Figure 8.

Determination of absolute –SH/–SOX ratios. A, IodoTMT labeling method for measurement of absolute –SH/–SOX ratios in Arabidopsis mitochondria (Nietzel et al., 2020). Free –SH and –SOX (red bold font) are distinctly tagged by different iodoTMT reagents by labeling –SHs prior to and after TCEP reduction, respectively. Six-plex-TMT quantification was used to measure absolute –SH/–SOX ratios. B, Cys-reactive phosphate tags (CPTs) method for absolute –SH/–SOX ratio quantification (Xiao et al., 2020). Protein extracts are divided into two fractions for labeling both –SHs and –SOXs (without IAM blocking before TCEP) and solely –SOXs (with IAM blocking before TCEP). Subsequently, immobilized metal affinity chromatography (IMAC) is used to capture phosphate tags, and phosphorylation is removed enzymatically. CPT-modified peptides in both fractions are differentially labeled with TMT. After the fractions have been mixed, a quantitative TMT analysis can determine the absolute –SH/–SOX ratios by MS. –SOX, reversible Cys OxiPTM.