title: A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma.
abstract: Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues are derivatised with biotin-hydrazide, enriched and characterised by tandem mass spectrometry. The strength of the method lies in an improved elution of biotinylated peptides from monomeric avidin resin using hot water (95°C) and increased sensitivity achieved by reduction of analyte losses during sample preparation and chromatography. For the first time MS/MS data analysis utilising diagnostic biotin fragment ions is used to pinpoint sites of biotin labelling and improve the confidence of carbonyl peptide assignments. We identified a total of 125 carbonylated residues in bovine serum albumin after extensive in vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein samples. The optimised workflow enabled detection of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine, valine, alanine, isoleucine, glutamine, lysine and glutamic acid (+14Da), an oxidised form of methionine - aspartate semialdehyde (-32Da) - and decarboxylated glutamic acid and aspartic acid (-30Da).
  
  
  
  Proteomic tools provide a promising way to decode disease mechanisms at the protein level and help to understand how carbonylation affects protein structure and function. The challenge for future research is to identify the type and nature of oxidised residues to gain a deeper understanding of the mechanism(s) governing carbonylation in cells and organisms and assess their role in disease.
authors.affiliation: Department of Biochemistry and Molecular Biology and VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark; Department of Molecular Biology and Genetics, Science and Technology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark.
authors.forename: Jesper F
authors.initials: JF
authors.lastname: Havelund
authors.affiliation: Department of Biochemistry and Molecular Biology and VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark; The Babraham Institute, CB22 3AT Cambridge, United Kingdom.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Wojdyla
authors.affiliation: Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Blegdamsvej 3, Copenhagen 2200, Denmark.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Davies
authors.affiliation: Department of Biochemistry and Molecular Biology and VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark.
authors.forename: Ole N
authors.initials: ON
authors.lastname: Jensen
authors.affiliation: Department of Molecular Biology and Genetics, Science and Technology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark.
authors.forename: Ian Max
authors.initials: IM
authors.lastname: Møller
authors.affiliation: Department of Biochemistry and Molecular Biology and VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark. Electronic address: adelinar@bmb.sdu.dk.
authors.forename: Adelina
authors.initials: A
authors.lastname: Rogowska-Wrzesinska
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D006801:Humans; D018384:Oxidative Stress; D050050:Protein Carbonylation; D012710:Serum Albumin, Bovine / Q000737:chemistry
chemical_list: D000596:Amino Acids; D001798:Blood Proteins; D012710:Serum Albumin, Bovine; D001710:Biotin
keywords: Biotin-hydrazide; Metal ion-catalysed oxidation; Oxidative stress; Protein oxidation