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

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A new "one-pot" SPROX workflow uses data-independent acquisition (DIA) and label-free quantification for protein folding stability measurements. This method achieves proteomic coverage comparable to DDA-SPROX without fractionation.

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Area of Science:

  • Proteomics
  • Biochemistry
  • Mass Spectrometry

Background:

  • The stability of proteins from the rates of oxidation (SPROX) technique measures protein folding stability on a proteomic scale.
  • Current SPROX methods primarily use quantitative bottom-up proteomics with data-dependent acquisition (DDA) and isobaric mass tags.
  • Isobaric tags enable multiplexing but limit proteomic coverage compared to data-independent acquisition (DIA).

Purpose of the Study:

  • To develop a novel "one-pot" SPROX workflow utilizing DIA and label-free quantification.
  • To assess the proteomic coverage and accuracy of the new DIA-SPROX strategy.
  • To identify ligand-induced changes in protein stability and target proteins, using cyclosporine A (CsA) as a model ligand.

Main Methods:

  • A "one-pot" SPROX workflow was established using DIA readout and label-free quantification.
  • Proteins from *E. coli* cell lysate were analyzed to calculate transition midpoints.
  • Proteins from *S. cerevisiae* cell lysate were assessed for CsA-induced changes in transition midpoints.
  • Data processing was performed using Spectronaut and DIA-NN software for DIA data analysis.

Main Results:

  • The DIA-SPROX strategy accurately calculated transition midpoints for *E. coli* proteins.
  • Known protein targets of CsA were identified in *S. cerevisiae* with a low false positive rate.
  • Proteomic coverage with DIA-SPROX was comparable to conventional DDA-SPROX.
  • Comparable coverage was achieved without fractionation strategies.

Conclusions:

  • The developed DIA-SPROX workflow offers a robust alternative to DDA-based methods for protein stability measurements.
  • This approach expands the applicability of SPROX to DIA strategies, potentially increasing proteomic coverage.
  • The method successfully identified CsA targets, demonstrating its utility in ligand-target interaction studies.