Sensitive and Quantitative Detection of MHC-I Displayed Neoepitopes Using a Semiautomated Workflow and TOMAHAQ Mass Spectrometry
| Autor: | Lélia Delamarre, Christopher M. Rose, Craig Blanchette, Martine Darwish, Romain Bouziat, Jennie R. Lill, Samuel B. Pollock |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Proteomics
TFF tangential flow filtration SPS synchronous precursor selection PTMs post-translational modifications Tandem mass tag TOMAHAQ Triggered by Offset Multiplexed Accurate-mass High-resolution and Absolute Quantification Biochemistry Mass Spectrometry Analytical Chemistry Workflow Epitopes Mice MS mass spectrometer PSMs peptide–spectrum matches Routine analysis 0303 health sciences biology β2M beta-2 microglobulin AMBIC ammonium bicarbonate pH 8 Chemistry 030302 biochemistry & molecular biology Technological Innovation and Resources Transporter associated with antigen processing DMP dimethyl pimelimidate Recombinant Proteins major histocompatibility complex TEA triethanolamine tandem mass tags (TMT) FWHM full width at half maximum IS-PRM internal standard-triggered parallel reaction monitoring FDR false discovery rate OG octyl-beta-d glucopyranoside Tumor cells Computational biology Major histocompatibility complex Mass spectrometry DSF differential scanning fluorimetry FAIMS high-field asymmetric waveform ion mobility spectrometry TFA trifluoroacetic acid 03 medical and health sciences Special Issue: Immunopeptidomics Cell Line Tumor MHC class I Escherichia coli Animals MHC major histocompatibility complex mIFNγ mouse interferon γ TAP transporter associated with antigen processing Molecular Biology 030304 developmental biology automation Histocompatibility Antigens Class I CV coefficient of variation ERAP endoplasmic reticulum aminopeptidases TMT tandem mass tag ACN acetonitrile BCA bicinchoninic acid TBS Tris-buffered saline TCEP Tris (2-carboxyethyl) phosphine Targeted mass spectrometry DTT dithiothreitol HCD higher-energy collisional dissociation biology.protein IAA iodoacetamide Adpgk ADP-dependent glucokinase |
| Zdroj: | Molecular & Cellular Proteomics : MCP |
| ISSN: | 1535-9484 |
| Popis: | Advances in several key technologies, including MHC peptidomics, have helped fuel our understanding of basic immune regulatory mechanisms and the identification of T cell receptor targets for the development of immunotherapeutics. Isolating and accurately quantifying MHC-bound peptides from cells and tissues enables characterization of dynamic changes in the ligandome due to cellular perturbations. However, the current multistep analytical process is challenging, and improvements in throughput and reproducibility would enable rapid characterization of multiple conditions in parallel. Here, we describe a robust and quantitative method whereby peptides derived from MHC-I complexes from a variety of cell lines, including challenging adherent lines such as MC38, can be enriched in a semiautomated fashion on reusable, dry-storage, customized antibody cartridges. Using this method, a researcher, with very little hands-on time and in a single day, can perform up to 96 simultaneous enrichments at a similar level of quality as a manual workflow. TOMAHAQ (Triggered by Offset, Multiplexed, Accurate-mass, High-resolution, and Absolute Quantification), a targeted mass spectrometry technique that combines sample multiplexing and high sensitivity, was employed to characterize neoepitopes displayed on MHC-I by tumor cells and to quantitatively assess the influence of neoantigen expression and induced degradation on neoepitope presentation. This unique combination of robust semiautomated MHC-I peptide isolation and high-throughput multiplexed targeted quantitation allows for both the routine analysis of >4000 unique MHC-I peptides from 250 million cells using nontargeted methods, as well as quantitative sensitivity down to the low amol/μl level using TOMAHAQ targeted MS. Graphical abstract Highlights • Semiautomated peptide immunoprecipitation on reusable antibody cartridges. • Application of TOMAHAQ for MHC-I detection and quantitation. • Routine analysis of >4000 unique MHC-I peptides from 250 million cells via automation. • Quantitative sensitivity down to the low amol/μl level using TOMAHAQ targeted MS. In Brief This manuscript highlights a new semiautomated MHC-I peptide immunoprecipitation method used in conjunction with the multiplexed quantitative technique TOMAHAQ (Triggered by Offset, Multiplexed, Accurate-mass, High-resolution, and Absolute Quantification) for the detection and quantitation of MHC-I peptides. This combination of techniques allows the routine analysis of >4000 unique MHC-I peptides from 250 million cells and in with targeted analysis, quantitative sensitivity down to the low amol/μl level. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|