ProteoViz: a tool for the analysis and interactive visualization of phosphoproteomics data

Storey, Aaron J.; Naceanceno, Kevin S.; Lan, Renny S.; Washam, Charity L.; Orr, Lisa; Mackintosh, Samuel G.; Tackett, Alan J.; Edmondson, Rick D.; Wang, Zhengyu; Li, Hongyu; Frett, Brendan; Kendrick, Samantha; Byrum, Stephanie D.

doi:10.1039/c9mo00149b

Cited by 23 publications

(20 citation statements)

References 19 publications

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“…Due to the length of the ratio heatmap of the entire dataset, an interactive heatmap is provided and is available to be examined interactively online (details on File S2). For PTM signature enrichment analysis (PTM-SEA), we adopted the methodology developed by Krug et al 38 and R scripts from Storey et al 39 using 7 amino acids flanking each pTyr site with the following modifications. The input for PTM-SEA was modified to be a vector of log-transformed q values multiplied by the sign of the mean log2 reporter ion ratio, instead of p values.…”

Section: Experimental Methodsmentioning

confidence: 99%

Ovalbumin antigen-specific activation of T cell receptor closely resembles soluble antibody stimulation as revealed by BOOST phosphotyrosine proteomics

Chua

Salomon

2021

Preprint

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Activation of T cell receptors (TCR) leads to a network of early signaling predominantly orchestrated by tyrosine phosphorylation in T cells. TCR are commonly activated using soluble anti-TCR antibodies, but this approach is not antigen-specific. Alternatively, activating the TCR using specific antigens of a range of binding affinities in the form of peptide-major histocompatibility complex (pMHC) is presumed to be more physiological. However, due to the lack of wide-scale phosphotyrosine (pTyr) proteomic studies directly comparing anti-TCR antibodies and pMHC, a comprehensive definition of these activated states remains enigmatic. Elucidation of the tyrosine phosphoproteome using quantitative pTyr proteomics enables a better understanding of the unique features of these activating agents and the role of ligand binding affinity on signaling. Here, we apply the recently established Broad-spectrum Optimization Of Selective Triggering (BOOST) to examine perturbations in tyrosine phosphorylation of TCR triggered by anti-TCR antibodies and pMHC. Our data reveals that high-affinity ovalbumin (OVA) pMHC activation of the TCR triggers a largely similar, albeit potentially stronger, pTyr-mediated signaling regulatory axis compared to anti-TCR antibody. Signaling output resulting from OVA pMHC variants correlates well with their weaker affinities, enabling affinity-tunable control of signaling strength. Collectively, we provide a framework for applying BOOST to compare pTyr-mediated signaling pathways of T cells activated in an antigen-independent and antigen-specific manner.

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Section: Experimental Methodsmentioning

confidence: 99%

Ovalbumin antigen-specific activation of T cell receptor closely resembles soluble antibody stimulation as revealed by BOOST phosphotyrosine proteomics

Chua

Salomon

2021

Preprint

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Section: Methodsmentioning

confidence: 99%

“…Both buffers adjusted to pH 10 with ammonium hydroxide for offline separation. Protein identification, normalization and statistical analysis were performed as previously described by Storey et al 34 Real-time PCR RNA was purified with an Arum Total RNA mini kit (Bio-rad #732-6820). Reverse transcription was carried out using the iScript Advanced cDNA Synthesis kit (Bio-rad #1725037).…”

Section: Methodsmentioning

confidence: 99%

DNA-PKcs kinase activity stabilizes the transcription factor Egr1 in activated immune cells

Waldrip

Burdine

Harrison

et al. 2021

Preprint

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DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is known primarily for its function in DNA double-stranded break repair and non-homologous end joining (NHEJ). However, like other DNA damage repair kinases (DDR), DNA-PKcs also has a critical yet undefined role in immunity impacting both myeloid and lymphoid cell lineages spurring interest in targeting DNA-PKcs for therapeutic strategies in immune-related diseases. To gain insight into the function of DNA-PKcs within immune cells, we performed a quantitative phosphoproteomic screen in T cells to identify first order phosphorylation targets of DNA-PKcs. Results indicate that DNA-PKcs phosphorylates the transcription factor Egr1 (early growth response protein 1) at S301. Expression of Egr1 is induced early upon T cell activation and dictates T cell response by modulating expression of cytokines and key costimulatory molecules. Mutation of serine 301 to alanine via CRISPR-Cas9 resulted in increased proteasomal degradation of Egr1 and a decrease in Egr1-dependent transcription of IL2 (interleukin-2) in activated T cells. Our findings identify DNA-PKcs as a critical intermediary link between T cell activation and T cell fate and a novel phosphosite involved in regulating Egr1 activity.

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“…A protein and phosphopeptide analysis was performed as previously described in Storey et al 24 Briefly, 100 mg of protein from each cell line was reduced, alkylated, and purified by chloroform/methanol extraction prior to digestion with sequencing grade trypsin and LysC (Promega). The resulting peptides were labeled using a tandem mass tag 10-plex isobaric label reagent set (Thermo) and enriched using High-Select TiO 2 and Fe-NTA phosphopeptide enrichment kits (Thermo) following the manufacturer's instructions.…”

Section: Phosphoproteomics Analysismentioning

confidence: 99%

Multi-omics data integration reveals correlated regulatory features of triple negative breast cancer

et al. 2021

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ProteoViz: a tool for the analysis and interactive visualization of phosphoproteomics data

Abstract: Quantitative proteomics generates large datasets with increasing depth and quantitative information.

Cited by 23 publications

References 19 publications

Ovalbumin antigen-specific activation of T cell receptor closely resembles soluble antibody stimulation as revealed by BOOST phosphotyrosine proteomics

Ovalbumin antigen-specific activation of T cell receptor closely resembles soluble antibody stimulation as revealed by BOOST phosphotyrosine proteomics

DNA-PKcs kinase activity stabilizes the transcription factor Egr1 in activated immune cells

Multi-omics data integration reveals correlated regulatory features of triple negative breast cancer

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