Traditional data-dependent acquisition (DDA) approaches have been widely employed for LFQ experiments, but they suffer from run-to-run inconsistencies due to intensity-based stochastic triggering of precursors, often leading to undersampling of low-abundant proteins. Missing values become more likely as sample size increases, so data-independent acquisition (DIA) has emerged as a popular technique for large scale quantitative analyses. A suitable analytical workflow addresses the need for reproducible sample preparation, robust separations, high-quality quantitative measurements, and reliable data analysis. In this study, we developed a high-throughput Velocity DIA workflow on the Orbitrap Exploris 480 mass spectrometer with in-depth proteome coverage and highly accurate label-free quantitation. As an evaluation of performance, HeLa digest and three-proteome mixtures were loaded onto a 50 cm μPAC Neo HPLC column in direct injection mode using a Vanquish Neo UHPLC system over 9 min, 30 min, and 60 mins active LC gradients, respectively, before being transferred into the Orbitrap Exploris 480 mass spectrometer. Acquired data has been processed by Spectronaut (Biognosys, v18) using a directDIA approach, DIA-NN (v1.8.1) or Proteome Discoverer software (v3.1.0.638) using CHIMERYS™ intelligent search algorithm by MSAID.

With a 30 min active gradient, 6,300+ proteins and 40,000+ peptides were identified, along with a protein group CV of approximately 5%, suggesting that the 30 min active gradient method provides the strongest analytical balance to achieve throughput while maximizing identification and quantitative performance. We extended this workflow to a 60 min active gradient and successfully identified close to 7,200 proteins and >60,000 peptides, highlighting that deeper proteome coverage can be achieved in the Velocity DIA workflow by using a longer gradient. To test the robustness and reproducibility of quantitation accuracy, in this data set, we used two samples with different amounts of spiked microbial proteins to mimic biological samples where proteins might be up- or downregulated under different conditions. The Velocity DIA workflow used for relative quantitation of e.coli and Yeast proteins in a high amount of human peptides as background yields excellent quantitation accuracy across all ratios, with median values exhibit a high degree of proximity to the theoretical ratios, as well as a narrow distribution of all data points around the median values, indicating high quantitative accuracy and precision of the workflow.

Additionally, the three-proteome mix experiment further highlights the proteome depth that can be achieved with an Orbitrap Exploris 480 MS. In the 60 min active gradient, >10,000 protein groups were identified. In conclusion, we demonstrate the high-throughput, high-resolution and high quantitative precision, and accuracy of the Velocity DIA workflow for label-free quantitation on an Orbitrap Exploris 480 MS.