Protein chips and functional proteomics

Introduction

A protein microarray, sometimes referred to as a protein binding microarray, provides a multiplex approach

1. to identify protein–protein interactions,2. to identify the substrates of protein kinases,3. to identify transcription factor protein-activation,4. to identify the targets of biologically active small molecules.

The array is a piece of glass on which different molecules of protein or specific DNA binding sequences (as capture probes for the proteins) have been affixed at separate locations in an ordered manner thus forming a microscopic array.The most common protein microarray is the antibody microarray, where antibodies are spotted onto the protein chip and are used as capture molecules to detect proteins from cell lysate solutions.

Types

The two main types of protein chips are analytical and functional. With analytical protein chips, the proteins being studied are in the solution that

is washed over the chip. Analytical chips are primarily used to identify the contents of an analyte

With functional protein chips, the proteins being studied are attached to the chip.[1] Functional chips are primarily used to study interactions between the protein of interest and other molecules.

1. Analytical Chips

Example of an analytical protein chip.

Analytical chips are classified according to the capture molecule that is affixed to the chip. The molecule can be very specific as to the types of proteins it binds to.

Examples of these specific molecules include antibodies, antigens, enzymatic substrates, nucleotides and other proteins.

Analytical chips can also contain molecules that bind to a range of proteins. These molecules are similar to the ones used in liquid chromatography. The techniques include reverse phase, cation exchange and anion exchange.

Reverse phase protein chips, also known as reverse phase protein arrays (RPA), are related to analytical microarrays and are used to identify different levels of expression of proteins. RPAs have become well used to the point where they may even be considered a separate type of protein chip all together.

RPAs are high-throughput technology that involves the use of two pre-existing technologies known as laser capture microdissection (LCM) and microarray fabrication.

LCM visualizes stained tissue cells of interest under a microscope in real time. Once visualized, the cells are then isolated and lysed and placed into the spots of the microarray. An antibody that can be detected by a fluorescent is used to probe the slides. RPA allows for the protein to be immobilized in order to be analyzed instead of the typical protein microarrays which immobilizes the antibody probe. This is how it got the name ‘reverse phase’. This process allows that the protein need not be labeled since the protein lysate has already been denatured.

Advantages of RPAs over other types include

the ability to run different test samples in each individual array spot and only require a single antibody to probe an entire array slide.

This use of a single antibody eliminates the need to run multiple analytes, and instead, a single one is measured and then compared to the different test samples that were applied to the individual spots.

This process is optimal for cell populations with low cell count due to its ability to run a single analyte for a greater number of spots in the array, for example, all of the proteins present in the cell.

A major use of RPAs is to view

the different stages of progressing cancer along with studying signaling transduction pathways.

They can be used to determine different activation statuses of proteins over a set amount of time or due to certain treatment conditions.

2. Functional Chips

Example of a functional protein chip.

Unlike analytical chips, there is only one type of functional chip.

Functional chips are used to discover additional information and properties about a particular protein. These properties include binding strength, biochemical functions and protein-protein interactions.

The major methods used to characterize an organism's proteome often result in the denaturing of the sample thus ruling out any functional studies. Current functional analysis methods are mostly in vivo techniques which have inherent variabilities.

The benefits of functional analysis using these chips is that proteins can be identified and studied in vitro while they are still biochemically active and in their multimeric complexed form.

There are many challenges when developing a functional protein array including

creating an expression clone library,

actual protein production which includes isolation and purification,

adaptation of microarray technology,

stabilizing the proteins on the array, and keeping the concentrations of the protein constant between slides as well as between spots on the same slide.

Functional arrays have many uses including the complete characterization of an organism's whole proteome.

Protein chips enable us to study biochemical interactions on an unprecedented scale.

Thousands of proteins can be screened for protein-protein, protein-nucleic acid, and protein-small molecule interactions simultaneously.

The in vitro nature of the method ensures that it has advantages over current functional assay methods, and its parallel, quantitative format propels it above many other techniques in the field.

Common Detection Methods

ELISA (Enzyme-Linked Immunosorbent Assay)

ELISA assays are useful tool in the detection of antigens or proteins.

A specific antibody is used to target the desired antigen or protein.The complex that forms from the antibody-antigen binding is bound by another antibody which recognizes such complexes. The latter antibody is attached to an enzyme thus 'enzyme-linked' is part of the acronym, ELISA. The binding of the antigen will usually trigger a reaction that can be observed and qualified or quantified.

Useful as ELISA assays may be, they are unfortunately prone to false positives as many non-specific protein-antibody interactions can occur.

Sandwich Immunoassay

Sandwich immunoassays, a version of an ELISA assy, use fluorescently labeled antibodies for the probe and laser scanning for collecting the data.

Isotopic labeling

Isotopic labeling involves using a radio isotope-labeled analyte for the probe and X-ray film for collecting the data

The attachment of unusual isotypes provides the information needed to identify specific markers, or in this case, proteins.

Molecules containing these isotopes can be distinguished by either mass spectrometry or infrared spectroscopy. There are various isotopes and each has a different mass therefore leading to the utilizaion of mass spectrometry.

Infrared spectroscopy can be used because the various isotopes have different vibrational modes.

Planar Waveguide

Planar waveguides involve using fluorescently labeled antibodies for the probe and a charge-coupled device for collecting the data.

