efforts are underway to harness this information so that it can be used for early diagnosis of diseases such as cancer. The physicochemical malleability and high surface areas of
technologies, a signi\ufb01cant goal is to tailor nanoparticle surfaces to selectively bind a subset of biomarkers, sequestering them for later study using high sensitivity proteomic tests. To date, applications of nanoparticles have largely focused on imaging systems and drug delivery vectors. As such, biomarker
This review comes from a themed issue on
Proteomics and genomics
Edited by Garry P Nolan and Emanuel F Petricoin
Recent application of highly sensitive mass spectrometry tools to study the proteomic content of blood has revealed a new subset of low abundance, low molecular weight (LMW) proteins that had been previously overlooked. Initial studies utilized mass spectrometry to survey the LMW serum proteome for the presence of m/z value patterns, or \ufb01ngerprints, linked to disease states or even particular stages of a disease, such as ovarian cancer . Since this early approach, a signi\ufb01cant effort has been focused on the identity of LMW proteins and polypep- tides in the blood, as re\ufb02ected in a recent report of an atlas of 6929 distinct peptides identi\ufb01ed in plasma . The
major protein components in blood are high abundance, high molecular weight molecules (99% of the mass) that number fewer than 30 proteins . It is in the LMW, low abundance population of blood proteins that the potential biomarker-rich population is currently thought to reside. Recent studies have demonstrated that these LMW spe- cies are associated non-covalently with the high abundance blood proteins [3\u20135] (Figure 1). Most proteomic studies have relied on the discarding of high abundance molecules in the blood such as albumin, to reduce the signal-sup- pressive effects of these proteins . An alternative approach capitalizes on the carrier functions of these macromolecules through isolating proteins such as albu- min, which then have their low abundance, LMW cargo eluted and measured by mass spectrometry [3\u20135]. This approach was recently used to identify potential disease- related biomarkers, such as proteolytic BRCA2 fragments, present in the blood of patients with ovarian cancer [7\ue000].
Detection of LMW cargo bound to albumin is changing the way that high abundance proteins are viewed from a proteomic perspective. Now, the goal is to \ufb01nd strategies that fully utilize this LMW information for the purposes of biomarker discovery and detection. One strategy for achieving this is to generate harvesting platforms out of nanoscale particles that bind and preserve low abun- dance, LMW biomarkers. In one scenario, the nanopar- ticle could be engineered to out-compete the native carrier proteins for the LMW biomarkers (Figure 2). Alternatively, the particles could be designed as func- tional biomolecule\u2013nanoparticle conjugates, thereby creating selective af\ufb01nity surfaces for the capture of LMW proteins (Figure 3). The LMW proteins could then be eluted from the biomolecule\u2013nanoparticle con- jugates. Because the application of nanoparticles to bio- marker harvesting is in its infancy, examination of the previous uses of nanoparticles provides insight into the potential harvesting applicability of this technology. To date, these platforms have been used largely to release a therapeutic payload or as imaging agents (references described below). This review focuses on how these uses of nanoparticles, found in manuscripts published over the past 11 years, provide insight into the biomarker harvest- ing potential of nanoparticles.
Tools for reproducible isolation and preservation of low abundance, labile disease-related biomarkers will be increasingly required. Organic and inorganic materials
and biochemical substances offer a platform that will allow researchers to develop systems for biomarker har- vesting from the vascular compartment [8,9]. Such new technologies will \ufb01ll the need forex vivo biomarker harvesting systems that could be used in the physician\u2019s of\ufb01ce. It can also be envisioned that particles designed for
A wide variety of materials and biomolecules can be used to fashion nanometer scale particles, including inorganic sub- stances and aggregates of biomolecules [10,11,12\ue000]. These materials provide a rich palette for the creation of nanopor- ous, nanoscale substances, in the size range of macromo- lecular complexes, which have speci\ufb01c molecular interaction characteristics. On the basis of the surface topology, charge and chemical functional groups, a given particle would be expected to have selective molecular interactive properties. Hence, it is likely that nanoparticle preparations can be created with selective speci\ufb01city to isolate certain fractions of the LMWproteome of the blood.
2. Particles conjugated with proteins or other chemical groups that function as bait molecules for LMW proteomic species (Figure 3).
Prototypical carrier protein. A limited number of proteins in the blood
represent the majority of its proteomic content. These high abundance
proteins, such as albumin, have known binding properties for LMW
proteins and fragments of proteins that have considerable potential as disease biomarkers. A critical need within clinical proteomics research is to develop harvesting platforms that retrieve the low abundance, low molecular weight proteomic content of the blood for testing using tools such as mass spectrometry.
Nanoparticle harvesting platform. Nanoparticle surfaces can be engineered with properties such as varying porosity, surface charges and functional groups. Further, the nanoparticle may contain an intrinsic label, such as a quantum dot, which functions as a tracer element. The surface properties of the particle provides a binding site for selected proteins or protein fragments that circulate in the blood bound to other carrier proteins, such as albumin.
A simple con\ufb01guration for a biomarker harvesting platform is to design a set of nanoparticles with distinct physico- chemical properties. For example, the relative surface area, presence or absence of size-selective pores, and surface chemistries of nanoparticles can be tailored to provide a physicochemically based fractionation tool for blood pro- teins. One scenario for use of these particles is to apply them to a serum sample. Following association of mole- cules with the particles, the particles are washed, removing the subset of unbound molecules. The depleted serum may then be analyzed, or alternatively, the particles may be treated with an eluant to remove bound molecules for subsequent analysis. There are a wide variety of substrates that could be used for such a platform, including silica particles. As an introductory approach, nanoporous silica particles have been used to fractionate serum proteins before subsequent mass spectrometry (DH Gehoet al., manuscript submitted for review). This approach can be extended in the future to include metal, polymeric and biomolecule-based particles as well.
A more complicated, and perhaps more selective, harvest- ing approach is to conjugate af\ufb01nity proteins onto the surface of nanoparticles to develop harvesting platforms
that speci\ufb01cally target certain biomarkers for isolation from serum. For example, the biomarker associates with the af\ufb01nity molecule attached to the nanoparticle. The complex is then isolated, followed by elution and detec- tion of the targeted biomarker. This approach for bio- marker harvesting remains to be validated; however, there is signi\ufb01cant evidence that proteins can be attached to nanoparticles and still retain their native binding functions (see below).
Of particular interest are bioconjugated particles that contain a reporter system, which functions as an intrinsic tracer element. For example, quantum dots are size- tunable nanoparticles that have been widely used as labels for biological probes . In one instance, they have been conjugated to streptavidin, creating a reporter complex for molecular studies such as protein microarrays . Moreover, the quantum dot\u2013streptavidin complexes were further modi\ufb01ed with polyethylene glycol. This demonstrates the versatility of nanoparticle-based plat- forms in forming higher-order structures with speci\ufb01c biomolecular activities. Quantum dots are just one exam- ple of an inorganic nanoparticle that can be attached to functional proteins; the composite organic\u2013inorganic nanoparticles (COINs) are an alternative nanoparticle class that have been used as labels for immunoassays
Nanoparticle bioconjugate harvesting platform. Nanoparticle surfaces can be engineered with functional groups that enable affinity groups such as proteins (e.g. antibodies) to be added to their surfaces. The nanoparticle can contain an intrinsic label, such as a quantum dot, which functions as a tracer element. The affinity molecules on the surface of the particle provide binding sites for selected proteins or protein fragments that circulate in the blood bound to other carrier proteins, such as albumin.
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