New methodology to monitor the oxidation of Met due to LC separation

In the context of biomarker discovery and their absolute quantification in complex samples, we are developing a standardization strategy aiming to control the entire sample preparation process before LC-MS analysis. The strategy involves the use of a chimeric protein and different levels of its heavy peptides spiked at opportune moment in the sample during sample processing. Among these peptides, some containing a methionine are inserted. Methionine oxidation is a well-known protein modification occurring in vivo but also in vitro and may result in structural and biological changes of the protein. Controlling these artefactual modifications is then of great interest and will be possible thanks to our strategy.
To address the origin of methionine oxidation, we sought differential oxidation levels comparing direct infusion, LC-MS using new columns and LC-MS using old ones. For the last two, an Acquity UPLC M-Class system with a nanoEase MZ symmetry C18 trap column and a NanoEase MZ HSS T3 analytical column has been used.
The direct infusion of pure synthetic peptides in a Q ExactiveTM Hybrid Quadrupole-OrbitrapTM showed a low percentage of oxidized peptide (less than 2%). Once injected onto the M-Class system coupled online with the Q Exactive, this percentage jumps to higher value (50-60% dependant of the peptide injected). The same peptides have been injected on the same M-Class system on which both trapping and analytical columns were replaced by new ones. Methionine oxidation in this case, was as low as in the direct infusion, meaning that the previous use of columns has a huge influence on the oxidation of methionine in pure synthetic peptides. This effect is certainly due to the presence of metal ions in the columns that promote methionine oxidation during chromatography separation. Furthermore, when looking at the chromatogram, one can see a difference between the shape of the oxidized peak from new columns and old columns. When using new columns, the shape of the oxidized peptide peak is sharp whereas on old columns its shape is broadened and extended. In both cases, the peak of the native peptide stays sharp, proving that this modification occurs during the chromatographic separation. The same effect was seen on peptides coming from the injection of a complex sample (i.e. commercial HeLa protein digest). Indeed, the percentage of oxidized peptides varies from 1 to 8% when using the new columns and increases to 20% to 80% when injecting the same HeLa digest on the old columns.
The use history of a column greatly impacts the oxidation degree of peptides and has to be taken into account when studying methionine oxidation. Future works will be dedicated to the implementation of strategies that could stabilize the oxidation during chromatography separation.