There is an intense interest in understanding the pathogenesis of diseases, and to develop new biomarkers for their diagnosis. In particular, early detection can lead to significant benefits in terms of efficient and timely treatment1. The blood contains a multitude of unstudied and unknown biomarkers that could reflect the ongoing physiologic state of tissues and organs2. The low-molecular weight (LMW) region of the blood proteome is an important source of diagnostic markers3. The approach used to identify markers of potential diagnostic importance relies on matrix-assisted laser desorption (MALDI) mass spectrometry3. In case of serum analysis, the interference of abundant proteins (>90% of total serum proteins), limit the sensitivity of the MALDI detection of low abundant species in serum3.
Our group recently developed a novel three step size-exclusion strategy based on nanoporous silica chips for the efficient removal of the high molecular weight proteins and for the specific isolation and enrichment of LMW species present in complex biological mixtures (figure 1)4. In the Nanoporous Silica Chip Technology, tunable pore sizes and surface chemistries act as integrated "processors" for the selective depletion of the High MW protein content in serum samples and for the enrichment of LMW peptides and proteins. By tuning the chemo-physical properties of nanoporous silica surfaces we demonstrated for the first time the correlation between pore size and molecular cut-off (figure 2). Reproducibility, sensitivity and protein profiles were assessed in relation to the physical (pore size, distribution, density and structure) and chemical (surface charge, hydrophobicity, hydrophilicity) properties of nanoporous silica (figure 2). Finally, we applied the Nanoporous Silica Chip Technology to the analysis of human serum and developed different proteomic chips to specifically target regions of the human LMW circulating peptidome. Harvested peptides were subjected to MALDI analysis and profiles consisting of more than 300 peaks in the range 800-20 000 m/z were generated (figure 3). Our results demonstrate that nanoporous silicon chips are valuable tools for the detection of low abundant, LMW peptides in complex solutions such as serum. We envision that screenings based on our nanoporous silica chip technology may serve as a complement to histopathology, molecular imaging and other state of the art diagnostic techniques. This approach may help in the selection of individualized therapeutic combinations that target the entire disease-specific protein network, in the real-time assessment of therapeutic efficacy and toxicity, and in the rational modulation of therapy based on changes in the protein network associated with prognosis and drug resistance.