Pre-analytical operating procedures for serum Low Molecular Weight protein profiling

Biological specimen collection and storage are an integral component of serum proteomics research. Although many efforts have been posed to address the effects of pre-analytical procedures, standardized protocols for collection and storage of samples for Low Molecular Weight (LMW) proteome profiling are still needed. Here we report a systematic analysis on the influence of pre-analytical factors [clotting times, temperature and time storage, addition of protease inhibitor (PI)] on serum LMW proteome profiling. Moreover, a comparison between manual versus automated peptide purification by functionalized magnetic bead-based MALDI-MS approach was performed. The results demonstrated best serum LMW proteins recovery and stability using a clotting time between 1 and 2h, with serum stored up to 2h either at room temperature or at 4 degrees C, independently of PI addition. PI addition to whole blood resulted in a lower number of LMW peaks detected. Finally, minimal effects on serum proteome profiles were observed after 1-month storage at -80 degrees C, independently of PI addition on whole blood and/or serum. In conclusion, the use of standardized pre-analytical and storage procedures together with an automated peptide purification might minimize potential bias on serum LMW profiling results, thus allowing a better homogeneity and reproducibility in future proteomics studies.