Waveguides typically involve the detection of electromagnetic waves. The term planar simply refers to the geometry of the system resembling a plane.

Planar waveguides may offer the exciting ability to analyze systems in real-time thus enabling the study of protein interaction kinetics.

Electro-Chemical

Electro-chemical detection involves using metal-coupled analyte for the probe and a conductivity measurement for collecting the data. 2

Data

Like DNA microarrays, protein chip experiments using fluorescent labeling provide data in the form of images with spots of varying intensities. These images are then analyzed using software packages similar to those used for DNA microarray analysis. Two examples of the types of analysis performed are the quantification of spot intensities and the comparison of intensities between control and experimental conditions.

Software

Software packages used for analysis of labeled protein chips include:

Protein Microarray Analysis Tool (ProMAT) is a freely available software package used to evaluate the intensity of the spots. ProMAT was developed at Pacific Northwest National Laboratory.

ZeptoVIEW PRO is a commercially available software package from Zeptosens that allows quantification of spot intensity and is used with their protein chips.

Label-Free

This type of analysis takes advantage of the properties of the proteins and includes mass spectrometry, surface plasmon resonance (SPR) and atomic force microscopy (AFM).

Mass Spectrometry

Certain chips, such as the ProteinChips by Ciphergen, can be coupled to a MALDI-TOF mass spectrometer. 2 Improved resolution can be obtained by using SELDI mass spectrometry. Data collection and analysis are performed in the same manner as mass spectrometry performed after protein separation techniques such as liquid chromatography.

Surface Plasmon Resonance (SPR)

Surface plasmon resonance is an optical effect produced when polarized light is shone on a specially designed protein chip.

The chip needs to contain a thin metal film that will cause the light to refract.

The angle of refraction depends on the mass of the molecule bound to the chip so a protein with a substrate bound to it will cause the light to refract at a different angle than a protein with no substrate.

The different angles can be measured by a photodiode array.

Additionally, surface plasmon resonance can be coupled to mass spectrometry for protein identification using microrecovery from the chip surface.

Surface plasmon resonance has the ability to provide real-time analysis.

The consequences of such are that it becomes possible to study the kinetics of protein interactions. Detection resolution, a challenge discussed previously on this page, represents a drawback to SPR analysis.

Planar waveguides may provide for a real-time analysis with improved detection resolution.

Atomic Force Microscopy (AFM)

Atomic force microscopy uses changes in surface topology to detect protein interactions. It is a high resolution technique and the data is collected in the form of topographical maps

Manufacture

Protein chips and DNA microarrays are manufactured in a similar fashion. Both involve spotting a biological component (such as DNA or proteins) onto a coated glass slide or other substrate such as gel pads and microwells.

For protein chips, the slide must be coated with a substance that will bind the proteins without denaturing them. There are variety of binding methods available including absorption, cross-linking and hybridization.

Chip Formats

Glass Slide Chips

Glass slide chips are advantageous because they can be used with standard microarray equipment and are inexpensive.

They are prone, however, to evaporation of samples and cross contamination.

The first methods for creating protein chips on glass slides involved placing the proteins in small gel pockets that were attached to the glass surface.

The process then evolved to attaching the proteins with a crosslinking agent that coated the glass. This agent would bind to the primary amines on the proteins, while a 40% glycerol solution would prevent dehydration due to evaporation.

Most array spotting is now done in a high humidity enviornment to control evaporation.

Well Chips

Using wells instead of coated glass slides reduces the problem of evaporation and cross contamination between spots. However, this method is more expensive and is not compatible with DNA microarray spotting equipment.

3D Matrix Gels

A third method is to use a 3D matrix created by polyacrylamide gel pads.

This method limits evaporation and allows the proteins to remain in an aqueous environment.

Additionally, this method allows for stronger binding of the proteins.

Like the microwell method, this method is more expensive and can't be used with traditional DNA microarray spotting equipment.

Protein Attachment Methods

The surface of the chip must be modified to allow the proteins to bind properly. An attachment layer which usually consists of a sugary gel such as dextran-based hydrogel is coated onto the chip. The noise level for these types of attachment layers is high, however, because of the non-specificity of the medium. Cross-linkers can be used that covalently bind to certain groups on proteins, allowing for much more specific binding. The cross-linkers could, however, effect the conformation or activity of the proteins.It is important to remember that unlike nucleic acids, different proteins will be effected in different ways by the surface chemistry of the chip, i.e. a certain treatment will tightly bind some proteins but may denature others.

Protein Delivery Methods

Many protein chips made currently are spotted using automated technology, with spot densities greater than 30,000 spots per chip, though some low density chips may still be hand spotted. In addition to spotting, peptides can be synthesized on the chip via photolithography

Applications

Protein chips are a powerful resource which offers high-throughput methodology approaching that of DNA microarrays thus narrowing the gap between genomics and proteomics.

Protein chip experiments, like the field of proteomics itself, can be divided into two types of questions.

1. Qualitative - What is it? What does it do? Does it interact with anything?2. Quantitative - How much is there?

Protein chip applications are numerous and span the entire field. They include:

Probing a tissue extract for disease markers Investigating protein-protein, protein-nucleic acid, and protein-drug interactions Determining the amount of a protein in a particular sample Identifying antibodies in a particular sample

Some specific examples of protein chip applications are:

Looking for hepatitis B antibodies in a human blood sample Identifying biomarkers for ovarian cancer Diagnosing SARS