Immunopeptidomics focuses on identifying and quantifying peptides presented by major histocompatibility complex (MHC) molecules, crucial for immune system activation. Discovery mass spectrometry methods have successfully identified immunogenic and tumor-specific peptides. Translating these discoveries into high-throughput, targeted mass spectrometry (MS) methods is essential for precise quantitation of immunopeptide copy numbers, supporting therapeutic development. Traditional triple quadrupole mass spectrometers face limitations in noise and extensive method development. The Thermo Scientific™ Stellar™ mass spectrometer, a quadrupole ion trap, addresses these challenges by enabling ultra-sensitive, high-throughput quantitation of immunopeptides using parallel reaction monitoring (PRM) and tMS3. This technology facilitates the transition from discovery to targeted workflows while providing high sensitivity for absolute quantitation. Here, we present a targeted proteomics method that enables ultra-sensitive, high-throughput immunopeptide absolute quantitation.
A dilution series of 48 isotopically labeled AQUA peptides was spiked into a background of 1% immunopeptide sample from 1e8 HCT116 cells, equivalent to the immunopeptide material from 1e6 cells. The heavy peptides were spiked at concentrations ranging from 0.001 to 100 fmol. Peptides were quantified using parallel reaction monitoring (PRM). Method development and data acquisition was on the Stellar mass spectrometer and involved initial setup with unscheduled data-independent acquisition (DIA) windows, wide-window acquisition, narrow-window acquisition, and final method establishment, totaling nine injections. Advanced mass spectrometry capabilities, including PRM and tMS3, facilitated the efficient monitoring of all transitions in one scan event, reducing the need for extensive transition selection and optimization. Data was processed using Skyline software daily.
Method development was completed in less than one week, with fewer than nine injections, representing an 11-fold time saving compared to traditional triple quadrupole SRM workflows. The comprehensive approach enabled monitoring all transitions without extensive optimization, accommodating precursor and transition variability in diverse patient samples and tumor microenvironments.