ASMS 2026: DIA Isolation Strategies for Host Cell Protein Detection Sensitivity
Comparing Narrow, Overlapped, Mixed and Wide m/z windows for low-abundance HCP detection in complex matrices
About this Poster
Data-independent acquisition keeps gaining ground in proteomics, especially for host cell protein analysis where low-abundance detection is the whole game. This study compares four DIA isolation strategies — Narrow (8 m/z), Overlapped (8 m/z with 4 m/z step), Mixed (8 + 15 m/z), and Wide (15 m/z) — using an E. coli whole-cell lysate spiked with USP1 standard from 0.1 to 50 fmol. All four strategies detect USP1 at the lowest 0.1 fmol spike, but real differences emerge in the middle of the range: narrower windows outperform when quantifying low-abundance targets against a complex background. Match-between-runs across the full sample set is shown to rescue sensitivity at the lowest concentrations.
Key Learnings:
- Compare four DIA isolation strategies (Narrow, Overlapped, Mixed, Wide) for HCP detection in an E. coli matrix spiked with USP1 from 0.1 to 50 fmol.
- See how narrower isolation windows deliver superior sensitivity at intermediate concentrations, while all strategies tie at the 0.1 fmol detection floor.
- Learn how automatic match-between-runs with %RSD filtering rescues quantitative information at low concentrations without inflating false positives.
- Understand how chimeric spectral matches in DIA enable confident identification of co-eluting peptides — including subtle PTMs like deamidation (N → D, +0.984 Da).
E. coli lysate dataset from Gotti et al. Data acquired on Orbitrap Fusion Tribrid MS (Thermo Fisher Scientific) with Dionex Ultimate 3000 RSLC nano-LC.