Methylenetetrahydrofolate reductase (MTHFR) is an enzyme that catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH2-H4folate) to 5-methyltetrahydrofolate (CH3-H4folate). Its active form is a homotetramer that contains both a symmetrical and asymmetrical interface. Using gel filtration chromatography, previous research has found that MTHFR dissociates into dimers at low concentrations. To better understand the nature of this dissociation, we utilized hydrogen-deuterium exchange (HDX) in conjunction with liquid chromatography and mass spectrometry (LC-MS) to characterize and compare the dynamics of the MTHFR protein at the peptide level. A challenge in this work is the low concentration of protein required to study the dimer creates requires rapid desalting of digested HDX samples prior to mass spectrometry analysis. In this study, we present data on optimizing the LC methods to allow for the analysis of HDX samples with high salt loads. Preliminary analysis showed us that the differences in deuterium exchange were small between the dimer and tetramer for the most easily detected peptides.

Development of techniques for the comparison of tetramers and dimers of MTHFR with hydrogen-deuterium exchange liquid chromatography and mass spectrometry

255th National Meeting (2018)

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