High-throughput high-resolution data-independent acquisition workflow on an Orbitrap ascend MultiOmics tribrid mass spectrometer for accurate label-free quantitation

Quantitative proteomics is an essential tool for understanding global protein expression and the mechanisms of biological processes and disease states. Accurately quantifying the abundances of proteins of interest in complex samples is a prerequisite for developing suitable statistical models to gain biological insights from experimental data sets. Statistical significance is improved by decreasing variability in measurements and/or increasing the sample set. A suitable analytical workflow addresses the need for reproducible sample preparation, robust separations, high-quality quantitative measurements, and reliable data analysis. In the present study, we developed a high-throughput Velocity LFQ-DIA analysis workflow on the Orbitrap Ascend MultiOmics Tribrid mass spectrometer for comprehensive proteome coverage and excellent quantitation accuracy. To evaluate the performance of the Velocity DIA workflow, HeLa digest and three-proteome mixtures were loaded onto a 50 cm μPAC Neo HPLC column and separated using a Vanquish Neo UHPLC system over 9 min, 30 min, and 60 mins active LC gradients, respectively, before being transferred into the Orbitrap Ascend MultiOmics 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.

Various run times of 9, 30, and 60 minutes were evaluated for a Hela digest standard to meet different throughput needs. With a 30 min active gradient, 7,000+ proteins and 47,000+ peptides were identified, along with a protein group CV of approximately 5%, suggesting that the 30 min active gradient method enables relatively high throughput while maximizing identification and quantitative performance. We extended this workflow to a 60 min active gradient and successfully identified close to 7,800 proteins and >76,000 peptides, highlighting that deeper proteome coverage can be achieved in the Velocity DIA workflow by using a longer gradient.

To test the quantitative accuracy of the Velocity DIA workflow, we created a 3-proteome mix to mimic biological samples where proteins might be up- or down-regulated under different conditions. The Velocity DIA workflow yielded excellent quantitative accuracy across a wide dynamic range, with median values extremely close 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 mass spectrometer. In the 60 min active gradient experiment, >11,000 protein groups were identified. Together, our data demonstrated deep proteome coverage and excellent quantitation accuracy afforded by the Orbitrap Ascend MultiOmics mass spectrometer.