Higher-throughput and sensitive immunopeptidomics profiling

ABSTRACT: We developed a refined sample preparation workflow that is fully optimized to process low-input and crude samples in 96-well plates, aiming to combine high-throughput with maximal sensitivity. We implemented a miniaturized and automated workflow in a 96-well format using the Tecan Resolvex® A200 liquid handling positive pressure device. We optimized lysis in small volumes to achieve more sensitive and efficient IPs in 96-well plates under static conditions, to minimize peptide losses due to surface contact, and to eliminate the need for plate rotation and unnecessary sample transfers. Additionally, using an appropriate pore-size for the 96-well filter plate for IP prevented sample leakage and ensured efficient processing. This workflow reduces the required sample input from 500 million to as few as 16 million cells for routine experiments, and is compatible with input amounts going down to 20,000 cells. It minimizes reagent volumes, accommodates up to 96 samples simultaneously, and enables the ultrasensitive pull down from low input samples with near-complete capture of MHC peptides. Furthermore, we achieved high sensitivity by combining the sample preparation with advanced ion mobility mass spectrometry using a timsTOF SCP. This improved workflow achieves robust immunopeptidome coverage from as few as 20,000 JY cells, identifying approximately 13,000 distinct MHC-I peptides and over 5,000 MHC-II peptides from 16 million JY cells by injecting only 25% of the enriched peptides into the mass spectrometer.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Patrick Willems  

LAB HEAD: Francis Impens

PROVIDER: PXD062352 | Pride | 2026-04-03

REPOSITORIES: Pride