DIA-SPROX E.coli and S.cerevisiae Midpoints

ABSTRACT: The stability of proteins from rates of oxidation (SPROX) technique is a mass spectrometry-based approach for making protein folding stability measurements on the proteomics scale. The development and application of SPROX, to date, has involved 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, use of such isobaric mass tags is restricted to DDA strategies, which can be limited in their proteomic coverage compared to data independent acquisition (DIA) strategies. Reported here is a new “one-pot” SPROX workflow that employs a DIA readout and a label-free quantitation strategy. Analysis of the proteins in an E.coli cell lysate using our DIA-SPROX strategy allowed for calculating transition midpoints with reasonable accuracy. The proteins from a S.cerevisiae cell lysate were also assessed for ligand induced changes in their transition midpoints upon the introduction of cyclosporine A (CsA) to identify protein targets of this well-studied ligand. Using DIA-SPROX, we 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 using a methionine-containing peptide enrichment strategy in DIA-SPROX.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Natalie Labbe  

LAB HEAD: Michael C.

PROVIDER: PXD066697 | Pride | 2026-03-03

REPOSITORIES: Pride