Scanning SWATH acquisition enables high-throughput proteomics with chromatographic gradients as fast as 30 seconds

Abstract

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.

Acquiring and Analyzing Data Independent Acquisition Proteomics Experiments without Spectrum Libraries

“…Other applications of compressed sensing to achieve higher specificity include higher order multiplexing with MSX, 28 and using a scanning quadrupole. 29,30 These windowing schemes take Most quadrupoles do not have perfectly rectangular isolation efficiency over the complete isolation window and DIA methods must account for the fact that transmission efficiency is lower at the window boundaries. The easiest way to account for this is to add window margins (typically ± 0.5 m/z) to each precursor isolation window, and this works well for normal or variable width windows.…”

Clinical classifiers of COVID-19 infection from novel ultra-high-throughput proteomics

“…Firstly, on this type of fast chromatography, conventional mass spectrometric acquisition schemes do not reach sufficient sampling velocity in data-dependent mode (peaks elute too fast). Secondly, when using data-independent acquisition schemes which do not sample each peak individually, conventional software cannot deconvolute the interference-rich short-gradient data produced (Demichev et al, 2020;Messner et al, 2019) . We were able to overcome these issues, and present an acquisition scheme that bases on 5-minute water to acetonitrile chromatographic gradients at a flow rate of 800 μl/min.…”

“…The high-flow setup used achieved a median peak Full Width at Half Maximum (FWHM) of 3 seconds with a 20-minute gradient length. For comparison, an extensively optimized micro-flow LC (Demichev et al, 2020;Messner et al, 2019) , achieved a FWHM of 5 seconds, at the same gradient length ( Figure 2c). Thus, high-flow gradients as fast as 5 minutes resulted in peak capacities comparable to the highly optimized 20-minute micro-flow setup (Demichev et al, 2020;Messner et al, 2019) (Figure 2d).…”

Data‐Independent Acquisition Mass Spectrometry‐Based Proteomics and Software Tools: A Glimpse in 2020

“…Various schemes of DIA strategies have been implemented since the beginning of this century, including but not limited to Shotgun CID, [9] DIA, [5] MS E , [10] PAcIFIC, [11] AIF, [12] SWATH-MS, [6] MSX, [13] SONAR, [14] WiSIM, [15] BoxCar DIA, [16] Scanning SWATH, [17] diaPASEF, [18] and PulseDIA [19] (Figure 1, Table 1), but not all schemes have been widely revisited. In Shotgun collision-induced dissociation (Shotgun CID) [9] induces in-source fragmentations where all ions co-eluting from chromatographic separation into the mass spectrometer are fragmented in parallel.…”

“…Scanning quadruple DIA (SONAR) [14] uses wide continuously sliding precursor windows for fragmentation, strengthening the correlation of precursor and fragment ions. Similarly, scanning SWATH [17] also uses continuous scanning with first quadruple and fast duty cycles with new analysis tool DIA-NN, [25] enabling high-flow liquid chromatography for high throughput proteomic analysis. Wide selected-ion monitoring (WiSIM)-DIA [15] combines conventional DIA with wide selectedion monitoring windows to partition the precursor m/z space to produce high-quality precursor ion chromatograms.…”