ReCap enables deep, copy number-scaled cysteine redox proteomics with minimal exogenous oxidation

Abstract

Cysteine oxidation analyses require the preservation of the redox state present at harvest and quantitative scaling to relate oxidation to protein copy numbers, rather than only providing fractional oxidation data. Here, we present ReCap, a Redox Capture workflow combining Oxi-DIA, an enrichment-free isotope-encoded DIA workflow, with Oxi-Stop, a simple oxygen-exclusion strategy for cryopreserved tissue. In mouse brains, Oxi-DIA quantified 17,809 cysteine sites belonging to 6,085 protein groups in every sample, enabling matched measurements of residue-resolved oxidation and protein abundance. Atmospheric oxygen exposure during 14 days of cryopreservation distorted the measured cysteine redox state. The resultant increase of an estimated 5.3176 x 1011 {micro}g-1 oxidised cysteine molecules was mitigated by Oxi-Stop, which minimised exogenous oxidation during cryopreservation. Copy-number scaling altered the interpretation of cysteine oxidation values. Although cysteine oxidation was detected across 2,371 sites and 1,439 proteins, 20 sites on abundant proteins accounted for 44% of the oxidised signal. ReCap advances redox proteomics from providing a site catalogue into a biologically weighted map of redox information, revealing cysteine oxidation as a sparse, ordered and quantitatively concentrated signal.