PURPOSE: Proteomic methodologies offer promise in elucidating the systemwide cellular and molecular processes that characterize normal and diseased thyroarytenoid (TA) muscle. This study examined methodological issues central to the application of 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE) to the study of the TA muscle proteome using a rat model. METHOD: 2D SDS-PAGE was performed using 4 chemically skinned rat TA muscle samples. Gel images were analyzed and compared. Protein spot detection and matching were performed using computational image analysis algorithms only and computational image analysis followed by visual inspection and manual error correction. A synthetic master gel, constructed to control for uninteresting biological variation and technical artifact due to differences in protein loading and staining, was evaluated against its constituent gels. RESULTS: Manual error correction resulted in a consistent increase in the number of protein spots detected (between 5.8% and 40.9%) and matched (from 25.8% to 70.8%) across all gels. Sensitivity and specificity of the automatic (computational) spot detection procedure, evaluated against the manual correction procedure, were 74.1% and 97.9%, respectively. Evaluation of protein quantitation parameter values revealed statistically significant differences (p < .0001) in optical density, area, and volume for matched protein spots across gels. The synthetic master gel successfully compensated for these intergel differences. CONCLUSIONS: Valid and reliable proteomic data are dependant on well-controlled manageable variability and well-defined unmanageable variability. Manual correction of spot detection and matching errors and the use of a synthetic master gel appear to be useful strategies in addressing these issues. With these issues accounted for, 2D SDS-PAGE may be applied to quantitative experimental comparisons of normal and disease conditions affecting voice, speech, and swallowing function.
PURPOSE: Proteomic methodologies offer promise in elucidating the systemwide cellular and molecular processes that characterize normal and diseased thyroarytenoid (TA) muscle. This study examined methodological issues central to the application of 2-dimensional sodium dodecyl sulfatepolyacrylamide gel electrophoresis (2D SDS-PAGE) to the study of the TA muscle proteome using a rat model. METHOD: 2D SDS-PAGE was performed using 4 chemically skinned rat TA muscle samples. Gel images were analyzed and compared. Protein spot detection and matching were performed using computational image analysis algorithms only and computational image analysis followed by visual inspection and manual error correction. A synthetic master gel, constructed to control for uninteresting biological variation and technical artifact due to differences in protein loading and staining, was evaluated against its constituent gels. RESULTS: Manual error correction resulted in a consistent increase in the number of protein spots detected (between 5.8% and 40.9%) and matched (from 25.8% to 70.8%) across all gels. Sensitivity and specificity of the automatic (computational) spot detection procedure, evaluated against the manual correction procedure, were 74.1% and 97.9%, respectively. Evaluation of protein quantitation parameter values revealed statistically significant differences (p < .0001) in optical density, area, and volume for matched protein spots across gels. The synthetic master gel successfully compensated for these intergel differences. CONCLUSIONS: Valid and reliable proteomic data are dependant on well-controlled manageable variability and well-defined unmanageable variability. Manual correction of spot detection and matching errors and the use of a synthetic master gel appear to be useful strategies in addressing these issues. With these issues accounted for, 2D SDS-PAGE may be applied to quantitative experimental comparisons of normal and disease conditions affecting voice, speech, and swallowing function.
Authors: Naila Cannes do Nascimento; Andrea Pires Dos Santos; Rodrigo Mohallem; Uma K Aryal; Jun Xie; Abigail Cox; M Preeti Sivasankar Journal: J Proteomics Date: 2021-11-23 Impact factor: 4.044