In this study, we introduced a multiplex acquisition strategy at the precursor level, called "Multiple Accumulation Precursor Mass Spectrometry" (MAP-MS). This technique enhances the dynamic range by accumulating multiple precursor mass ranges while the Orbitrap (OT) analyzes MS/MS spectra without increasing the cycle time.

Our results confirm that peptide detection in OT-based data-independent acquisition (DIA) analysis can be improved by extending the precursor dynamic range using MAP-MS. This is achieved through a software package that combines precursor and fragment information, demonstrating that MAP-MS can serve as an alternative procedure for precursor analysis in OT-based mass spectrometers.

By analyzing 125 HeLa tryptic peptides, we identified 99,056 peptides and 7,721 protein groups using MAP-MS, compared to 82,336 peptides and 6,736 protein groups identified with the standard DIA method on an Orbitrap Exploris^TM 480 mass spectrometer with a 110-minute gradient.

Additionally, we introduced a true positive validation method based on gas-phase fractionation (GPF). This method can be applied to verify post-acquisition method optimization using retention times (RT) from GPF-DIA as a reference.