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Absolute quantification of tumor antigens using embedded MHC-I isotopologue calibrants

ABSTRACT: SureQuant-MHC utilizes a series of isotopologues and internal standard-triggered targeted mass spectrometry to generate an embedded multipoint calibration curve to determine the endogenous pMHC concentrations for a panel of 18 antigen targets. We apply this methiod to profile changes in pMHC expression levels in melanoma cell lines treated with binimetinib, a MEK inhibitr, and traslate the approach to profiling antigen levels in human tumor punch biopsies.

INSTRUMENT(S): Orbitrap Exploris 480, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Melanocyte, Skin

SUBMITTER: Lauren Stopfer

LAB HEAD: Forest White

PROVIDER: PXD024917 | Pride | 2021-09-21

REPOSITORIES: Pride

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			Action	DRS
	1000fmol_1.raw	Raw
	1000fmol_2.raw	Raw
	1000fmol_3.raw	Raw
	100amol_1.raw	Raw
	100amol_2.raw	Raw

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Publications

Absolute quantification of tumor antigens using embedded MHC-I isotopologue calibrants.

Stopfer Lauren E LE Gajadhar Aaron S AS Patel Bhavin B Gallien Sebastien S Frederick Dennie T DT Boland Genevieve M GM Sullivan Ryan J RJ White Forest M FM

Proceedings of the National Academy of Sciences of the United States of America 20210901 37

Absolute quantification measurements (copies per cell) of peptide major histocompatibility complex (pMHC) antigens are necessary to inform targeted immunotherapy drug design; however, existing methods for absolute quantification have critical limitations. Here, we present a platform termed SureQuant-IsoMHC, utilizing a series of pMHC isotopologues and internal standard-triggered targeted mass spectrometry to generate an embedded multipoint calibration curve to determine endogenous pMHC concentra ...[more]

PMID: 34497125

Similar Datasets

Absolute quantification of tumor antigens using embedded MHC-I isotopologue calibrants.

Project description:Absolute quantification measurements (copies per cell) of peptide major histocompatibility complex (pMHC) antigens are necessary to inform targeted immunotherapy drug design; however, existing methods for absolute quantification have critical limitations. Here, we present a platform termed SureQuant-IsoMHC, utilizing a series of pMHC isotopologues and internal standard-triggered targeted mass spectrometry to generate an embedded multipoint calibration curve to determine endogenous pMHC concentrations for a panel of 18 tumor antigens. We apply SureQuant-IsoMHC to measure changes in expression of our target panel in a melanoma cell line treated with a MEK inhibitor and translate this approach to estimate antigen concentrations in melanoma tumor biopsies.

| S-EPMC8449407 | biostudies-literature

Breast cancer and liver tissue biopsies used to develop and validate an index for liver contamination in metastatic breast cancer

Project description:PURPOSE: To develop an index capable of detecting and quantifying the extent of liver RNA contamination in liver biopsies of metastatic breast cancer METHODS: We developed a microarray-based gene expression liver index by comparing the expression levels of genes in liver tissue biopsies and in primary breast cancer biopsies. The predictive performance of the index was then evaluated in defined mixtures of liver and breast cancer RNA. A calibration curve was established to allow estimation of the liver RNA content in unknown metastatic breast cancer samples. A set of liver biopsies and primary breast cancer biopsies were used to identify genes that were expressed significantly higher in liver tissue. These genes were used in a liver index, which was validated in a new set of liver and primary breast cancer biopsies. A set of liver/breast cancer RNA mixtures was used to establish a calibration curve to allow quantification of liver contamination in metastatic breast cancer samples. The prediction error of the calibration curve was assessed in a new set of liver/breast cancer RNA mixtures and applied to estimate the liver content in a set of unknown metastatic breast cancer biopsies.

2013-01-05 | E-GEOD-33116 | biostudies-arrayexpress

Antigen perception in T cells by long-term Erk and NFAT signaling dynamics [scRNAseq]

Project description:Immune system threat detection hinges on T cells’ ability to perceive varying peptide major-histocompatibility complex (pMHC) antigens. As the Erk and NFAT pathways link T cell receptor engagement to gene regulation, their signaling dynamics may convey information about pMHC inputs. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that, together, enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs. Both pathways initially activate uniformly across various pMHC inputs, but diverge only over longer (9+ hrs) timescales, enabling independent encoding of pMHC affinity and dose. These late signaling dynamics are decoded via multiple temporal and combinatorial mechanisms to generate pMHC-specific transcriptional responses. Our findings underscore the importance of long timescale signaling dynamics in antigen perception, and establish a framework for understanding T cell responses under diverse contexts.

