conditions were harvested, cleared by centrifugation and spin concentrated (using Amicon, 15-ml 10-kDa spin filters) to a final volume of 1 ml.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were harvested, centrifuged (300 g , 3 min), and washed with PBS. Cell pellets were resuspended in lysis buffer (2 ml: 10 mM HEPES, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 0.5% Triton X-100, 1× protease inhibitors, 1 μM thiamet G), swelled for 5 min on ice, and disrupted by Dounce homogenization using a tight glass hand pestle (20–30 strokes) as described previously [19,20]. Insoluble material was pelleted by centrifugation (3700 g , 10 min, at 4 °C).…”

Section: Methodsmentioning

confidence: 99%

“…The supernatant was collected as the “soluble fraction” while the pellet was kept as the “insoluble fraction.” The insoluble fraction was resuspended in 1% RapiGest/PBS and briefly sonicated. The conditioned medium and soluble fractions were adjusted to 1% RapiGest/PBS as described before [19,20]. Protein concentration from the three fractions was measured by bicinchonic acid assay (Pierce).…”

Section: Methodsmentioning

confidence: 99%

“…Western blots were performed as described in [13]. Affinity purification via copper-catalyzed azide-alkyne cycloaddition (CuAAC) with a biotin–alkyne probe was next performed as previously shown ([19,20]; see also [13]). Briefly, following CuAAC, protein aliquots were precipitated and solubilized.…”

Section: Methodsmentioning

confidence: 99%

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A metabolic labeling approach for glycoproteomic analysis reveals altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells

Sheta

Woo

Roux‐Dalvai³

et al. 2016

Journal of Proteomics

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Epithelial ovarian cancer (EOC) is a disease responsible for more deaths among women in the Western world than all other gynecologic malignancies. There is urgent need for new therapeutic targets and a better understanding of EOC initiation and progression. We have previously identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene, a member of the GalNAc-transferases (GalNAc-Ts) gene family, as hypomethylated and overexpressed in high-grade serous EOC tumors, compared to low malignant potential EOC tumors and normal ovarian tissues. This data also suggested for a role of GALNT3 in aberrant EOC glycosylation, possibly implicated in disease progression. To evaluate differential glycosylation in EOC caused by modulations in GALNT3 expression, we used a metabolic labeling strategy for enrichment and mass spectrometry-based characterization of glycoproteins following GALNT3 gene knockdown (KD) in A2780s EOC cells. A total of 589 differentially expressed glycoproteins were identified upon GALNT3 KD. Most identified proteins were involved in mechanisms of cellular metabolic functions, post-translational modifications, and some have been reported to be implicated in EOC etiology. The GALNT3-dependent glycoproteins identified by this metabolic labeling approach support the oncogenic role of GALNT3 in EOC dissemination and may be pursued as novel EOC biomarkers and/or therapeutic targets. Biological significance: Knowledge of the O-glycoproteome has been relatively elusive, and the functions of the individual polypeptide GalNAc-Ts have been poorly characterized. Alterations in GalNAc-Ts expression were shown to provide huge variability in the O-glycoproteome in various pathologies, including cancer. The application of a chemical biology approach for the metabolic labeling and subsequent characterization of O-glycoproteins in EOC using the Ac4GalNAz metabolite has provided a strategy allowing for proteomic discovery of GalNAc-Ts specific functions. Our study supports an essential role of one of the GalNAc-Ts — GALNT3, in EOC dissemination, including its implication in modulating PTMs and EOC metabolism. Our approach validates the use of the applied metabolic strategy to identify important functions of GalNAc-Ts in normal and pathological conditions.

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A Dual‐Purpose Bromocoumarin Tag Enables Deep Profiling of the Cellular Cysteinome

et al. 2019

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Chemical proteomics enables comprehensive profiling of small molecules in complex proteomes. A critical component to understand the interactome of a small molecule is the precise location on a protein where the interaction takes place. Several approaches have been developed that take advantage of bio-orthogonal chemistry and subsequent enrichment steps to isolate peptides modified by small molecules. These methods rely on target identification at the level of mass spectrometry making it difficult to interpret an experiment when modified peptides are not identified. Herein, an approach in which fluorescence-triggered two-dimensional chromatography enables the isolation of small molecule-conjugated peptides prior to mass spectrometry analysis is described. In this study, a bromocoumarin moiety has been utilized that fluoresces and generates a distinct isotopic signature to locate and identify modified peptides. Profiling of a cellular cysteinome with the use of a bromocoumarin tag demonstrates that two-dimensional fluorescence-based chromatography separation can enable the identification of proteins containing reactive cysteine residues. Moreover, the method facilitates the interrogation of low abundance proteins with greater depth and sensitivity than a previously reported isotope-targeted approach. Lastly, this workflow enables the identification of small-molecule modified peptides from a protein-of-interest.

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Isotope Targeted Glycoproteomics (IsoTaG) to Characterize Intact, Metabolically Labeled Glycopeptides from Complex Proteomes

Cited by 15 publications

References 33 publications

Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars

Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars

A metabolic labeling approach for glycoproteomic analysis reveals altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells

A Dual‐Purpose Bromocoumarin Tag Enables Deep Profiling of the Cellular Cysteinome

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