This DIA workflow runs global protein quantification. Phosphorylation, HLA, isoform and drug-interaction analyses are not included. Your DDA choices remain saved for DDA studies.
Choose any additional analyses. Low coverage does not fail the study: results include nonsignificant features, and tests with insufficient evidence are labeled in the output tables.
Gene-level counts and comparisons
Use peptides assigned unambiguously to one gene. Peptides shared between genes are excluded from these counts only; protein counts remain unchanged. These are proteomics measurements, not RNA expression.
Raw counts, normalized matrices and z-scores appear under Optional analyses → Gene counts. With differential analysis enabled, gene comparisons use the methods, reference condition, FDR and count filters selected in DEPs, with separate model fitting and FDR correction. Modified-peptide counts per gene follow this option too. If drug-interaction testing is enabled, this option also includes its gene-level tests, using the drug-interaction methods and FDR.
Immunopeptidomics
Optional dedicated workflow for HLA-enriched, undigested peptides measured by DDA-PASEF.
Choose Yes for HLA-enriched, undigested peptide samples. Choose No for ordinary global or phosphoproteomics samples.
Both classes use a 7–25 amino-acid search. Class selection labels the report and sets the length range of the sequence export for external binding prediction.
Dedicated HLA search preset
- Nonspecific digestion; no fixed cysteine modification.
- Variable oxidation, protein N-terminal acetylation and cysteinylation.
- DDA search with MSBooster / Percolator; 1% PSM FDR. Peptide and protein FDR are not restricted to 1%.
- IonQuant enabled; match-between-runs off.
This selects DDA only and replaces the global or phosphorylation search settings. DEPs methods, comparisons and optional low-count filtering apply to peptide spectral counts only.
Results include peptide counts, sample summaries, peptide-length distributions and sequences for external HLA binding prediction. Binding prediction and allele assignment are not run here.
Based on Benchmarking Software for DDA-PASEF Immunopeptidomics, adapted to the installed FragPipe 24 tools. Validate with representative HLA-enriched samples before scientific use.
Drug experiments
Use DEPs to compare individual drug treatments with your control. Combination-interaction testing is inactive for single-drug studies.
Statistical comparisons for optional analyses
Compare selected evidence between conditions independently of the DEPs switch. Drug-combination interaction has its own four-condition design, method choices (edgeR, limma-voom and Monte Carlo) and FDR setting in Drugs.
Results include effect sizes, p-values, FDR, condition means, detection counts, significant subsets and an overall condition test. Each cohort and DDA/DDA+ search is analyzed separately.