Data-Independent Acquisition (DIA) Strategy for Measuring Protein Stability Using Stability of Proteins from Rates of Oxidation (SPROX)

The stability of proteins from the rates of oxidation (SPROX) technique is a mass spectrometry-based approach for making protein folding stability measurements on the proteomic scale. The development and application of SPROX, to date, have primarily relied on the use of quantitative bottom-up proteomics and data-dependent acquisition (DDA) strategies using isobaric mass tags. Use of isobaric mass tags is attractive, as it enables the mass spectrometry readout in SPROX to be highly multiplexed. However, the use of such isobaric mass tags is restricted to DDA strategies, which can be limited in their proteomic coverage compared with data-independent acquisition (DIA) strategies. Reported here is a new "one-pot" SPROX workflow that employs a DIA readout and a label-free quantification strategy. Analysis of the proteins in an <i>E. coli</i> cell lysate using the DIA-SPROX strategy allowed for the calculation of transition midpoints with reasonable accuracy. The proteins from a <i>S. cerevisiae</i> cell lysate were also assessed for ligand-induced changes in their transition midpoints upon the introduction of cyclosporine A (CsA) to identify the protein targets of this well-studied ligand. The DIA-SPROX strategy developed here successfully identified known protein targets of CsA with a low false positive rate using a combination of two different software, Spectronaut and DIA-NN, for DIA data processing. We also find that the proteomic coverage obtained using DIA-SPROX is comparable to the coverage obtained in conventional DDA-SPROX experiments. Significantly, this comparable coverage can be achieved without a fractionation strategy (e.g., methionine-containing peptide enrichment) in DIA-SPROX.