SICyLIA-TMT: a multi-step labelling strategy for global redox proteomics

High levels of reactive oxygen species can cause oxidation of different classes of molecules causing profound consequences on cellular functions. ROS have also been shown to be a signal intermediates that modulate enzyme activity and trigger signalling cascades. Notably, Cysteines are an important stress sensor that swiftly respond to external oxidative stimuli. We have previously developed SICyLIA, a method for accurate measurement of Cysteine oxidation levels in cells and tissues using light or stable-isotope labelled, heavy, iodoacetamide (IAA) as readout for cysteine thiol oxidation. Here we further developed the method, taking advantage of TMT technology (SICyLIA-TMT). SICyLIA-TMT improved the throughput and additionally enabled the measurement of reversibly oxidised cysteines and total protein levels within the same sample. Using SICyLIA-TMT, we also reduce by half both sample preparation and analysis at the mass spectrometry.

To swiftly block free thiols, we add light IAA to lysis buffer. Reversibly-oxidised Cysteines are then reduced, and newly generated thiols blocked with heavy IAA. This creates two labelled populations of free and reversibly-oxidised thiols, which are further labelled with TMT, fractionated and analysed by mass spectrometry. In normal conditions, high levels of intracellular Glutathione maintain the redox balance. This keeps the quantity of reversibly oxidised thiol to a minimum level, which results in making their detection very difficult. To overcome this issue, we use one of the TMT channels for a heavy Iodoacetamide-labelled sample, to act as "carrier channel" to boost the signal of the low-level reversibly oxidised peptides.

We benchmarked the SICyLIA-TMT method against the same samples analysed with the original SICyLIA method. SICyLIA-TMT outperformed the old method in respect to the number of peptides robustly quantified (>9,000 with SICyLIA; >12,000 light and >1,000 heavy with SICyLIA-TMT). Depth of the redox proteome was also evaluated using a new generation mass spectrometer, Thermo Astral.

Finally, SICyLIA-TMT can determine the oxidation state of both free and reversibly-oxidised thiol fraction. . This enables to calculate the oxidation stoichiometry of each Cysteine-, under the assumption that, for each Cysteine residue, the summed intensities of free and reversible oxidised thiol fraction represent 100% of the residue.

In conclusion, we have developed SICyLIA-TMT as a new simple method for proteome-wide analysis of oxidative signalling and proteome dynamics.