In-depth characterization of Mycolicibacterium smegmatis MC2155 phosphoproteome based on the Ti4+-IMAC enrichment strategy

Abstract:

[Objective] Mycolicibacterium smegmatis was used to explore more efficient strategies for enriching phosphorylated peptides in prokaryotes.[Methods] We evaluated the efficiency of three different methods,TiO₂,Fe³⁺-NTA and Ti⁴⁺-IMAC,for the enrichment of phosphopeptides.Further,we employed two mass spectrometers with different resolutions,Orbitrap Velos and Orbitrap Fusion Lumos,to assess the enrichment stability.[Results] Ti⁴⁺-IMAC was the optimum enrichment method,with the number of phosphopeptides and sites enriched seven-fold that of TiO₂ or Fe³⁺-NTA.TiO₂ and Fe³⁺-NTA showed no significant difference in the number of phosphorylation sites enriched,which was similar to the results of the published works about TiO₂.In addition,the detection results of two different mass spectrometers showed that Ti⁴⁺-IMAC enrichment was stable in two biological duplicate samples.The average phosphorylation sites detected by Lumos was 2.6-fold that by Velos.[Conclusion]Ti⁴⁺-IMAC technique can efficiently accomplish high enrichment of phosphorylation events in M.smegmatis.We identified a total of 2 280 phosphorylated proteins,10 880 phosphorylated peptides and 4 433 credible phosphorylation sites.Ti⁴⁺-IMAC method can be widely used in the phosphoproteomics of other microorganisms.