Complex Nature of Protein Carbonylation Specificity After Metal-Catalyzed Oxidation.
Protein carbonylation is an irreversible modification of Lys, Arg, Thr and Pro amino acids under conditions of oxidative stress. Previous studies have reported specific carbonylated residues in purified recombinant albumins, albeit with a lack of agreement between the studies. Currently, structural factors that determine site-specific protein carbonylation are not well understood.



In this study, we utilized metal-catalyzed oxidizing conditions to generate carbonylation in recombinant human serum albumin (HSA) and granulocyte-colony stimulating factor (G-CSF), two proteins with distinct metal-binding abilities. To estimate predictability of HSA carbonylation sites, the same oxidative reaction was repeated based on the previously reported conditions. For G-CSF, oxidative conditions were gradually adjusted to achieve substantial levels of protein carbonylation. Corresponding accumulation of specific oxidized residues was identified and confirmed with high-resolution mass spectrometry.



Our HSA dataset contained 55 carbonylated residues and showed a significant overlap with the previously published pooled data, indicating a certain level of carbonylation site specificity for albumins. Oxidation of G-CSF under multiple oxidative conditions consistently showed a highly specific carbonylation at position Pro45. We also detected a previously unreported, oxidation-induced cleavage site in G-CSF between His44 and Pro45, which might be attributed to a presence of a potential metal-binding site near residue Pro45.



Our results show distinct patterns of protein carbonylation for HSA and G-CSF. Thus, specificity of protein carbonylation induced by metal-catalyzed oxidation is protein dependent and might be predicted by availability of transition metal binding site(s) within the protein.
Laboratory of Applied Biochemistry, Division of Biotechnology Products Research and Review III, Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Silver Spring, MD, 20993, USA.
Dmitry
D
Kryndushkin
Office of the Director, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, 20993, USA.
Wells W
WW
Wu
Laboratory of Applied Biochemistry, Division of Biotechnology Products Research and Review III, Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Silver Spring, MD, 20993, USA.
Ramesh
R
Venna
Laboratory of Applied Biochemistry, Division of Biotechnology Products Research and Review III, Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Silver Spring, MD, 20993, USA.
Michael A
MA
Norcross
Office of the Director, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, 20993, USA.
Rong-Fong
RF
Shen
Laboratory of Applied Biochemistry, Division of Biotechnology Products Research and Review III, Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Silver Spring, MD, 20993, USA. ashutosh.rao@fda.hhs.gov.
V Ashutosh
VA
Rao
D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D055162:Biocatalysis; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D006801:Humans; D008670:Metals / Q000737:chemistry*; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D011485:Protein Binding; D050050:Protein Carbonylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry*
D000596:Amino Acids; D008670:Metals; D011994:Recombinant Proteins; D012709:Serum Albumin; D016179:Granulocyte Colony-Stimulating Factor
carbonylation; protein oxidation; specificity; transition metal