Enhanced Identifications and Quantification Through Retention Time Down-Sampling in Fast-Cycling Diagonal-PASEF Methods

Data-independent acquisition (DIA) mass spectrometry is essential for comprehensive quantification of proteomes, enabling deeper insights into cellular processes and disease mechanisms. On the timsTOF platform, diagonal-PASEF acquisition methods have recently been introduced to directly and continuously cover the observed diagonal shape of the peptide precursor ion distributions. Diagonal-PASEF has already shown great promise, and its adaptation as a routine workflow can be further pushed with improved method development as well as enhanced algorithmic solutions. Here, we conducted a systematic and comprehensive optimization of diagonal-PASEF for 17-min gradients on the timsTOF HT in conjunction with Spectronaut. We demonstrate that Spectronaut fully supports all tested diagonal-PASEF methods independent of the number of slices or overlaps and with minimal user intervention required. We derive an optimized analysis strategy where we coupled diagonal-PASEF acquisitions to retention time down-sampling by summation (RTsum) and thereby exploit the fast-cycling nature of diagonal-PASEF methods. Through the combination of RTsum with diagonal-PASEF, we demonstrate that this strategy yields higher signal-to-noise ratios while retaining the peak shape for analytes of interest ultimately improving overall number of peptide and protein identifications of diagonal-PASEF. Importantly, combining RTsum with diagonal-PASEF improved overall identifications and quantitative precision when compared to dia-PASEF with variable quadrupole isolation widths and across different input amounts for cell line injections. We also tested the performance of diagonal-PASEF in controlled quantitative experiments where diagonal-PASEF outperformed dia-PASEF in the overall number of retained candidates below 1% or 5% error-rate, quantitative precision, and identifications on peptide level and protein level. These data indicate that RTsum demonstrates a positive use case of the high sampling rate of diagonal-PASEF and might therefore be a valuable addition to existing analysis pipelines. Collectively, our findings imply that diagonal-PASEF is developing into a competitive alternative to dia-PASEF and that the data analysis options are making fast progress.