Achieving close-to-complete proteome coverage in single-shot deep dive DIA workflows

Throughout the history of LC-MS based proteomics, comprehensive identification and quantification of all proteoforms present in biological samples has been the ultimate ambition. Several approaches have been close to achieving this goal, with the deepest coverage commonly obtained by fractionation of digested proteins using a separation mechanism orthogonal to low-pH reversed phase separation (SCX, high-pH C18) and subsequent LC-MS analysis of isobaric labeled peptide samples. As recent innovations in LC-MS instrumentation have leveraged significant increases in analysis speed, resolution and sensitivity, we investigate the possibility to achieve similar coverage using single-shot label-free analyses with ultra-high peak capacity LC-MS. Hereby significantly reducing the labor-intensiveness, overall cost and required analytical skill needed to achieve close to fulll proteome coverage.

Human proteome samples were analyzed using an Orbitrap mass spectrometer coupled to a Vanquish Neo UHPLC instrument. For spectral library generation, protein digest samples were fractionated into 8 concatenated fractions at pH 5.5. For single-shot analyses, lyophilized and unfractionated cell digests were resuspended in 40 µl 0.1 % FA to obtain a stock solution of 500 ng/µl. Reversed phase LC separation was performed on a 110 cm µPAC Neo column with gradient lengths up to 180 min. Both DDA and DIA acquisition strategies were used to collect data. LC-MS data were searched using Spectronaut 17 or a beta version of Thermo Scientific Proteome Discoverer 3.1.

In single-shot deep dive proteomic experiments, highest possible peak capacities are key to reduce sample complexity as much as possible prior to MS/MS analysis. To increase separation performance for long gradient analyses, a 110 cm long microfluidic channel uniformly filled with 2.5 µm pillars was used, significantly increasing peak capacities to well above 1600 in single-shot analyses. Median peak widths as low as 4.6 and 7.2 s have been obtained for respective gradient lengths of 90 and 180 min, which is 30% better than reported up to now.

Evaluating the effect of increased separation performance on proteome depth for single species tryptic digests,following systematic optimization of MS acquisition settings, demonstrated that significant increases in depth were achieved by operating in DIA mode, with up 20% deeper coverage as compared to the best performing DDA method we tested. Using a library free approach, close to 10.000 protein groups were consistently identified with 180 min gradient times. We will further optimize LC-MS methods towards increased throughput and evaluate the impact of high-field asymmetric waveform ion mobility spectrometry (FAIMS) on the proteome coverage that can be achieved within single-shot LC-MS analyses.