Effect of storage temperature as a preanalytical variable on the lens crystallins protein quality for proteomic studies

Abstract

Purpose: To study the effect of storage temperature on lens crystallins quality for proteomic analysis, using αA-crystallins as internal marker.

Experimental design: Lenses were stored at -40°C, -10°C and ice for up to 10 days. Protein extracts were prepared from samples stored at -40°C and -10°C on completion of 10 days; for samples kept under ice-storage, lenses were taken out at every 24 h, extracts prepared and stored. Fresh lens extracts served as the control.

Results: SDS-PAGE analysis of proteins from lens stored at -40°C and -10°C for 10 days did not show any appreciable change in profiles; however, two protein bands of 27 and 29 kDa disappeared from lens stored in ice. A time-course analysis showed that such changes in ice-stored lens occurred beyond six days of storage. Appearance of additional αA-crystallin fragments on 1-D and 2-D immunoblots confirmed degradation of αA-crystallins. Protein spots altered in abundance and identified by peptide mass fingerprinting include αA-, αB-, βB1-, βB2- and γ-crystallins.

Conclusions and clinical relevance: For proteomic studies, quality of the starting material must be ensured to avoid erroneous and misleading interpretation of results. Under field conditions where deep freezing or immediate preparations of samples are not the options, eye lens can be transported under ice-storage for about six days without deterioration in protein quality.