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Mass Spectrometry-based immunopeptidomics
The immunopeptidome is the key to the immune system’s ability to discriminate self from non-self, which creates the basis for immunotherapy. Identifying and quantifying the immunopeptidome is crucial for understanding the immune system and advancing precision medicine and cancer vaccines. Thermo Fisher Scientific mass spectrometers enable deep coverage with accurate and precise quantitation of the immunopeptidome at scale.
Immunopeptidomics encompasses the identification and quantification of immunopeptides presented on the cell surface. The small peptides, also known as epitopes, are presented by major histocompatibility (MHC) proteins, where they are recognized by T cells to initiate an immune response.
Mass spectrometry (MS)-based immunopeptidomics methods have the unique ability to prove that an epitope is presented on the surface of the cell. However, measuring the immunopeptidome presents unique challenges due to the low sample abundance, limited sample input, and complexity and diversity of epitopes.
5 step workflow for immunopeptidomics
This 5-step workflow provides information on the basic steps for an immunopeptidomics experiment.
Pillars of a successful immunopeptidomics platform
What you get from Thermo Scientific immunopeptidomics platform
The Thermo Scientific immunopeptidomics platform meets the pillars of successful immunopeptidomics technology, supporting immunopeptide discovery, characterization, and validation.
Our immunopeptidomics platform provide the sensitivity, dynamic range of analysis, accuracy of annotation, and reproducibility of detection for characterization of antigen peptides being presented to the immune system by the MHC.
Sensitivity and dynamic range of detection
Tumor specific neoantigens are expected to be present in relatively low abundance in samples that contain a very high dynamic range of immunopeptide intensities. Thermo Scientific Orbitrap-based mass spectrometers can support simultaneous detection of very high and very low-level analytes.
Orbitrap-based mass spectrometers, including the Thermo Scientific Orbitrap Astral MS, support simultaneous detection of very high and very low-level analytes. They offer exceptional sensitivity and dynamic range to detect high and low abundance immunopeptides in samples, which is crucial to fully characterize tumor-associated and tumor-specific neoantigens.
Accuracy of annotation
To accurately annotate the amino acid sequence of neoantigens that may be composed of novel mutations or that may derive from non-canonical regions of the genome, it may require de novo sequence interpretation. Accurate de novo sequencing requires mass spectral quality with accurate mass, high resolution, and a complete series of fragment ions, as well as auxiliary information ions such as immonium ions.
Orbitrap-based mass spectrometers offer full m/z transmission of MS/MS scans to enable detection of the complete b/y ion series and immonium ions without discrimination to promote unambiguous annotation with de novo sequencing. Automatic gain control and normalized collision energy assure reproducible spectral quality within and across sample cohorts.
Workflow reproducibility
Validation of tumor specific or other neoantigens will require consistent performance and robust operation across cohorts of biological samples or technical replicates. The high reproducibility offered by the Thermo Scientific HRAM Orbitrap MS and liquid chromatography systems enables accurate comparisons across samples.
Thermo Scientific Vanquish Neo UHPLC systems offer highly reproducible chromatographic separations and supports a wide range of operation from nanoflow to high microflow.
Thermo Scientific immunopeptidomics workflows
Workflow for comprehensive discovery and characterization of immunopeptides
Thermo Scientific Orbitrap mass spectrometry-based discovery workflow provides sensitivity for a full dynamic range of analysis to enable comprehensive immunopeptide detection and annotation. The discovery workflow leverages sample preparation, highly reproducible chromatographic separations, and high-resolution, accurate mass Orbitrap mass spectrometers that provide high spectral quality for both MS and MS/MS scans, necessary for confident annotation or de novo sequencing of neoantigens.
Detection of immunopeptides regardless of the abundance
Experience exceptional sensitivity and dynamic range to detect both high and low abundance immunopeptides in the sample, crucial to fully characterize both tumor-associated and tumor-specific neoantigens.
Wide dynamic range detection in MS1 scan.
What are customers saying about Orbitrap Astral MS
"The sensitivity, mass accuracy, and speed of acquisition of the Orbitrap Astral enhance the comprehensiveness of immunopeptidomic analyses and enable the detection of low abundance antigens in primary cancer specimens.
