ScanningSWATH enables ultra-fast proteomics using high-flow chromatography and minute-scale gradients

Rapidly emerging applications in data-driven biology and personalised medicine call for the development of fast and reliable proteomic methods. However, the use of fast chromatographic gradients is limited by the mass spectrometers’ sampling rate and signal interferences. Here we present scanningSWATH, a data-independent acquisition method, in which the DIA-typical stepwise windowed acquisition is replaced by continuous scanning with the first quadrupole. ScanningSWATH enables ultra-fast duty cycles as well as to assign precursor masses to the MS2 fragment traces. Furthermore, we have implemented the support for scanningSWATH in DIA-NN, a fully-automated software suite designed to deconvolute fast DIA experiments, which corrects for signal interferences. We show that the combination of scanningSWATH and DIA-NN enables the efficient application of high-flow liquid chromatography (800 μL/min flow rate) to proteomics. High-flow scanningSWATH increases proteomic sample throughput to the minute-scale, while the use of high-flow chromatography hardware improves reliability and robustness. Benchmarking on yeast and human cell lysates, we demonstrate that the proteomic depth achieved with five-minute high-flow scanningSWATH gradients is comparable to that obtained with several times slower nano- and microflow chromatographic gradients, even if compared to most recent studies that used microflow-SWATH or Evosep-DIA methods. The combination of scanningSWATH, advanced data processing, and industry-standard high-flow LC hardware paves a way for a new generation of cheaper and highly consistent proteomic methods. These allow the recording of hundreds of precise quantitative proteomes per day on a single instrument.