Library-free data-independent acquisition mass spectrometry enables comprehensive coverage of the cyanobacterial proteome.

Cyanobacteria have played a leading role in elucidating the fundamental mechanisms behind oxygenic photosynthesis, carbon fixation, the circadian clock, and phototaxis. Such molecular processes rely on proteins at their core. Thus, proteomics has become an indispensable tool in building our understanding of these processes. Amongst the proteomic approaches used, "shotgun proteomics", where complex protein mixtures are enzymatically digested into peptides and analyzed by liquid chromatography-mass spectrometry, has become the go-to technique for whole-proteome analysis. In this study, we introduce shotgun workflows that excel in speed, throughput, and sensitivity, and allow an in-depth description of the cyanobacterial proteome. The main features of these workflows are the improvement of sample cleanup and digestion through single-pot solid phase-enhanced sample preparation (SP3), the adoption of a previously validated trifluoroacetic acid lysis strategy, and the application of library-free data-independent acquisition. Using the established model organism Synechococcus elongatus PCC 7942, we show that these workflows exhibit high quantitative reproducibility and enable the detection of 83% to 85% of all open reading frames, the greatest single-shot coverage achieved so far for a cyanobacterium. These workflows require only a couple of hours of hands-on time and should be applicable to most, if not all, cyanobacterial species. Together with the rapid advancements in mass spectrometry technologies, this work has the potential to accelerate cyanobacterial proteomics.