Thunder-DDA-PASEF enables high-coverage immunopeptidomics and is boosted by MS2Rescore with MS2PIP timsTOF fragmentation prediction model

Abstract

Human leukocyte antigen (HLA) class I peptide ligands (HLAIps) are key targets for developing vaccines and immunotherapies against infectious pathogens or cancer cells. Identifying HLAIps is challenging due to their high diversity, low abundance, and patient individuality. Here, we develop a highly sensitive method for identifying HLAIps using liquid chromatography-ion mobility-tandem mass spectrometry (LC-IMS-MS/MS). In addition, we train a timsTOF-specific peak intensity MS2PIP model for tryptic and non-tryptic peptides and implement it in MS2Rescore (v3) together with the CCS predictor from ionmob. The optimized method, Thunder-DDA-PASEF, semi-selectively fragments singly and multiply charged HLAIps based on their IMS and m/z. Moreover, the method employs the high sensitivity mode and extended IMS resolution with fewer MS/MS frames (300 ms TIMS ramp, 3 MS/MS frames), doubling the coverage of immunopeptidomics analyses, compared to the proteomics-tailored DDA-PASEF (100 ms TIMS ramp, 10 MS/MS frames). Additionally, rescoring boosts the HLAIps identification by 41.7% to 33%, resulting in 5738 HLAIps from as little as one million JY cell equivalents, and 14,516 HLAIps from 20 million. This enables in-depth profiling of HLAIps from diverse human cell lines and human plasma. Finally, profiling JY and Raji cells transfected to express the SARS-CoV-2 spike protein results in 16 spike HLAIps, thirteen of which have been reported to elicit immune responses in human patients.

High-coverage immunopeptidomics using timsTOF mass spectrometers with Thunder-DDA-PASEF boosted by MS2Rescore

“…In Hystar, create a new list by clicking on New [1] and save it by clicking Save as… [2]. Enter a representative name [3], and click OK [4].…”

“…4a-d): Open PEAKS XPro, then click on Window/Con guration/Database [1,2]. Click on Browse… [3], navigate to the location of your local FASTA database, and select it [4]. Select the proper FASTA format database using the drop-down menu [5].…”

“…However, it is challenging due to the high diversity, extensive dynamic range, and individual-speci city of the immunopeptidome (2,3). To address these challenges, we previously optimized a process enabling the identi cation of more than 10,000 MHC class I ligands per cell line by exploiting the additional resolution, speed, and sensitivity provided by ion mobility separation (IMS) in timsTOF mass spectrometers (4).…”

“…(I) Immunopeptide -MHC complexes are enriched by antibody-based immunoprecipitation (IP) and immunopeptides are subsequently isolated. We use a previously optimized method (5) with slight modi cations (4,6). (II) The peptides are analyzed by LC-MS. We use a nanoElute 2 coupled with timsTOF Pro2 with an LC-IMS-MS method called Thunder-Data-Dependent Acquisition with Parallel Accumulation-SErial Fragmentation (Thunder-DDA-PASEF) (4,7).…”

“…We use a previously optimized method (5) with slight modi cations (4,6). (II) The peptides are analyzed by LC-MS. We use a nanoElute 2 coupled with timsTOF Pro2 with an LC-IMS-MS method called Thunder-Data-Dependent Acquisition with Parallel Accumulation-SErial Fragmentation (Thunder-DDA-PASEF) (4,7). To maximize immunopeptide coverage, this method exploits an extended ion mobility resolution, the "high sensitivity mode" in timsTOF instruments, and a semi-selective fragmentation of precursors corresponding to possible immunopeptides.…”