Methionine sulfoxides in serum proteins as potential clinical biomarkers of oxidative stress

Abstract

Oxidative stress contributes to the pathophysiology of a variety of diseases, and circulating biomarkers of its severity remains a topic of great interest for researchers. Our peptidomic strategy enables accurate and reproducible analysis of circulating proteins/peptides with or without post-translational modifications. Conventional wisdom holds that hydrophobic methionines exposed to an aqueous environment or experimental handling procedures are vulnerable to oxidation. However, we show that the mass spectra intensity ratio of oxidized to non-oxidized methionine residues in serum tryptic proteins can be accurately quantified using a single drop of human serum and give stable and reproducible results. Our data demonstrate that two methionine residues in serum albumin (Met-111 and Met-147) are highly oxidized to methionine sulfoxide in patients with diabetes and renal failure and in healthy smokers versus non-smoker controls. This label-free mass spectrometry approach to quantify redox changes in methionine residues should facilitate the identification of additional circulating biomarkers suitable for predicting the development or progression of human diseases.

Oxidised Met147 of human serum albumin is a biomarker of oxidative stress, reflecting glycaemic fluctuations and hypoglycaemia in diabetes

“…Improved methodology for the quantification of oxidised and non-oxidised Met-containing serum tryptic peptides. We first sought to improve the accuracy and reproducibility of our previous methodology for the quantification of the oxidation of Met residues in serum tryptic proteins 28 . We previously found that the ratio of trypsin-digested serum albumin fragments containing oxidised and a non-oxidised Met residues at position 147 of human serum albumin, Alb(Met 147 O) and Alb(Met 147 ), is one of the most promising potential clinical biomarker of intravascular redox status among the Met-containing tryptic serum proteins identified using a proteomic strategy 28 .…”

“…We first sought to improve the accuracy and reproducibility of our previous methodology for the quantification of the oxidation of Met residues in serum tryptic proteins 28 . We previously found that the ratio of trypsin-digested serum albumin fragments containing oxidised and a non-oxidised Met residues at position 147 of human serum albumin, Alb(Met 147 O) and Alb(Met 147 ), is one of the most promising potential clinical biomarker of intravascular redox status among the Met-containing tryptic serum proteins identified using a proteomic strategy 28 . Because the use of stable isotope-labelled peptides is known to enable the accurate quantification of peptide concentrations in biological samples, we synthesized two stable isotope-labelled peptides, SI-Alb(Met 147 ) and SI-Alb(Met 147 O), corresponding to the tryptic peptides, Alb(Met 147 ) and Alb(Met 147 O), respectively.…”

“…Finally, we assessed the advantage of this updated methodology over our previous method that simply measured signal intensity ratios of Alb(Met 147 O) and Alb(Met 147 ) ([Alb(Met 147 O]/[Alb(Met 147 )]) without the use of stable isotope-labelled peptides 28 . We reanalysed all clinical samples pretreated with L-Met and L-Cys to determine [Alb(Met 147 O]/[Alb(Met 147 )] and performed exactly the same statistical analyses as described above.…”

“…(LC-MS), irrespective of the time the blood sample is left to clot at room temperature before centrifugation or repeated freeze/thaw cycles 28 . This may be because of the absence of MetO reductase activity in human blood 29 , although these have not been well characterised to date.…”

Identification of plasma binding proteins for glucose-dependent insulinotropic polypeptide

“…For the LC-MS/MS analysis, an ultra-sensitive LC-MS system combining LC (Easy-n LC 1000, Thermo Fisher Scientific) and Q-Exactive (Thermo Fisher Scientific) was used as described [14]. Database searches were performed using the SEQUEST algorithm incorporated into Proteome Discoverer 1.4.0.288 software (Thermo Fisher Scientific).…”