Fig. 1.

Instrument schematic and explanation of narrow-bin DIA methods.A, Orbitrap Astral instrument schematic. B, MS¹ extracted ion chromatograms for two peptides of similar m/z which coelute and are coisolated, even with narrow DIA bins during the 15-min method. The monoisotopic and ¹³C isotope extracted ion chromatograms are shown for both peptides. C, MS¹ spectrum at 16.76 min with inset showing two peptides coisolated within 2 Th gray box. D, MS/MS extracted ion chromatograms of product ions of the two coisolated peptides. E, MS/MS spectrum at 16.75 min with annotation of product ions for two identified peptide sequences. Vertical bars indicate frequency of Orbitrap MS¹ and MS/MS scans in B and D, respectively. F–H, the complexity of DIA MS/MS spectra is greatly reduced using narrow quadrupole isolation. Each dot represents a unique MS/MS scan and color corresponds to the number of PSMs in each scan, as identified by Spectronaut; 4 Th and 6 Th bins are theoretical, generated via in silico widening of bins of the 2 Th data processing. DIA, data-independent acquisition; MS, mass spectrometry; MS/MS, tandem mass spectrometry; PSM, peptide-spectrum match.