Purchase this article with an account.
Hector Gonzalez-Iglesias, Amanda Suárez-Fernández, Ana González-Antuña, Lydia Álvarez, Montserrat Garcia, Pedro Pablo Rodríguez-Calvo, Pablo Rodríguez-González, J. Ignacio García-Alonso, Miguel Coca-Prados; Validation of Candidate Glaucoma Biomarkers by Quantitative Proteomics. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3676. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Recent studies have identified a panel of protein candidate-biomarkers in serum of patients with primary open angle glaucoma (POAG) and pseudoexfoliation glaucoma (PEXG) (Gonzalez-Iglesias et al., J. Proteomics, 2014, 98, 75-68). The purpose of the present work was the application of an analytical method that utilizes stable isotope-labeled peptides tandem mass spectrometry (González-Antuña, J. Proteomics, 2015, 112, 141-155), for the accurate and simultaneous quantitation of biomarkers in serum.
Three groups of participants were recruited in the present study: i) patients with POAG; ii) patients with PEXG; and iii) patients undergoing cataract surgery, as controls. Sera from blood samples, collected from the three groups, were processed and stored at -80oC. For each biomarker candidate, two proteotypic peptides were identified by LC-MS/MS (Agilent 6460), and their minimally 13C2-labeled analogues were synthesized (Liberty Blue synthesizer, CEM Corporation). Absolute quantification of biomarkers was carried out by Isotope Dilution Mass Spectrometry (IDMS) and Multiple Reaction Monitoring (MRM), in the LC-MS/MS instrument equipped with an electrospray source (ESI).
Three biomarker candidates [Apolipoprotein A-IV (APOA4), Complement C3 (C3) and Vitronectin (VTN)], were selected based on their high discrimination power to classify POAG/PEXG cases from healthy controls, and used to optimize the MRM technology for their quantitation. The enzymatic hydrolysis of APOA4, C3, and VTN, yielded distinctive “proteotypic peptides”, and the MRM transitions and their corresponding collision energies were optimized, to select both the precursor and the product ions (Table 1). The absolute quantitation of the candidate biomarkers by IDMS was carried out by adding 13C-labeled proteotypic peptides to each of the studied serum samples prior to their enzymatic digestion. The accuracy and precision of the proposed methodology was evaluated with recovery studies of proposed biomarkers fortifying blank human serum samples.
A powerful-targeted MS approach for the quantitation of biomarkers in serum by IDMS has been optimized for its application in a large cohort of patients, in a multiplexing fashion. The methodology offers some specific advantages over regular IDMS procedures, including the direct determination of several proteins in a single sample without resorting to extra calibration runs as usually needed.
This PDF is available to Subscribers Only