title: A novel (18)O inverse labeling-based workflow for accurate bottom-up mass spectrometry quantification of proteins separated by gel electrophoresis.
abstract: In the present work we report on a novel and fast protocol for accurate bottom-up protein quantification that overcomes the drawbacks of in-gel digestion and MALDI analysis, while maintaining their benefits. It relies on the following steps: (i) gel electrophoresis separation of proteins, (ii) fast in-gel protein digestion with trypsin, (iii) (18)O-labeling through the decoupled method, (iv) quantification through selected peptides previously chosen using the (18)O inverse labeling approach and that, finally, (v) it takes advantage of software specifically developed to select the peptides that will drive the quantification of the protein in an automated mode. We have accurately quantified the following six proteins: glycogen phosphorylase, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor, and α-lactalbumin. As a case study we have quantified the protein vitellogenin in plasma of Cyprinus carpio exposed to high levels of estrogens. The proposed new protocol was validated against the traditional ELISA method; both were found to provide comparable results (non-parametric Mann-Whitney U-test).
authors.affiliation: REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa, Caparica, Portugal.
authors.forename: Hugo M
authors.initials: HM
authors.lastname: Santos
authors.forename: Daniel
authors.initials: D
authors.lastname: Glez-Peña
authors.forename: Miguel
authors.initials: M
authors.lastname: Reboiro-Jato
authors.forename: Florentino
authors.initials: F
authors.lastname: Fdez-Riverola
authors.forename: Mário S
authors.initials: MS
authors.lastname: Diniz
authors.forename: Carlos
authors.initials: C
authors.lastname: Lodeiro
authors.forename: José-Luis
authors.initials: JL
authors.lastname: Capelo-Martínez
mesh_terms: D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D005399:Fishes; D007553:Isotope Labeling / Q000379:methods*; D008956:Models, Chemical; D010103:Oxygen Isotopes / Q000032:analysis* / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D018709:Statistics, Nonparametric; D014357:Trypsin / Q000378:metabolism
chemical_list: D010103:Oxygen Isotopes; D010446:Peptide Fragments; D011506:Proteins; D014357:Trypsin