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Interactive, Symbiotic and you may Parasitic – The step step three tic(k)s of every work-matchmaking

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Profile the first step portrays the brand new SICyLIA workflow so you’re able to on your own evaluate cysteine oxidization in two ranged trials toward an entire proteome level

Figure the initial step illustrates this new SICyLIA workflow so you’re able to oneself contrast cysteine oxidization in two varied samples to your a whole proteome measure

Proteomic measurement from globally cysteine oxidization

Control and oxidatively stressed cells or tissue samples were extracted separately in the presence of either light ( 12 C₂H_cuatroINO) or stable isotope-labelled heavy ( 1step three C₂D₂H₂INO) IAM to alkylate reduced cysteine thiols (SH), coupling a carbamidomethyl (CAM) group to the cysteine residue. After labelling , equal amounts of protein extracts were mixed using a label-swap replication strategy and treated with dithiothreitol (DTT) to reduce reversibly oxidised thiols, which were subsequently blocked with n-ethylmaleimide (NEM). Proteomes were then digested and peptides fractionated using off-line high pH reversed phase chromatography prior to UHPLC-MS/MS analysis on a Q-Exactive HF. Cysteine oxidation ratios are calculated using the MaxQuant computational platform 20 based on the abundance of light and heavy CAM-modified peptide pairs for each cysteine-containing unique peptide. As IAM reacts with reduced cysteine thiols, a -modification for a given peptide indicates increased cysteine oxidation. Whereas changes in the levels of reduced cysteine between samples undergoing a short-term treatment can be compared immediately (Fig. 1a ), different cell lines or tissues derived from different mice have distinct proteomes and require normalisation for protein levels. For relative protein quantification, stable isotope dimethyl labelling 26 was used in con labelling (Fig. 1b ). This method follows a comparable workflow as described above, streamlining these parallel procedures. As shown in Fig. 1b , a fraction of the lysates used to prepare IAM-labelled samples are digested and dimethylated with either light (H 12 CHO/NaBH₃CN) or heavy (D 13 CDO/NaBD₃CN) formaldehyde/sodium cyanoborohydride, mixed in equal ratios using a label-swap replication strategy for independent replicates, and subjected to high pH reversed phase chromatography fractionation before UHPLC-MS/MS analysis. Continue Reading