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

ABSTRACT: 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.

SUBMITTER: Stopfer LE

PROVIDER: S-EPMC8449407 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description: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.

2021-09-21 | PXD024917 | Pride

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Absolute quantification of proteins in the fatty acid biosynthetic pathway using protein standard absolute quantification.

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Absolute quantification of miRNAs

2009-10-27 | GSE15835 | GEO

Absolute quantification of miRNAs

Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. Total liver RNA was mixed with 2.5 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes and subsequently fluorescently labelled by 3â ligation. Total RNA mix was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool (n = 6). The synthetic miRNA pool consisted of 2.5 fmol of each of 891 non redundant miRNAs sequences and miRControl 3 sequences. The array data was normalized by calculating the median of the miRControl 3 present in the liver and UR sample. The miRNA amount was calculated with respect to the corresponding miRNA in the UR.

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2017-03-08 | GSE83734 | GEO

Absolute quantification of miRNAs

Project description:Absolute quantification of miRNAs

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Absolute quantification of microRNAs by using a universal reference.

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