Proteomics/Protein Chips/Analysis

Analysis:

Analysis

Within the realm of protein chip analysis, there are several hurdles presently being cleared by scientists experimenting at the fringe of this technology. Several of the main challenges facing scientists include: dynamic protein concentrations, almost overwhelming numbers of unique proteins, and creation of specific probes for each protein.

Dynamic protein concentration refers to the reality that the exact number of any given protein within a cell can be drastically different than the exact number of another protein within the same cell. In fact, there are current estimates that place the difference in protein numbers between those expressed at the lowest levels and those expressed at the highest levels at an order of magnitude of roughly 10¹². In other words, a million million fold difference can exists between the rarest and the most abundant proteins.

To further complicate the issue of dynamic protein concentrations, the exact number of any given protein will fluctuate based on which type of cell is being considered. Of course, there are some proteins, known as housekeeping proteins, that are highly expressed in every cell. These proteins are needed for the basic structure and functions that keep the cell alive. Housekeeping proteins aside, cells of different tissues will often have varying protein concentration profiles. For example, proteins that are expressed at high concentrations in heart tissue will most likely be expressed at different concentrations in liver tissue. Therefore, methods that detect a protein in one cell type may not work for a different cell type.

Unfortunately, scientists are often interested in those proteins that are rare and present in low concentrations. One method devised in order to overcome this limitation has been the removal of abundant proteins. Usually, there are a very small number of proteins that are present in copious quantities. Through the removal of these proteins, scientists can reduce the order of magnitude differences between the lowest and highest expressed proteins. However, protein purification can be a very laborious and time consuming process and many scientists recognize that it may be prudent to run several protein chip experiments, each analyzing a different range of protein concentrations. ¹

Another massive challenge exists in determining a specific probe for each protein. Currently, one attempt to solve this problem is in the designing of specific antibodies. In the past, antibodies have been created through the immunization of animals. This method, however, is not high-throughput enough to be considered effient for protein chip experimental design. Instead, methods are being developed in order to create antibody libraries. ¹ A few examples from Molecular Station are listed below.

• Phage antibody display
• Ribosome display
• SELEX (Systomatic Evolution of Ligands by Exponential Enrichment)
• mRNA display
• Affibody display

Antibody approaches can be narrowed somewhat by creating antibodies that bind specific protein groups and not specific proteins. An alternative to antibody usage exists in aptamers, or short oligonucleotides which are much easier to create, but can be difficult to select for a specific protein. ¹

In order to analyze the results of the protein chip it is necessary to determine the types or numbers of proteins that were bound to the capture agents (for analytical chips) or the number of interactions the proteins had (for functional chips). Analysis of protein chips can be broken into two categories: Labeled and Label-Free. Analysis of protein chips can be a difficult and tricky process. Many intuitive methods have been developed, but each has its own limitations. Short descriptions of these methods and their limitations exists below.

Labeled

This type of analysis involves labeling either with radioactivity or fluorescence. ² The label can be attached directly to the protein or capture agent (such as an enzyme or substrate) and detected immediately. The label can also be attached to a different molecule (such as an antibody, antigen or substrate) that is washed over the chip as part of a second step. This prevents the label from altering the conformation of the proteins being studied.

However there are several issues that arise with labeled protein chips. Although methods exist to prevent a label from altering protein conformation, a probability of interference with protein interactions still exists. ¹ Label influence on binding properties, along with a knack for having a lack of reproducability ¹, provide for a couple of significant issues which can be seen as sufficient reasoning for using a label-free approach.

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.

A very useful animation of both a positive and negative ELISA reaction can be found here.

Useful as ELISA assays may be, they are unfortunately prone to false positives as many non-specific protein-antibody interactions can occur. ¹

Sandwich Immunoassay

Sandwich immunoassays, a version of an ELISA assy, use fluorescently labeled antibodies for the probe and laser scanning for collecting the data. ²

Isotopic labeling

Isotopic labeling involves using a radio isotope-labeled analyte for the probe and X-ray film for collecting the data. ² 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. ³ 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. ² 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. ¹

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). ²

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. ² 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. ² The chip needs to contain a thin metal film that will cause the light to refract. ² 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. ² The different angles can be measured by a photodiode array. ² Additionally, surface plasmon resonance can be coupled to mass spectrometry for protein identification using microrecovery from the chip surface. ²

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. ¹

Atomic Force Microscopy (AFM)

Atomic force microscopy uses changes in surface topology to detect protein interactions. ² It is a high resolution technique and the data is collected in the form of topographical maps. ⁴

References

1. Protein Microarray Chips. 2005-6. Molecular Station. (accessed) April 1, 2006 <http://www.molecularstation.com/protein-microarrays/>.

2. Twyman R.M. Principles of Proteomics; BIOS Scientific Publishers: Oxon, U.K., 2004; Chapter 9.

3. "Isotopic labeling." Wikipedia, The Free Encyclopedia. 5 Feb 2007, 03:19 UTC. Wikimedia Foundation, Inc. 9 Apr 2007 <http://en.wikipedia.org/w/index.php?title=Isotopic_labeling&oldid=105717372>.

4. Atomic Force Microscopy - Davis Heart Lung Research Institute. http://heartlung.osu.edu/6161.cfm (accessed May 20, 2006).

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