Sample Preparation Strategies for Mass Spectrometry-based Proteomics
By: Lee Pey Yee (leepeyyee@yahoo.com)
Sample preparation is a critical step that has a profound impact on the quality and outcome of every experiment. The human blood is widely used for routine clinical diagnostic assays and biomarker discovery work due to its accessibility and ability to reflect underlying biological states. However, proteomic analysis of the blood is very challenging due to the fact that it is highly heterogeneous in terms of its constituents, with a vast array of proteins that vary greatly in their concentrations. In particular, the most prominent protein species that constitute the major fraction of the total protein content frequently obscure the detection of proteins that present at lower concentrations. Hence, depletion of the abundant proteins is essential to allow the detection and identification of the less abundant proteins as disease biomarkers. In this study, the performance of two antibody-based immunocapture affinity columns for the removal of two and twelve (respectively) highest abundant proteins was evaluated. This immunodepletion approach was found to yield favorable specificity and efficiency in removal of targeted abundant proteins. Depletion of the top twelve high abundant proteins was demonstrated to enhance the detection of additional less abundant proteins and led to an increase in protein sequence coverage. In addition, the sample processing protocol was also optimized for serum proteomic analysis work. Our results indicated that protein digestion in the presence of urea increased the yield of tryptic peptides and improved the identification of low abundant proteins. Furthermore, we also optimized other parameters such as dithiothreitol and iodoacetamide concentrations, and evaluated the performances of different grades of trypsin and trypsin stability. These findings are important in establishing an optimal method that could be applied for future proteomics analysis.
