Despite technological advances, two-dimensional electrophoresis (2DE) of biological fluids, such as vitreous, remains a major challenge. In this study, artificial neural network was applied to optimize the recovery of vitreous proteins and its detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer), temperature, and total voltage. The highest protein recovery (94.9% ± 4.5) was achieved using 4% (w/v) CHAPS, 0.1% (v/v) Genapol, 20 mM DTT, and 2% (v/v) IPG buffer. Two iterations were required to achieve an optimized response (580 spots) using 4% (w/v) CHAPS, 0.2% (v/v) Genapol, 60 mM DTT, and 0.5% (v/v) IPG buffer at 35 kVh and 25 °C, representing a 2.4-fold improvement over the standard initial conditions of the experimental design. The analysis of depleted vitreous using the optimized protocol resulted in an additional 1.3-fold increment in protein detection over the optimal output, with an average of 761 spots detected in vitreous from different vitreoretinopathies. Our results clearly indicate the importance of combining the appropriate amount of solubilizing agents with a suitable control of the temperature and voltage to obtain high-quality gels. The high-throughput of this model provides an effective starting point for the optimization of 2DE protocols. This experimental design can be adapted to other types of matrices.
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This project was supported by the University of Beira Interior—Health Sciences Research Centre (CICS). Santos FM received a fellowship (CENTRO-07-ST24-FEDER-002014) and a doctoral fellowship (SFRH/BD/112526/2015) from FCT. Gaspar LM received a fellowship from Novartis Farma-Produtos Farmacêuticos, SA. This work is supported by FEDER funds through the POCI—COMPETE 2020—Operational Programme Competitiveness and Internationalisation in Axis I—Strengthening research, technological development and innovation Project (POCI-01-0145-FEDER-007491) and National Funds by FCT—Foundation for Science and Technology Project (UID/Multi/00709/2013). This work was also supported by the Applied Molecular Biosciences Unit- UCIBIO which is financed by national funds from FCT/MCTES (UID/Multi/04378/2019). CNB-CSIC proteomics lab is a member of Proteored, PRB2-ISCIII and is supported by grant PT13/0001, of the PE I +D+i 2013–2016, funded by ISCIII and FEDER.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
The protocol for sample collection was approved by the Ethics Committee of Leiria-Pombal Hospital, Portugal (Code: CHL-15481). An informed consent from all patients was obtained in agreement with the Declaration of Helsinki.
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