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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.
Enhanced immunopeptide confidence with EThcD fragmentation
Achieve superior sequence coverage and increased confidence in peptide identification with EThcD fragmentation. The alternative fragmentation method provided by EThcD generates additional c- and z-ions, resulting in significantly better sequence coverage compared to HCD alone.
Sequence coverage of all PSMs from GR cell line analysis using either HCD or EThcD fragmentation on Orbitrap Excedion Pro mass spectrometer.
What are customers saying about Orbitrap mass spectrometers
"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
"The Orbitrap Astral Zoom mass spectrometer improves throughput, identification depth, and sensitivity to comprehensively profile immunopeptidomics samples, facilitating analyses from 10-100 times fewer cells per experiment. This advancement not only enables the investigation of more biologically relevant precious samples, but also allows for testing more experimental conditions per study to open up new opportunities for profiling drug-induced perturbations to the immunopeptidome."
Greg Potts, Principal Research Scientist, AbbVie
"EThcD fragmentation on the Orbitrap Excedion Pro mass spectrometer boosts peptide sequence coverage, minimizing detrimental sequence gaps, even for peptides with unfavorable physicochemical properties, allowing less error-prone immunopeptidomics, glycoproteomics, and de novo sequencing."
Albert Heck, Professor of Pharmaceutical Sciences, Biomolecular Mass Spectrometry and Proteomics, Utrecht University, the Netherlands
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 Thermo Scientific Stellar mass spectrometer supports ultra-sensitive, high-throughput peptide analysis with PRM, allowing comprehensive monitoring of all peptide fragments for precise identification and quantification. Its advanced MS3 capabilities reduce noise, enabling efficient and accurate data acquisition. The Orbitrap Excedion Pro MS offer unparalleled selectivity with high resolution accurate mass precision for PRM.
SureQuant workflow
The Thermo Scientific Orbitrap Excedion Pro 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 the Stellar mass spectrometer
The Stellar mass spectrometer addresses the needs of targeted immunopeptidomics by enabling absolute quantitation leveraging advanced technology. This mass spectrometer offers PRM for streamlined method development and built-in confirmation of sequence identity for immunopeptides, and MS3 fragmentation capabilities to reduce noise and increase sensitivity. Achieve ultra-sensitive, high-throughput peptide quantification down to 1 amol with efficiency and precision.
Innovative Features of the Stellar mass spectrometer for targeted immunopeptidomics. A) Demonstrates the Stellar MS capability to detect and quantify peptides from 100fmol (A) and 1 amol (B). B) Showcases the advanced MSn capabilities, providing improved selectivity and sensitivity for peptide quantitation. C) Highlights the significant reduction in method development time, achieving an 11-fold increase in efficiency.
Adaptive RT routine for comprehensive targeted compounds across large sample cohorts
To maximize data completeness and reproducibility across samples, Adaptive RT is supported on Stellar MS and Thermo Scientific Orbitrap Excedion Pro Mass Spectrometers. By dynamically adjusting retention times in real time at the LC timescale, Adaptive RT enables narrow MS² triggering windows for highly confident targeted analyses.
Benefit of adaptive RT for narrow window tMS2 yields reproducible measurements. With adaptive RT (left) and without RT adjustment (right).
What customers are saying about SureQuant targeted quantitation
“The SureQuant targeted quantitation method on the Orbitrap instruments 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
The LC-MS are not intended for in vitro diagnostic purposes in accordance with our product documentation, manuals, and labels. They are designated for General Laboratory Use Only.
Our Products have not been tested or validated for such applications and their use for in vitro diagnostic purposes may result in inaccurate results and, more seriously, health and safety risks. They are designated for Research Use Only Applications.