Fig. 2: Overview of DIA phosphoproteomics on the Orbitrap Astral MS.

A Illustration of DIA acquisition scheme. B Average number of localized phosphorylation sites identified using DIA method with various isolation widths for n = 3 injection replicates. Error bars indicate maximum and minimum observed values. C Evaluation of phosphopeptide loading mass on performance. Data was collected with a 30-min active gradient and 2 m/z DIA isolation width. D Effect of gradient length on performance. Points represent the average across n = 3 injection replicates. Error bars indicate maximum and minimum observed values. E Evaluation of phosphosite identification reproducibility (for results from (D)). F Evaluation of phosphosite quantitative precision. The relative standard deviation of phosphosite quantities is shown for phosphosites detected across triplicate injections with the median value displayed for each active gradient length. G Comparison of External (maize/human entrapment experiment) and Internal (Spectronaut) FDR on precursor level. H Phosphoproline Decoy Search to test reliability of localization algorithm. The cumulative distribution of localization probabilities is shown for phosphorylation events at different amino acids. I Average site localization error rate as a function of localization probability cutoff. Synthetic phosphopeptide standards spiked into a yeast phosphopeptide background were used as a ground truth for error rate determination. J The distribution of R² values for linear calibrations curves is shown for phosphopeptides standards detected in at least three concentrations across a five-point dilution series into a constant yeast phosphopeptide background. Source Data are provided as a Source Data file.