2.10. Affinitiy Chromatography and Mass Spectrometry
This protocol is extracted from research article:
Function and Regulation of Chloroplast Peroxiredoxin IIE
Antioxidants (Basel), Jan 21, 2021; DOI: 10.3390/antiox10020152

Reduced His-tagged PRX-IIE (3 mg) or PRX-IIE C146S (3 mg) were bound to 1 mL Ni-NTA resin (Qiagen, Hilden, Germany) and used as an affinity matrix. Ni-NTA matrix without PRX-IIE served as control. Leaves of about 5-week-old plants were homogenized in 50 mM Tris-HCl, pH 8.0, 1 mM PMSF, and afterward, filtrated through Miracloth. Clear protein extract was obtained via centrifugation (30 min at 20,000 rpm and 4 °C). The supernatant (about 40 mg protein) was applied to the matrix and incubated at RT with gentle agitation for 1.5 h. Non-bound material was removed by washing the column with 20 mL of 50 mM Tris-HCl, pH 8.0, and 20 mL of 50 mM Tris-HCl, pH 8.0, 200 mM NaCl. The first elution step was achieved with 1 mL of 50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 50 mM DTT, and incubation for 15 min at RT. The eluted fraction was collected and stored. Afterward, the columns were washed with 10 mL 50 mM Tris-HCl, pH 8.0, 200 mM NaCl. The second elution step was achieved by using a high concentration of imidazole, therefore 1 mL 50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 50 mM DTT, and 500 mM imidazole were applied to the column and incubated at RT for 15 min. The second elution step was also collected and stored. The samples of the first and second elution were trypsinated after chloroform/methanol precipitation [25] and dissolved in 0.1% formic acid, 1% acetonitrile. Peptides were separated by reverse-phase nano-LC and analyzed by electrospray ionization-mass spectrometry (ESI-QTOF-MS) as described [26]. Data were searched against the entries of UP000006548 3702 ARATH A. thaliana of the UniProt database using ProteinLynx Global Server 3.0.2. Proteins, which were found in two out of three biological experiments with at least two peptides were accepted for further analysis. In addition, proteins, which were identified in the control sample (nonspecific binding), were removed from protein lists. The LC-MS data are deposited using the e!DAL system of IPK Gatersleben [27] and available at http://dx.doi.org/10.5447/ipk/2021/0 [28].

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