Protein chip approach

In a more modified approach, differential display proteomics can also be done with no separation of proteins. This is called the protein chip approach. In this method, a variety of bait proteins such as antibodies, peptides, or protein fragments may be immobilized in an array format on specially treated surfaces. The surface is then probed with the samples of interest. Proteins that bind to the relevant target can then be analyzed by direct MALDI readout of the bound material (Nelson, 1997 Davies et ah, 1999). Lor example, well-characterized antibodies can be used as bait. Protein samples from two different cell states are then labeled by different fluorophores, mixed together, and used as probe. In such a case, the fluorescent color acts as an indicator for any change in the abundance of the protein that remains bound to the chip (Lueking et ah, 1999). A number of technical problems would still need to be overcome before applying this technique for large-scale analysis of proteins. 

So far the bottleneck in producing protein chips seems to be the preparation of the individual proteins, but for this heavily researched area solutions are on the horizon. The advantage of the protein chip approach is that a comprehensive set of individual proteins can be directly screened in vitro for a wide variety of activity, including protein-drug interactions, protein-lipid interactions, and enzymatic assays using a wide range of in vitro conditions - and faster and cheaper than with conventional methods. 

Other early work includes that of Moody et al. (2001) who spotted anticytokine monoclonals onto the bottom of polystyrene microtiter plates (Max-isorp, Nalge Nunc) and measured cytokine levels in stimulated peripheral blood mononuclear cells. Finally, although not strictly a microarray, the microwell array system developed by Michael Snyder s group at Yale University to measure kinase activity is a simple and elegant approach (Zhu et al., 2000). The "protein chip" is comprised of microwells fabricated in a flexible elastomer of PDMS [poly(dimethylsiloxane)] substrate by a molding process. 

Warren EN, Elms PJ, Parker CE, et al. Development of a protein chip a MS-based method for quantitation of protein expression and modification levels using an immunoaffmity approach. Anal. Chem. (2004) 76 4082-4092. 

Borrebaeck, C. A. Ekstrom, S. Hager, A. C. Nilsson, J. Laurell, T. Marko-Varga, G., Protein chips based on recombinant antibody fragments A highly sensitive approach as detected by mass spectrometry, Biotechniques 2001, 30, 1126-1132... 

These small devices have been used in a variety of ways. Liquid chromatography (LC) on-chip approaches are well represented in the literature (45,46). These chips have also been used to study the membrane proteome (47) and protein-protein interaction via fluorescence resonance energy transfer (48). Further examples of technology miniaturization can be found in journals dedicated to the on-chip methodology (i.e., Lab-on-a-Chip). 

Advantages of the on-chip approaches are that the systems are well established and very uniform processing is practicable. But these approaches can only be applied for the production of oligonucleotide microarrays. So far, only scientific but no commercial approaches have been undertaken to produce peptide microarrays by an on-chip approach. Additionally, depending on the specific application, low-density microarrays and only low microarray numbers are sufficient to answer particular biological questions thus, often small batches (hundreds) of microarrays with low density (a few hundred spots) are needed. In research applications the array layout needs to be flexible. The production of protein or cell microarrays is in high demand as well. 

In addition to on-chip approaches and contact and non-contact printing of agents onto microarrays, there are further methods for microarray fabrication. Two examples are the protein profiling chips offered by the company Zyomyx and the electronic microarrays offered by the company NanoGen. 

Whereas DNA is a relatively simple polyanion and can be modified and easily immobilized on solid surfaces based on electrostatic interactions or covalent bonding, protein bonding is much more delicate. The complexity derives from a multitude of biochemical properties. Protein molecules possess particular three-dimensional structures and varying chemical and physical properties (e.g., hydrophilic and hydrophobic domains, ionic interactions), and the activity and function as well as the partial charge of domains depend on the local physical and chemical microenvironment. Additionally, complexity is further increased by posttranscriptional modifications of protein conformation hence the well-established on-chip approaches of oligonucleotide microarrays are not applicable to protein microarrays. For protein microarray production, four major requirements have to be fulfilled ... 

Surface-enhanced laser desorption ionization (SELDI) uses so-called protein chips for the detection of peptides and proteins from complex biological fluids such as blood or urine, often for the identification of diagnostic biomarkers for specific carcinomas [156, 157]. These protein chips can contain various media for positive or negative ion exchange or reverse-phase chromatography, as well as specific antibodies or DNA. The functionaUzed surface is immobihzed on a MALDl sample plate for the selective enrichment of constituents of the complex mixture applied, whereas the not bound supernatant is removed by washing. Unfortunately, a large number of unsubstantiated claims for the detection of disease-related biomarkers has discredited this approach, mostly as a result of poor mass spectrometric performance. 

In 1993, Hutchens and co-workers described surface-enhanced laser desorption/ionization (SELDI) technique, an affinity technology, which has progressed over the last decade to become a powerful analytical, an on-plate approach (Hutchens and Yip 1993). SELDI is a distinctive form of laser desorption/ionization (LDI) mass spectrometry in which the EDI probe plays an active role in the homogenization, preconcentration, amplification, purification, extraction, enrichment digestion, derivatization, synthesis, separation, and detection with complementary techniques, prior to the desorption and ionization of the analytes by MALDI (Merchant and Weinberger 2000). The principle of this approach is very simple. Biomolecules are captured by adsorption, partition, electrostatic interaction, or affinity chromatography on a solid-phase protein chip surface. Although SELDI provides a unique sample preparation platform, it is similar to MALDI-MS in that a laser... 

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