A novel 18O inverse labeling-based workflow for accurate bottom-up mass spectrometry quantification of proteins separated by gel electrophoresis

Hugo M. Santos, Daniel Glez-Peña, Miguel Reboiro-Jato, Florentino Fdez-Riverola, Mário S. Diniz, Carlos Lodeiro, José Luis Capelo-Martínez

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

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) 18O-labeling through the decoupled method, (iv) quantification through selected peptides previously chosen using the 18O 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).

Original languageEnglish
Pages (from-to)3407-3419
Number of pages13
JournalElectrophoresis
Volume31
Issue number20
DOIs
Publication statusPublished - 1 Oct 2010

Keywords

  • Biomarker
  • DPD software
  • Gel electrophoresis
  • Protein quantification
  • Vitellogenin

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