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 treatment [1]. The blood contains a multitude of unstudied and unknown biomarkers that could reflect the ongoing physiologic state of tissues and organs [2]. The low-molecular weight (LMW) region of the blood proteome is an important source of diagnostic markers [3]. The approach used to identify markers of potential diagnostic importance relies on matrix-assisted laser desorption (MALDI) mass spectrometry[3]. 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 serum[3].

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.

1. Etzioni, R., et al., The case for early detection. Nat Rev Cancer, 2003. 3(4): p. 243-52.
2. Liotta, L.A., M. Ferrari, and E. Petricoin, Clinical proteomics: written in blood. Nature, 2003. 425(6961): p. 905.
3. Liotta, L.A. Petricoin, E.F. Serum peptidome for cancer detection: spinning biologic trash into diagnostic gold. J. Clin. Invest.116, p26 (2006)
4. Gaspari, M. Ming-Cheng Cheng, M. Terracciano, R. Liu, X. Nijdam, A,J. Vaccari, L. di Fabrizio, E. Petricoin, E.F. Liotta, L.A., Cuda, G. Venuta, S. Ferrari, M. Nanoporous surfaces as harvesting agents for mass spectrometric analysis of peptides in human plasma. J Proteome Res. 5, p1261 (2006)

Figure 1. (a-c)Schematization of the fractionation protocol. After the serum is spotted on the surface (a), LMW proteins and peptides are trapped into the pores while the larger species remained outside the pores and are removed during the washing steps (b). The enriched small molecules are then eluted from the nanopores (c) and analyzed by mass spectrometry.(d-j) Nanoporous Silica Chip Technology. (d) Block copolymer self-assembly of porous silica. (e) asssembly of porous silica on substrate. (f) definition of chip by silicone coating. (g-i) Transmission electron micrographs of chip surface showing the porous structure. The numbers indicate pore size. Scalebar is 10 nm  (j) Photograph of a NSC with the SU-8 photoresist and (k) detail of the areas defined by the SU-8 mask respectively (note the thickness of the SU-8 layer); (l) A top view of different SU-8 patterned chips with decreasing fractionation areas show the uniformity of the surfaces (the scale bar is 10mm).

Figure 2. Correlation between Nanoporous Silica Chip properties and peptide enrichment range. (a) correlation to pore size. (b) correlation to hydrophilic state. (c) correlation to pore morphology.

Figure 3. Comparison of MALDI analysis of crude serum (top) and serum fractionated by means of Nanoporous Silica Chip (bottom). Peptide enrichment in the 800 to 3500m/z range is observed.