TY - JOUR
T1 - A novel 18O inverse labeling-based workflow for accurate bottom-up mass spectrometry quantification of proteins separated by gel electrophoresis
AU - Santos, Hugo M.
AU - Glez-Peña, Daniel
AU - Reboiro-Jato, Miguel
AU - Fdez-Riverola, Florentino
AU - Diniz, Mário S.
AU - Lodeiro, Carlos
AU - Capelo-Martínez, José Luis
N1 - H. M. Santos acknowledges the doctoral grant SRFH/BD/38509/2007 provided by FCT (Science and Technological Foundation) of Portugal. D. Glez-Pena acknowledges the Xunta de Galicia (Spain) for the program Maria Barbeito. Dr Jose-Luis Capelo-Martinez and Dr Carlos Lodeiro are grateful to the Xunta de Galicia (Spain) for the program Isidro Parga Pondal and for financial support given under project 09CSA043383PR. University of Vigo is thanked for financial support under projects In Ou-Univ. Vigo 2009-K915 and K914. M. Diniz acknowledges financial support given by REQUIMTE (Portugal). The research findings here reported are protected under patent pending PCT/IB2006/052314 and PT 103 303.
PY - 2010/10/1
Y1 - 2010/10/1
N2 - 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).
AB - 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).
KW - Biomarker
KW - DPD software
KW - Gel electrophoresis
KW - Protein quantification
KW - Vitellogenin
UR - http://www.scopus.com/inward/record.url?scp=77958184199&partnerID=8YFLogxK
U2 - 10.1002/elps.201000251
DO - 10.1002/elps.201000251
M3 - Article
C2 - 20882554
AN - SCOPUS:77958184199
SN - 0173-0835
VL - 31
SP - 3407
EP - 3419
JO - Electrophoresis
JF - Electrophoresis
IS - 20
ER -