A Platform for High-throughput and Ultrasensitive Immunopeptidomics.

Area of Science:

• Immunology
• Proteomics
• Mass Spectrometry

Background:

• Mass spectrometry (MS)-based immunopeptidomics identifies peptides on major histocompatibility complex (MHC) molecules for vaccine and immunotherapy development.
• Traditional workflows are low-throughput and require large cell numbers.
• Existing methods often sacrifice sensitivity for throughput or vice versa.

Purpose of the Study:

• To develop a semi-automated immunopeptidomics platform combining high sensitivity and high throughput.
• To optimize conditions for isolating MHC class I and II peptides.
• To enable robust immunopeptide detection from low-input samples.

Main Methods:

• A 96-well positive-pressure device was used for optimized MHC class I and II peptide isolation.
• Lysis in small volumes (100 μl) facilitated efficient MHC capture and automated processing.
• Analysis was performed on a timsTOF SCP mass spectrometer using DDA-PASEF mode.

Main Results:

• The platform identified over 13,500 MHC I and 6,000 MHC II peptides from 16 million cells.
• High sensitivity was demonstrated by identifying up to 1,000 MHC I peptides from as few as 20,000 cells.
• Known and novel bacterial immunopeptides were identified from infected macrophages.

Conclusions:

• The optimized platform achieves high sensitivity and high throughput for immunopeptidomics.
• It enables robust immunopeptide detection from limited sample amounts.
• This facilitates biological discovery in applications with sample constraints.