click to enlarge Figure 1. Structure of Nanotrap particle and harvested biomarkers. (Figures: Shimadzu Scientific Instruments)

Biomarkers for early disease detection and therapeutic development are in high demand. However, the discovery process is hindered by several complex challenges, including wide sample dynamic range, limited instrument sensitivity, and labor-intensive data mining. Such barriers require researchers to implement various sample fractionation and enrichment strategies. These strategies must address several concerns.

Biological fluids (including human plasma, sera, urine, and saliva) contain a wide range of protein concentrations. High-abundance, less-relevant proteins mask the presence of disease-specific biomarkers in biomarker discovery experiments. Additionally, potential biomarkers are susceptible to degradation by proteases, enzymes, or analytical processes, potentially removing these biomarkers as candidates for disease detection. Additionally, the fractionation strategies must be scalable to a wide variety of protein concentrations as well as biological sample volumes and types.

A key strategy for addressing these concerns is applying nanotechnology to biomarker discovery. Nanotrap (Ceres Nanosciences, Manassas, Va.) particles are core-shell hydrogel particles¹ with porous interiors containing various affinity-selector baits and size-exclusion exteriors. These particles deliver affinity selection and size exclusion in a single step, and offer simplified fractionation and enrichment, serving as molecular mops that purify, concentrate, size-exclude, and preserve the low-abundance proteome.

click to enlarge Figure 2. One-step enrichment of platelet derived growth factor (PDGF) peptide (1930.0 Da) using Nanotrap particles.

Nanotraps exclude the more abundant, less interesting carrier proteins while simultaneously harvesting low-level proteins and peptides. They also allow unique amplification and protection capabilities. Biomarkers that were once immeasurable can now be harvested directly from complex biofluids, protected from enzymatic degradation and amplified. These Nanotrap properties offer exciting advantages to biomarker researchers, enabling them to tap into and mine the low-level proteome.

Once samples are enriched, they are eluted from the nanoparticles and spotted directly onto a MALDI plate with matrix. Data is acquired through automated acquisition using a Shimadzu AXIMA series MALDI-TOF mass spectrometer, which can then be analyzed using Progenesis MALDI software (Nonlinear Dynamics, Durham, N.C.). Progenesis MALDI provides a fast and reliable means of comparing complicated data sets to obtain statistically significant and objective results.

Shown in Figure 2 is an example of one-step enrichment of platelet derived growth factor (PDGF) peptide (1930.0 Da) using Nanotrap particles. PDGF (60 ng) in the presence of albumin (600 ng) was processed with Nanotraps to sequester and enrich the extremely labile PDGF protein. Comparison of MALDI mass spectra of a control (A) to a sample processed with Nanotraps (B) demonstrates the capability to exclude abundant, high-molecular-weight proteins, for example, albumin, and simultaneously amplify a low-level PDGF peptide (highlighted in blue), resulting in an 8-fold increase in signal intensity (left).

Nanotrap technology enables researchers to tap mine the low-level proteome and address many of the challenges facing researchers, such as complex fractionation and enrichment; and difficult-to-analyze low level compounds and samples that may contain many abundant proteins.

At the same time, Nanotrap technology is not limited to biomarker discovery. Nanotrap particles can be utilized in research targeting the discovery and quantification of known low abundance analytes present in a range of complex biofluid matrices, including cell culture supernatant, serum, urine, cerebrospinal fluid, and environmental water samples. This technology can also be used in the fields of environmental chemistry, metabolomics, food safety, clinical diagnostics research, and toxicology.

The Nanotrap Biomarker Discovery Platform speeds discovery, identification, and validation, making early disease detection possible.

About the Author
Scott Kuzdzal joined Shimadzu as Life Science Business Leader in January 2008. He received his PhD in Analytical Chemistry in 1997 from the University of California at Riverside, and previously served as a postdoctoral fellow at the Johns Hopkins University School of Medicine.

References
1. Longo C, et al. Core-shell hydrogel particles harvest, concentrate, and preserve labile low-abundance biomarkers. PLoS ONE. 2009; 41(3):e4763.

This article was published in Drug Discovery & Development magazine: Vol. 13, No. 4, May 2010, p. 21.