Short Gradient Multi-CV FAIMSpro BoxCar DIA Facilitates Quantitative Time-Course Analysis of Osmotic and Salt Stress in Arabidopsis thaliana – Technical Dataset

A major limitation in quantitative time-course proteomics is the tradeoff between depth-of-analysis and speed-of-analysis. In high complexity, high dynamic samples such as plant extracts, this is tradeoff is especially apparent. To address this, we evaluate multiple composition voltage (CV) High Field Asymetric Waveform Ion Mobility Spectrometry (FAIMSpro) settings using the latest label-free, single-shot Orbitrap-based DIA acquisition workflows for their ability to deeply-quantify the Arabidopsis thaliana seedling proteome at microliter per minute flow rates. Using a micro-flow BoxCar DIA acquisition workflow with -30, -50, -70 CV FAIMSpro settings we are able to consistently quantify >5000 Arabidopsis seedling proteins over a 21-minute gradient, facilitating the analysis of ~48 samples per day. Utilizing this acquisition approach, we next performed an abiotic stress time-course experiment, whereby we quantified proteome-level changes occurring in Arabidopsis seedling shoots and roots over 24 h of salt and osmotic stress. Here, we successfully quantify over >6400 shoot and >8500 root protein groups, respectively, quantifying nearly 9700 unique protein groups total across the study. Collectively, we pioneer a fast-flow, multi-CV FAIMSpro BoxCar DIA acquisition workflow that represents an exciting new analysis approach for quantitative time-course proteomics experimentation in plants.