title: The efficiency of trypsin digestion for mass-spectrometry-based identification and quantification of oxidized proteins: evaluation of the digestion of oxidized bovine serum albumin.
abstract: In bottom-up proteomics approaches, the enzymatic proteolysis step before mass spectrometry (MS) analysis is of crucial importance, as only the efficient digestion of the protein will ensure its accurate quantification. The structural and chemical alterations occurring upon protein oxidation may decrease the efficiency of trypsin digestion, compromising the ensuing MS analysis. Herein, the efficiency of the trypsin digestion of oxidized bovine serum albumin (BSA) was assessed by protein-sequence coverage and the exponentially modified protein abundance index (emPAI) algorithm, allowing a comparison of protein abundance in samples with different levels of oxidation. Despite the extensive oxidation induced to BSA, verified by analysis of protein carbonyls, no significant difference in the yield of tryptic peptides from oxidized samples could be observed by nano-high-performance liquid chromatography (HPLC) and nano-HPLC7-electrospray ionization-MS analysis. After a database search, similar protein-sequence coverage rates were obtained for both treated and control samples. Thus, exponentially modified protein abundance index scores confirmed that, regardless of being oxidized, the same amount of BSA was present in the sodium dodecyl sulfate/polyacrylamide gel electrophoresis bands excised for digestion. The obtained results show that the digestion of the control and oxidized samples were similar, leading to the conclusion that in-gel proteolysis is not a main hindrance for the identification and quantification of oxidized proteins by MS.
authors.affiliation: Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal.
authors.forename: Duarte D
authors.initials: DD
authors.lastname: Gouveia
authors.affiliation: REQUIMTE/Departmento de Química e Bioquimica, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre S/N, 4169-007 Porto, Portugal.
authors.forename: André M N
authors.initials: AM
authors.lastname: Silva
authors.affiliation: Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal.
authors.forename: Rui
authors.initials: R
authors.lastname: Vitorino
authors.affiliation: Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal.
authors.forename: M Rosário M
authors.initials: MR
authors.lastname: Domingues
authors.affiliation: Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal. p.domingues@ua.pt.
authors.forename: Pedro
authors.initials: P
authors.lastname: Domingues
mesh_terms: D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D010084:Oxidation-Reduction; D040901:Proteomics / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000032:analysis* / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D014357:Trypsin / Q000378:metabolism*
chemical_list: D012710:Serum Albumin, Bovine; D014357:Trypsin