Accurate Determination of Protein Methionine Oxidation by Stable Isotope Labeling and LC-MS Analysis

Abstract

Methionine (Met) oxidation is a major modification of proteins, which converts Met to Met sulfoxide as the common product. It is challenging to determine the level of Met sulfoxide, because it can be generated during sample preparation and analysis as an artifact. To determine the level of Met sulfoxide in proteins accurately, an isotope labeling and LC-MS peptide mapping method was developed. Met residues in proteins were fully oxidized using hydrogen peroxide enriched with (18)O atoms before sample preparation. Therefore, it was impossible to generate Met sulfoxide as an artifact during sample preparation. The molecular weight difference of 2 Da between Met sulfoxide with the (16)O atom and Met sulfoxide with the (18)O atom was used to differentiate and calculate the level of Met sulfoxide in the sample originally. Using a recombinant monoclonal antibody as a model protein, much lower levels of Met sulfoxide were detected for the two susceptible Met residues with this new method compared to a typical peptide mapping procedure. The results demonstrated efficient elimination of the analytical artifact during LC-MS peptide mapping for the measurement of Met sulfoxide. This method can thus be used when accurate determination of the level of Met sulfoxide is critical.

Convergent signaling pathways—interaction between methionine oxidation and serine/threonine/tyrosine O-phosphorylation

“…This likely explains the strong preference for a negatively charged residue at the −1 position from MetSO observed by Ghesquière et al (2011). Using 18 O stable-isotope labeling to discriminate analytical artifacts, Liu et al (2013) have verified the occurrence of bona fide, physiological MetSO in target proteins.…”

“…However, the common occurrence of Met adjacent to Ser/ Thr/Tyr residues (Rao et al 2013) and the frequent occurrence of MetSO (Ghesquière et al 2011;Liu et al 2013;Salvato et al 2014) near O-phosphorylation-sites (Song et al 2012) suggests that crosstalk between these two PTM's is widespread. (Hardin et al 2009;Miernyk et al 2009), oxidation of Met might directly inhibit O-phosphorylation of a nearby Ser/Thr/Tyr-residue by interfering with the kinase recognition/binding (Fig.…”

Expansion for theBrachylophosaurus canadensisCollagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein

“…The oxidation of methionine is variably observed on one sequence (GQAGVM(OX)GFP(OH)GPK, Figure S9) and is known to occur as an artifact of sample preparation. 59 Hydroxylation of proline, however, is a crucial in vivo PTM for the structure and function of collagen I, as hydroxylated prolines play an integral role in the formation of its triple helical tertiary structure 60,61 and cannot be produced by bacteria. 62 Thus the presence of this modification in collagen sequences derived from fossils supports an endogenous origin.…”

Simultaneous monitoring of oxidation, deamidation, isomerization, and glycosylation of monoclonal antibodies by liquid chromatography-mass spectrometry method with ultrafast tryptic digestion

“…This method adopted an ultrafast trypsin digestion protocol we developed for assays for glycan analysis 44 and site-specific oxidation, 45 and avoided the assay artifacts of oxidation, deamidation, and isomerization induced during the cLC-MS method. 8,[34][35][36][37]39 With uLC-MS available for comparison, assay artifacts for Asn deamidation and Asp isomerization at certain sites were clearly shown in the longer digestion procedure. For example, deamidation at Asn316 of mAb A was »120-fold overestimated by an 18-hour digestion in the cLC-MS compared to the 5-min digestion procedure (60% vs 0.5%).…”

“…The sample digestion step for the method is a time consuming process that could take up to 24 hours at 37 C, a step that could potentially induce artifacts 34 such as asparagine deamidation, 8,[35][36][37] N-terminal glutamine cyclization, 38 and methionine oxidation. 39,40 In addition, the long preparation time hampers the high throughput analysis for a large number of samples in forced degradation studies and clone and media selection.…”