2023-09-06 | GSE242494 | GEO

Antigen perception in T cells by long-term Erk and NFAT signaling dynamics [RNAseq]

2023-09-06 | GSE242418 | GEO

MEK inhibition enhances presentation of targetable MHC-I tumor antigens in mutant melanomas [RNA-seq]

Project description:Combining multiple therapeutic strategies in NRAS/BRAF mutant melanoma – namely MEK/BRAF kinase inhibitors, immune checkpoint inhibitors, and targeted immunotherapies – may offer an improved survival benefit by overcoming limitations associated with any individual therapy. Still, optimal combination, order, and timing of administration remains under investigation. Here, we measure how MEK inhibition alters anti-tumor immunity by utilizing quantitative immunopeptidomics to profile changes the peptide MHC (pMHC) repertoire. These data reveal a collection of tumor antigens whose presentation levels are selectively augmented following therapy, including several epitopes present at over 1000 copies-per-cell. We leveraged the tunable abundance of MEKi-modulated antigens by targeting 4 epitopes with pMHC-specific T cell engagers and antibody drug conjugates, enhancing cell killing in tumor cells following MEK inhibition. These results highlight drug treatment as a means to enhance immunotherapy efficacy by targeting specific upregulated pMHCs and provide a methodological framework for identifying, quantifying, and therapeutically targeting additional epitopes of interest.

2022-11-15 | GSE185284 | GEO

TCR repertoire profiling of CMV specific and SARS-CoV2 Spike specific CD8+ T cells using single cell sequencing

Project description:We introduced single-chain trimer (SCT) technologies into a high throughput platform for pMHC library generation that can be used for screening antigen specific CD8+ T cells. We compared the diversity of T cell receptor repertoire captured by SCT and folded peptide-MHC multimer presenting HLA-A02:01 restricted CMV peptide. We then constructed SCT libraries designed to capture SARS-CoV-2 spike specific CD8+ T cells from COVID-19 participants and healthy donors. TCR sequencing with antigen specificity was analyzed. The immunogenicity of these epitopes was validated by functional assays of T cells with cloned TCRs captured using SCT libraries.

2022-01-01 | E-MTAB-11229 | biostudies-arrayexpress

Median Based Absolute Quantification using Fully Unlabelled Generic Internal Standard

Project description:By reporting molar abundances of proteins, absolute quantification determines their stoichiometry in complexes, pathways or networks and also relates them to abundances of non-protein biomolecules. Typically, absolute quantification relies either on protein- specific isotopically labelled peptide standards or on a semi-empirical calibration against the average abundance of peptides chosen from arbitrary selected standard proteins. Here we developed a generic protein standard FUGIS (Fully unlabelled Generic Internal Standard) that requires no isotopic labelling, synthesis of standards or external calibration and is applicable to proteins of any organismal origin. FUGIS is co-digested with analysed proteins and enables their absolute quantification in the same LC-MS/MS run. By using FUGIS, median based absolute quantification (MBAQ) workflow provides similar quantification accuracy compared to isotopically-labelled peptide standards and outperforms methods based on external calibration or selection of best ionized reporter peptides (Top3 quantification) with a median quantification error less than 15%

2021-11-30 | PXD026886 | Pride

Transcription profiling of rat REF52 cells expressing SV40 T antigens

Project description:SV40 transforms cells through the action of two oncoproteins, large T antigen and small t antigen. Small t antigen targets phosphatase PP2A, while large T antigen stimulates cell proliferation and survival by action on multiple proteins, including the tumor suppressors Rb and p53. Large T antigen also binds components of the transcription initiation complex and several transcription factors. We examine global gene expression in SV40-transformed REF52 cells. SV40 transformation alters the expression of approximately 480 cellular genes. Much of this regulation is consistent with T antigen action on the Rb-E2F pathway. Experiment Overall Design: We used the Affymetrix RGU34A microarrays to probe the global mRNA expression from two wild-type REF52neo and three REF52neoT stable cell lines. Cells were grown to two days post-confluence and harvested.

2009-09-12 | E-GEOD-7836 | biostudies-arrayexpress

Breast cancer and liver tissue biopsies used to develop and validate an index for liver contamination in metastatic breast cancer

2013-01-05 | GSE33116 | GEO

MEK inhibition enhances presentation of targetable MHC-I tumor antigens in mutant melanomas

2023-03-11 | PXD029860 | Pride