Unit 5

Introduction

Protienchip and functional proteomics

Different types of protein chips Detection and quantification of proteins bound to protein chips Emerging protein chip technology
Protein chips, also referred to as protein arrays or protein microarrays, are modeled after DNA microarrays. The success of DNA microarrays in large-scale genomic experiments inspired researchers to develop similar technology to enable large-scale, high-throughput proteomic experiments. Protein chips enable researchers to quickly and easily survey the entire proteome of a cell within an organism. They also allow researchers to automate and parallelize protein experiments. Protein chips were first developed in 2000 by researchers at Harvard University. Today there are many companies manufacturing protein chips using many types of techniques including spotting and gel methods. The types of protein chips available include "lab on a chip", antibody arrays and antigen arrays, as well as a wide range of chips containing "alternative capture agents" such as proteins, substrates and nucleic acids. Protein microarrays were developed due to the limitations of using DNA microarrays for determining gene expression levels in proteomics. The quantity of mRNA in the cell often doesnt reflect the expression levels of the proteins they correspond to. Since it is usually the protein, rather than the mRNA, that has the functional role in cell response, a novel approach was needed. Additionally post-translational modifications, which are often critical for determining protein function, are not visible on DNA microarrays. Protein microarrays replace traditional proteomics techniques such as 2D gel electrophoresis coupled with mass spectrometry or liquid chromatography, which were time consuming, labor intensive and ill-suited for the analysis of low abundant proteins. Immunoassays, the precursor to protein chips available since the 1980s, exploit the interactions between antibodies and antigens in order to detect their concentrations in biological samples. Their creation, however, is tedious and expensive. As a response to this, researchers at Harvard University combined the technologies of immunoassays and DNA microarrays to develop the protein chip. Array-based profiling techniques are conceptually simple. A protein microarray is a collection of miniaturized test sites, or spots, arranged on a solid substrate that permits many tests to be performed at the same time in order to achieve higher throughput and speed. Generally, all protein arrays are composed of a substrate, which constitutes the underlying core material of the array and a protein attachment layer (Figure 1). Preferred substrates include materials based on the following: glass; silicon (Zyomyx, Hayward, CA,

USA; Lumicyte, Fremont, CA, USA); plastic (Large Scale Biology, Vacaville, CA,USA); synthetic polymers (e.g., polystyrene, polyvi-nylidenedifluoride [PVDF], nitrocellulose).

Array substrates may have a coating layer of gold, aluminum or other metals. These coatings are applied using thin-film technologies, such as physical vapor deposition (PVD) or chemical vapor deposition (CVD). Chip surfaces may have adhesion interlayers (e.g., epoxy glue) that bind coatings to the substrate. An interlayer of titanium or chromium, may be used to fix a gold coating to a silicon wafer substrate. The protein attachment layer on the chip surface is typically an organic film < 20 nm thick. The thickness varies with the nature of application. The choice of material selected for attachment is based on the ability of a particular chemistry to immobilize proteins without denaturing them. Generally, hydrophilic materials perform well as they promote protein stability and binding. Materials that have been studied include agarose, dextran-based hydrogel (Biacore, Uppsala, Sweden) porous

polyacrylamide hydrogel (Packard Bioscience, Meriden,CT, USA), hydrophilic polymers and poly-amino
acids (e.g., poly-L-lysine coated micro-scopic slides) There is considerable interest in using selfassembled monolayers for the purpose of protein attachment. Monolayers investigated were composed of aldehyde-containing silanes and alkylthiols (Interactiva Biotechnology, Ulm,Germany). Aldehyde groups readily react withprimary amines on proteins to form Schiff sbase linkage. The thin film of the protein array XNA on Gold, developed by Interactiva, is based on a self-assembling monolayer of long chain alkylthiols adsorbed onto a 100 nm thick coating of 24-carat gold. Gold is typically used because it does not oxidize like other metals, which can affect attachment.The film surface can also be derivatized with

thiol-reactive male-imidyl groups, amine-reactive N-hydroxysuc-cinimidyl (NHS) groups or other functionalities that capture proteins. Amino acids or carbohydrate moieties inherent to proteins have been used to tether proteins directly to organic thin films. However, in many instances, affinity tags and adaptor molecules offer specific advantages in terms of protein immobilization. It should be noted that although affinity tags do not necessarily promote accessibility of the active or binding sites in proteins, they confer enhanced site specific attachment and oriented immobilization of capture proteins by binding to reactive groups of organic thin films. Table 1 outlines various tags used for protein capture. An affinity or epitope tag is usually an intact protein, a polypeptide or poly-amino acid (e.g., a poly-His tag for nickel bind-ing sites, polylysine for amide or Schiff base linkages, poly-cysteine for thioether linkages). For example, streptavidin based immobilization schemes have widely been employed to attach any biotinylated biological element to array surfaces. Biacore International Ltd and Interactiva Biotechnology have developed chip surfaces that employ streptavidin based immobilization schemes for the capture of biotinylated proteins.Protein G and protein A have been used as tags to immobilize the Fc regions of antibodies. Recombinant proteins have been genetically fused with glutathione-S-transferase (GST), maltose-binding protein (MBP), thioredoxin, green fluorescent protein (GFP) and poly-Hisfor convenient attachment, mediated by binding of the respective affinity tags to the correspond-ing antibodies imprinted on arrays. The affinity tag-thin film combination allows for gentle immobilization conditions that maintain protein stability and function. Affinity tags permit a common immobilization strategy that can be applied to a variety of different proteins.

There are three types of protein microarrays that are currently used to study the biochemical activities of proteins.

Analytical microarrays (also known as capture arrays) In this technique, a library of antibodies, aptamers or affibodies is arrayed on the support surface. These are used as capture molecules since each binds specifically to a particular protein. The array is probed with a complex protein solution such as a cell lysate (protien solution). Analysis of the resulting binding reactions using various detection systems can provide information about expression levels of particular proteins in the sample as well as measurements of binding affinities and specificities. This type of microarray is especially useful in comparing protein expression in different solutions. For instance the response of the cells to a particular factor can be identified by comparing the lysates of cells treated with specific substances or grown under certain conditions with the lysates of control cells. Another application is in the identification and profiling of diseased tissues. Functional protein microarrays (also known as target protein arrays) These are constructed by immobilising large numbers of purified proteins and are used to identify proteinprotein, protein-DNA, protein-RNA, protein-phospholipid, and protein-small molecule interactions, to assay enzymatic activity and to detect antibodies and demonstrate their specificity. They differ from analytical arrays in that functional protein arrays are composed of arrays containing full-length functional proteins or protein domains. These protein chips are used to study the biochemical activities of the entire proteome in a single experiment. Reverse phase protein microarray (RPA) These arrays involve complex samples, such as tissue lysates. Cells are isolated from various tissues of interest and are lysed. The lysate is arrayed onto the microarray and probed with antibodies against the target protein of interest. These antibodies are typically detected with chemiluminescent, fluorescent or colorimetric assays. Reference peptides are printed on the slides to allow for protein quantification of the sample lysates. RPAs allow for the determination of the presence of altered proteins or other agents that may be the result of disease. Specifically, post-translational modifications, which are typically altered as a result of disease can be detected using RPAs.

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