These capabilities facilitate the identification of tumor antigens originating from all genomic regions, ultimately leading to a better understanding of cancer immunology."
Pierre Thibault, Professor, Department of Chemistry
Faculty of Arts and Sciences, Université de Montréal
Principal Investigator, Institute for Research in Immunology and Cancer, Center for Advanced Proteomic Analyses
Simultaneous Quantitation and Discovery (SQUAD) for neoantigen targeted validation and concurrent unbiased immunopeptide monitoring
Thermo Scientific Orbitrap Tribrid Mass Spectrometers offer an exclusive workflow that allows for targeted screening with high sensitivity parallel reaction monitoring (PRM) and simultaneous unbiased data-dependent discovery acquisition in one method. This unique approach enables validation studies of defined neoantigens while allowing for concurrent surveys of undefined antigens during analysis in a single injection of precious samples.
The Orbitrap Ascend Tribrid Mass Spectrometer is ideal for SQUAD
Orbitrap Ascend Tribrid Mass Spectrometer is equipped with a high-resolution Orbitrap and sensitive linear ion trap mass analyzers. This means a high-resolution data-dependent experiment for untargeted analysis can be combined with highly sensitive targeted quantitative data in the ion trap (parallel-reaction monitoring (PRM)) using a single injection. The PRM part of the SQUAD workflow allows for simultaneous sequence confirmation, leveraging full MS/MS visibility of all fragments while quantifying target transitions. PRM-based quantitation can be label free but can also be absolute with the addition of external peptide standards.
Schematic representation of the SQUAD workflow.
Deep immunopeptidome profiling with SQUAD
The SQUAD workflow offers wide sensitivity and dynamic range to detect both high and low abundance immunopeptides in the sample.
Example of single shot analysis of 1e6 cell equivalents of HCT 116 cells and dynamic range of detection.
Absolute quantitation by the linear ion trap
The addition of heavy synthetic peptide standards during PRM acquisition can enable absolute quantitation of the immunopeptides.
Example of Heavy AQUA synthetic peptides spiked into 1e6 cell equivalents of HCT 116 cells enabling absolute quantitation.
High-throughput targeted screening and quantitation of immunopeptide neoantigens for validation
Thermo Scientific mass spectrometers offer robust, sensitive, and reproducible solutions for targeted quantitation. The Orbitrap Exploris series systems offer unparalleled selectivity with high resolution accurate mass precision for PRM. TSQ Plus triple quadrupole mass spectrometers leverage proven ion optics technology consistent with Orbitrap platforms for sensitive transmission of peptides and seamless transition between discovery and targeted quantitation for ultimate sensitivity.
SureQuant workflow
The Thermo Scientific Orbitrap Exploris 480 Mass Spectrometer offers unique SureQuant workflows that allow dynamic control of targeted acquisition, leveraging internal standards spiked into the sample to trigger targeted quantitation scans with the highest specificity and sensitivity for endogenous immunopeptides.
Schematic representation of the SureQuant workflow.
Targeted quantitation using a triple quadrupole mass spectrometer
The Thermo Scientific TSQ Altis Plus Triple Quadrupole Mass Spectrometer offers automatic dwell time optimization for easy method implementation, extended linear dynamic range of detection to prevent saturated calibration curves and narrow window isolation (NWI)-SRM capabilities to enhance selectivity and sensitivity.
Illustration of SRM approaches.
What are customers saying about SureQuant targeted quantitation
“The SureQuant targeted quantitation method on the Explores 480 enables peptide validation, through comparison of chromatography elution profiles and MS/MS spectra between synthetic and endogenous peptides, while simultaneously providing quantitative information regarding expression level of the endogenous peptide. When coupled with multiple synthetic isotopologues, this method provides absolute quantification of endogenous peptides, including tumor-associated antigens and tumor-specific neoantigens.”
Forest White, Ned C. and Janet C. (Bemis) Rice, Professor of Biological Engineering
Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology
For Research Use Only. Not for use in diagnostic procedures.