The lack of a reductase activity for strep-MBP-ROXY9 could be caused by the partial oxidation of the protein. Because glutathione is a reductant and is present in the reaction mixtures of all glutaredoxin activity assays, strep-MBP-ROXY9 purified in the presence of DTT should be dialyzed against buffer containing glutathione in order to prevent oxidation of the protein. Because glutathione interferes with the Ellman’s method, a first attempt to determine the oxidation state of strep-MBP-ROXY9 when dialyzed against glutathione by mass spectrometry was made. In addition, protein purified with DTT and protein from which DTT was removed by dialysis, as well as protein incubated for only 10 min with glutathione, was analyzed. At the end of the respective treatment, all samples were immediately precipitated with TCA and then labelled with IAM and iodoTMT: For this, first, all reduced cysteines were blocked with IAM. Subsequently, all oxidized cysteines were reduced with TCEP. Finally, all cysteine residues reduced by TCEP were labelled with iodoTMT. Thus, all originally reduced cysteine residues are marked by IAM whereas all originally oxidized cysteine residues are labelled with iodoTMT. For analysis of the MS data (Supplementary table SR1, part I to IV; Figures R15 and SR5), the number of independent identifications (PSMs) was normalized to the number of independent identifications of two different, unmodified peptides from the MBP tag. For normalization, only those peptides which were fully labelled and completely cleaved by trypsin were chosen, because they were
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present in all samples and corresponded to the most abundant peptides. This normalization allowed a comparison between different treatments for the same peptide. All other peptides, which were sometimes not found in all samples, were considered qualitatively.
Data analysis showed that protein containing DTT was the most strongly reduced preparation of all regarding the active site (Figure R15A to D and SR5A to D). Still, oxidized peptides were discovered in this sample, but their amount cannot be deduced. Removal of DTT, as well as introducing GSH by dialysis while removing DTT resulted in an increase of strep-MBP-ROXY9 with one or none reduced cysteine residues within the active site compared to the DTT-treated sample (Figure R15C and D and SR5C and D). The sample treated for 10 min with GSH after removal of DTT showed constantly low amounts of the different peptides (Figure R15A to D and SR5A to D). Cys49 was partially oxidized and partially reduced. The extent of reduction or oxidation did not vary much between the different treatments (Figure R15E and F and SR5E and F). Similar observations were made for Cys61 with exception of the sample which was treated for 10 min with glutathione (Figure R15G and H and SR5G and H). As for the active site peptides, this 10 min glutathione treatment led to the lowest normalized PSMs for all oxidation states analyzed. However, the effect was less pronounced compared to the active site peptides. Because the initial PSM values are normalized to the amount of protein analyzed by MS, these low numbers of peptides cannot result from lower protein amounts in general. Additionally, the peptides could be detected in other samples excluding difficulties with separation and detection of these peptides during MS. Instead, the low normalized PSM values might result from incomplete labelling and/or incomplete cleavage. Several not fully labelled or cleaved peptides were detected in the four samples (Supplementary table SR1).
Importantly, as not each of these peptides was detected throughout all samples, their number cannot be estimated. Thus, they represent an unknown variable in this analysis. Similarly, this analysis did not include a search for peptides linked by or containing disulphide bridges or cysteine residues oxidized to sulfonic acids. Whereas disulphide bridges can in principle be detected indirectly via reduction with TCEP and iodoTMT labelling, sulfonic acids cannot be reduced by TCEP and therefore escape labelling with iodoTMT [258]. However, enhanced sulfonic acid formation after 10 min treatment with the reducing agent glutathione seems unlikely.
Considering the whole dataset, the contribution of sulfonic acid formation to the number of oxidized peptides and of those modifications which cannot be
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Figure R15. Analysis of the strep-MBP-ROXY9 redox state by LC-MS/MS. Strep-MBP-ROXY9 was purified from insect cells in the presence of DTT and subjected to different oxidizing and reducing treatments: (1) Dialysis to remove DTT; (2) Dialysis to remove DTT and then incubation for 10 min with GSH; (3) Dialysis against buffer containing GSH, to remove DTT while simultaneously applying GSH. All samples were labeled with IAM (reduced cysteines) and iodoTMT (oxidized cysteines), separated in a 10 % non-reducing SDS-PAGE and analyzed by LC-MS/MS as described in Methods (pages 109-110 and 115-119). To allow quantification, the number of different PSMs for each peptide and modification was normalized to the number of PSMs of an unmodified peptide resulting from the MBP tag (LYPFTWDAVR [1267,64698 Da]). As this allows only comparison of the abundance of specific peptides between treatments, the values for the normalized PSMs were plotted accordingly: active site-containing peptides with none (A), one (B), two (C) or three (D) oxidized cysteines; peptides containing Cys49 in a reduced (E) or oxidized (F) state; peptides containing Cys61 in a reduced (G) or oxidized state (H). Please note that the diagrams show only the properly cleaved and fully labelled peptides of each sample. They are the most abundant peptides in each sample. All other peptides are listed in Supplementary table SR1.
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quantified is impossible to estimate. For these reasons, the entire the mass spectrometry analysis must be considered with caution. Despite these limitations, the data from the fully labelled and fully cleaved peptides suggested that strep-MBP-ROXY9 is only partially reduced, even in presence of DTT, and oxidizes more strongly in the active site, when DTT is removed or exchanged for glutathione. However, the nature of these redox modifications and the precise proportion of oxidized protein in all samples except for that from which DTT was removed by dialysis (Figure R11, sample A) remain elusive.