Liquid chromatography setup-dependent artefactual methionine oxidation of peptides: The importance of an adapted quality control process

Post-translational modifications (PTM) can be important indicators of biological activity at the individual protein and/or proteome level, but they can also occur during sample processing. For example, methionine oxidation is a reversible PTM that can occur as part of cellular redox-regulation, which may influence protein structure, stability, and function, and may play a role in disease and ageing. Alternately, it can also occur during LC-MS/MS during reversed-phase (RP) chromatographic separation. In a recent paper by Baumans et al. in The Journal of Chromatography A, researchers look at different RP variables, including column age, sample trapping time, gradient length, sample load, and types of stationary phases to determine if any of these factors influenced the amount of processing-related methionine oxidation in both a standardized peptide and complex lysates. Their result suggest that certain variables can be targeted to reduce undesired oxidation of methionine residues, and that the use of a quality control standard may be beneficial for differentiating between biologically relevant fluctuations in methionine oxidation and those that are an artefact of your LC system at the time of use.

How was PEAKS used?

The database search workflow in PEAKS Studio software v.10.5 was used to identify proteins in HeLa digests and plasma samples. In this workflow, oxidation of methionine was set as a variable modification to identify peptides that contained the target PTM. Following identification in PEAKS, the modified peptides were manually selected in Skyline software v. (MacCoss Lab Software, University of Washington, WA, USA) for further examination.

Baumans F, Hanozin E, Baiwir D, Decroo C, Wattiez R, De Pauw E, Eppe G, Mazzucchelli G. Liquid chromatography setup-dependent artefactual methionine oxidation of peptides: The importance of an adapted quality control process. J Chromatogr A. 2021 Sep 27;1654:462449. doi:10.1016/j.chroma.2021.462449. Epub 2021 Aug 5. PMID: 34399143.


In both biologics quality control experiments and protein post-translational modification studies, the analytical system used is not supposed to bring any artefactual modifications which could impair the results. In this work, we investigated oxidation of methionine-containing peptides during reversed-phase (RP) chromatographic separation. We first used a synthetic methionine-containing peptide to evaluate this artefactual phenomenon and then considered more complex samples (i.e., plasma and HeLa protein digests). The methionine oxidation levels of the peptides were systematically assessed and compared for the long-term use of the analytical column, the sample trapping time, the gradient length, the sample load and the nature of the stationary phase (HSS T3 from Waters, YMC Triart C18 from YMC Europe GmbH and BEH130 C18 from Waters). In addition to the oxidation of methionine in solution, we observed on the HSS T3 and the BEH130 stationary phases an additional broad peak corresponding to an on-column oxidized species. Considering the HSS T3 phase, our results highlight that the on-column oxidation level significantly increases with the age of the analytical column and the gradient length and reaches 56 % when a 1-year-old column set is used with a 180 min-long LC method. These levels go to 0 % and 18 % for the YMC Triart C18 and the BEH130 C18 phases respectively. Interestingly, the on-column oxidation proportion decreases as the injected sample load increases suggesting the presence of a discrete number of oxidation sites within the stationary phase of the analytical column. Those findings observed in different laboratories using distinct set of columns, albeit to varying degrees, strengthen the need for a standard of methionine-containing peptide that could be used as a quality control to appraise the status of the liquid chromatographic columns.