Fasting status as a consideration for human serum collection and preparation prior to depletion and analysis

The use of comparative serum proteomic analysis has the potential to reveal protein expression changes present at different stages of disease progression. Depletion strategies allow for the enrichment of low-abundance proteins, which are more likely to be clinically significant biomarkers. We have observed that patient serum samples filtered through 0.22 microm cellulose acetate spin filters prior to depletion showed a variable level of retention of patient material on the upper part of the filter. This could potentially be related to the fasting status of the patient as a reduction in the lipid content of samples through the incorporation of a centrifugation step prior to filtration was found to reduce this effect. In order to determine if proteins were being selectively retained during filtration, a 2-D difference gel electrophoresis (2-D DIGE) experiment was performed. This demonstrated no significant selective retention of protein within crude serum samples. However, as this analysis was carried out on crude serum, it must be emphasised that protein loss could be manifest in the low-abundance proteins which would be masked in our analysis. Depletion of the retentate was not possible due to technical limitations, however based on our results a centrifugation step might act as an alternative to filtration in serum processing prior to depletion.