Data independent acquisition - mass spectrometry (DIA-MS) is becoming widely utilised for robust and accurate quantification of samples in quantitative proteomics. Here, we describe the systematic evaluation of the effects of DIA precursor mass range on total protein identification and quantification. We show that a narrow mass range of precursors (~250 m/z) for DIA-MS enables a higher number of protein identifications. Subsequent application of DIA with narrow precursor range (from 400 to 650 m/z) on Arabidopsis sample with spike-in of known proteins identified 34.7% more proteins than in conventional DIA (cDIA) with a wide precursor range of 400-1200 m/z. When combining several DIA-MS analyses with narrow precursor ranges (i.e., 400-650, 650-900 and 900-1200 m/z), we were able to quantify 10,099 protein groups with a median coefficient of variation of <6%. These findings represent a 59.4% increase in the number of proteins quantified than with cDIA analysis. This is particularly important for low abundance proteins, as exemplified by the 6-protein mix spike-in. In cDIA only 5 out of the 6-protein mix were quantified while our approach allowed accurate quantitation of all six proteins.

The main goal of the paper is to compare the effects of the wave spectrum, computed using the Discrete Interaction Approximation (DIA) and the Webb–Resio–Tracy (WRT) methods, on statistical wave properties such as skewness and kurtosis. The statistical properties are obtained by integrating the three-dimensional free-surface Euler equations with a high-order spectral method combined with a phenomenological filter to account for the energy dissipation due to breaking waves. In addition, we investigate the minimum spatial domain size required to obtain meaningful statistical wave properties. The numerical simulations are performed over a physical domain of size 4.13 km × 4.13 km. The results indicate that statistical properties must be computed over an area of at least 4 km2. The results also suggest that selecting a more computationally expensive WRT method does not affect the statistical values to a great extent. The most noticeable effect is due to the energy dissipation filter that is applied. It is concluded that selecting the WRT or the DIA algorithm for computing the wave spectrum needed for the numerical simulations does not lead to major differences in the statistical wave properties. However, more accurate energy dissipation mechanisms due to wave breaking are needed.

The dynamic interplay of signaling networks in most major cellular processes is characterized by the orchestration of reversible protein phosphorylation. Consequently, analytic methods like quantitative phospho-peptidomics has been pushed forward from a highly specialized edge-technique to a powerful and versatile platform for comprehensively analyzing the phosphorylation profile of living organisms. Despite enormous progress in instrumentation and bioinformatics, a major problem remains a high number of missing values caused by the experimental procedure due to either a random phospho-peptide enrichment selectivity or borderline signal intensities, which both cause the exclusion for fragmentation using the commonly applied data dependent acquisition (DDA) mode. Consequently, an incomplete dataset reduces confidence in the subsequent statistical bioinformatic processing. Here, we successfully applied data independent acquisition (DIA) by using the filamentous fungus Magnaporthe oryzae as model organism and could prove that while maintaining data quality (such as phosphosite and peptide sequence confidence), the data completeness increases dramatically. Since the method presented here reduces the LC-MS/MS analysis from 3 h to 1 h and increases the number of phoshosites identified up to 10-fold in contrast to published studies in fungi, we pushed the phospho-proteomic technique beyond its current limits and could provide a sophisticated resource for investigation of signaling processes in filamentous fungi.