Background: This study examined the effect of storage temperature on the protein profile of human plasma. Plasma samples were stored for 13 days at -80°C, -20°C, +4°C and room temperature (20-25°C) prior to proteomic analysis. The proteomic comparisons were based on the differences of mean intensity values of protein spots between fresh plasma samples (named "time zero") and plasma samples stored at different temperatures. To better understand the thermally induced biochemical changes that may affect plasma proteins during storage we identified proteins with different expressions with respect to the time zero sample.
Results: Using two-dimensional electrophoresis followed by MALDI-TOF MS and /or LC-MS/MS 20 protein spots representing 10 proteins were identified with significant differences in abundance when stored at different temperatures. Our results, in agreement with various authors, indicate that during storage for a short period (13 days) at four different temperatures plasma proteins were more affected by degradation processes at +4°C compared to the other temperatures analysed. However, we founded that numerous protein spots (vitamin D binding protein, alpha-1-antitrypsin, serotransferrin, apoplipoprotein A-I, apolipoprotein E, haptoglobin and complement factor B) decrease in abundance with increasing temperature up to 4°C, but at room temperature their intensity mean values are similar to those of time zero and -80°C. We hypothesize that these proteins are labile at 4°C, but at the same time they are stable at room temperature (20-25°C). Furthermore we have grouped the proteins based on their different sensitivity to the storage temperature. Spots of serum albumin, fibrinogen gamma chain and haptoglobin are more resistant to the higher temperatures tested, as they have undergone changes in abundance only at room temperature; conversely, other spots of serum albumin, fibrinogen beta chain and serotransferrin are more labile as they have undergone changes in abundance at all temperatures except at -80°C.
Conclusions: Although there are many studies concerning protein stability of clinical samples during storage these findings may help to provide a better understanding of the changes of proteins induced by storage temperature.