However, no C598SEIWDR CL peptides were found, highlighting the need for YAP1C enrichment strategies

However, no C598SEIWDR CL peptides were found, highlighting the need for YAP1C enrichment strategies. redox sensitivity of Cys residues depends on its to more stable oxidation forms (Roos CL2A and Messens, 2011; Gupta and Carroll, 2014). For instance, -SOH can form intra- or intermolecular disulfides or mixed disulfides with another free thiol or glutathione (GSH), making it enzymatically reversible by the action of thioredoxins (TRXs) or glutaredoxins (GRXs), respectively (Roos and Messens, 2011; Akter et al., 2015b). Recently, extracellular H2O2 has been shown to be sensed through disulfide formation of extracellular cysteines in the plasma membrane receptor HYDROGEN PEROXIDE-INDUCED Ca2+ INCREASES 1 (HPCA1), leading to Ca2+ influx in guard cells (Wu et al., 2020). Conversely, besides disulfide formation, -SOH can further oxidize toward sulfinic (-SO2H) and sulfonic acid (-SO3H). Whereas -SO3H is generally considered as an irreversible modification associated with protein degradation (Huang et al., 2018), -SO2H can be reduced via sulfiredoxins (SRXs) (Biteau et al., 2003; Akter et al., 2018). Protein detection of and human cells (Leonard et al., 2009; Paulsen et al., 2012; Akter et al., 2015a). CL2A Further advancements in affinity-based enrichment strategies allowed the accurate identification of the sulfenylated cysteine residues within the proteins in both human and herb cells (Yang et al., 2014; Yang J. et al., 2015; Akter et al., 2018; Huang et al., 2019). In addition to these chemoproteomics approaches, a genetic construct based on the yeast ((Takanishi et al., 2007), yeast (Takanishi and Wood, 2011), and the legume model herb (Oger et al., 2012). In cells, we generated a Yap1-cCRD construct fused to a tandem affinity purification (TAP) tag for improved capture and downstream identification of cytosol and chloroplast, respectively (Waszczak et al., 2014; De Smet et al., 2019), but the sulfenylated cysteines remained unknown. Here, we describe how a tailored double affinity purification strategy enables the identification of sulfenylated cysteines in a noninvasive manner. Materials and Methods Herb Materials and Growth Conditions Transgenic cells expressing the YAP1C construct were generated as CL2A previously reported (Waszczak et al., 2014). In summary, the Yap1 C-terminal cysteine-rich domain name (cCRD) construct, entailing the Yap1-coding region corresponding to Asn565 to Asn650, was codon-optimized for expression in (L.) Heynh. and synthesized with introduction of the mutations Cys620Ala and Cys629Thr. This genetic construct was fused with an N-terminal TAP CL2A tag, made up of two IgG-binding domains of protein G and a streptavidin-binding peptide (SBP), separated by the Human Rhinovirus (HRV) 3C protease cleavage site. The YAP1C probe driven by a cauliflower mosaic virus 35S promoter was transformed in cells. YAP1C expression levels were assessed by western blot analysis (Waszczak et al., 2014). The PSB-D cell suspension cultures (NASC stock no. “type”:”entrez-protein”,”attrs”:”text”:”CCL84840″,”term_id”:”549381322″,”term_text”:”CCL84840″CCL84840) were maintained as described in the ABRC Cell Culture Handling Protocol1. For H2O2 treatments, 500 mL KLF4 of mid-log phase (3 days after culture refreshing, OD600 = 0.9) cells in 1-L glass flasks were treated with 20 mM H2O2 for 30 min before the cells were harvested through a vacuum filtration system (Pall Corporation, Port Washington, NY, United States) and snap-frozen in liquid nitrogen before storage at ?70C. Protein Extraction Frozen cell pellets harvested from approximately 1 L of suspension cultures were crushed with fine quartz granules (Merck, Darmstadt, Germany) with a precooled mortar and pestle in ice-cold lysis buffer (25 mM Tris, 15 mM MgCl2, 150 mM NaCl, 15 mM pNO2 PhenylPO4, 60 mM -glycerophosphate, 0.1% NP-40, 0.1 mM Na3VO4, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 M for 20 min; 4C), the supernatant was collected and protein concentrations were decided with the Bradford CL2A Protein Assay (He, 2011). Anti-C598SEIWDR Antibody Production and Its Coupling on Magnetic Beads The C598SEIWDR peptide was synthetized (purity 85%) and conjugated to Keyhole.