Dataset Information

Ovalbumin Antigen-Specific Activation of Human T Cell Receptor Closely Resembles Soluble Antibody Stimulation as Revealed by BOOST Phosphotyrosine Proteomics.

ABSTRACT: Activation of the T cell receptor (TCR) leads to a network of early signaling predominantly orchestrated by tyrosine phosphorylation in T cells. The TCR is 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 a 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 human TCR triggered by anti-TCR antibodies and pMHC. Our data reveal that high-affinity ovalbumin (OVA) pMHC activation of the human TCR triggers a largely similar, albeit potentially stronger, pTyr-mediated signaling regulatory axis compared to the anti-TCR antibody. The 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 human T cells activated in an antigen-independent and antigen-specific manner.

SUBMITTER: Chua XY

PROVIDER: S-EPMC8626127 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Publications

Ovalbumin Antigen-Specific Activation of Human T Cell Receptor Closely Resembles Soluble Antibody Stimulation as Revealed by BOOST Phosphotyrosine Proteomics.

Chua Xien Yu XY Salomon Arthur A

Journal of proteome research 20210521 6

Activation of the T cell receptor (TCR) leads to a network of early signaling predominantly orchestrated by tyrosine phosphorylation in T cells. The TCR is 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 a peptide-major histocompatibility complex (pMHC) is presumed to be more physiological. However, due to the lack of wide-scale phosphotyrosin ...[more]

PMID: 34018748

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Project description:Signal propagation mediating numerous cellular processed in T cells is orchestrated by tyrosine phosphorylation with a high degree of regulation1, 2. Because aberrant tyrosine phosphorylation can result in many diseases3, a method to systematically quantify this dynamic process can be helpful in understanding the mechanistic underpinnings of T cell functions. However, accurate quantitation of tyrosine phosphorylation is challenging due to its low abundance4. Mass spectrometry(MS) based phosphotyrosine proteomics has emerged to be an attractive solution for wide scale profiling of the tyrosine phosphoproteome5, 6. Many advances have been made in this field to improve the enrichment efficiency of phosphotyrosine(pTyr)-containing peptides to improve the coverage depth7-11. Separately, computational methods of Fourier transform-(FT)MS such as an Orbitrap analyzer have also made significantly progress in the quantitation of TMT samples. For example, the phase-constrained spectrum deconvolution method (ΦSDM) have been shown to substantially improve the spectral quality and speed of FT-MS instruments12. ΦSDM deconvolves FT spectra into frequency distributions to allow more efficient extraction of the harmonic components of the oscillating ions12. As a result, higher mass resolution and accuracy can be achieved with shorter transient times12. We recently developed the BOOST approach by coupling PV boost channel in a multiplexed TMT approach to significantly increase the quantitative depth of the tyrosine phosphoproteome13. While it has been shown to work in Jurkat cell line, it has not been demonstrated to work in scarce mice primary T cells. Here, we demonstrate the first phosphotyrosine proteomics performed in mice primary T cells using BOOST that enabled the identification of more than 6000 pTyr peptides using only 1 mg of protein. We further demonstrated the importance of ΦSDM, in which when disabled, enabled significantly deeper quantitation depth in low-abundance samples. Using samples with contrived ratios, disabling ΦSDM improved the precision of low-abundance peptides and allowed the quantitation of up to 2-fold more increase in statistically significant ratios.

Dataset Information

Ovalbumin Antigen-Specific Activation of Human T Cell Receptor Closely Resembles Soluble Antibody Stimulation as Revealed by BOOST Phosphotyrosine Proteomics.

Publications

Ovalbumin Antigen-Specific Activation of Human T Cell Receptor Closely Resembles Soluble Antibody Stimulation as Revealed by BOOST Phosphotyrosine Proteomics.

Similar Datasets

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