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 E. coli cell lysate
using the DIA-SPROX strategy allowed for the calculation of 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 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.