Enhanced Sensitivity and Reproducibility for HLA-Class I & II Immunopeptidomics Utilizing dia-PASEF Workflow

Significance of the Topic

Mass spectrometry based immunopeptidomics is essential for decoding antigen presentation mediated by HLA molecules. High sensitivity and reproducibility are critical for detecting low‐abundance peptides and ensuring robust label-free quantification in clinical and research settings.

Aims and Overview of the Study

This study compared data dependent acquisition (dda-PASEF) and data independent acquisition (dia-PASEF) workflows on the Bruker timsTOF Ultra 2 platform. The goal was to assess how library-based versus library-free (directDIA+) approaches affect identification of HLA Class I and Class II peptides across varying cell inputs.

Methodology and Instrumentation

The workflow included:

• Enrichment of HLA Class I and II peptides from IM9 B-lymphocyte cells.
• Separation on nanoElute with a 60 min gradient for dda-PASEF and 45 min for dia-PASEF.
• Acquisition on a trapped ion mobility quadrupole time-of-flight instrument (timsTOF Ultra 2).
• Processing of dda data with Bruker Proteoscape and TIMSRescore.
• Spectral library generation in Spectronaut 19 for library-based searches.
• Library-free directDIA+ analysis in Spectronaut 20.

Main Results and Discussion

Comparison across sample loads from 1×10⁶ to 5×10⁶ cells revealed:

• dda-PASEF identified 10 000–15 000 Class I ninemers depending on input.
• Library-based dia-PASEF achieved ~18 500 Class I precursors.
• DirectDIA+ identified ~17 000 Class I ninemers at 5×10⁶ cells, outperforming dda-PASEF at all dilutions.
• For HLA Class II, library-based dia-PASEF exceeded dda-PASEF in peptide IDs; directDIA+ for Class II remains to be evaluated.
• High overlap among workflows and consistent sequence motifs demonstrated reproducibility.
• Charge state and length distributions were similar across acquisition methods, confirming data quality.

Benefits and Practical Applications

The dia-PASEF approach on timsTOF Ultra 2 offers:

• Enhanced peptidome coverage with shorter gradients and lower sample inputs.
• Time savings and streamlined workflow by avoiding extensive library construction.
• Robust label-free quantification for immunopeptidomics studies.
• High confidence in peptide identification due to ion mobility separation and directDIA+ processing.

Future Trends and Possibilities of Use

Potential developments include:

• Extending directDIA+ to HLA Class II and other challenging peptide classes.
• Optimization of dia-PASEF window schemes for tailored immunopeptidome profiling.
• Integration with automated pipelines and cloud-based analysis for high-throughput studies.
• Application to clinical discovery of neoantigens and personalized immunotherapy targets.

Conclusion

The dia-PASEF workflow, especially in conjunction with directDIA+, substantially improves sensitivity, reproducibility, and throughput for HLA immunopeptidomics on the timsTOF Ultra 2. This method enables deeper peptidome coverage with reduced sample requirements and shorter analysis times.

Reference

Assis DM et al Enhanced Sensitivity and Reproducibility for HLA-Class I & II Immunopeptidomics Utilizing dia-PASEF Workflow ASMS 2022 ThP 454