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NDR White Paper Issue 21: Peptide Array Technology Provides a Cornerstone for ALS Research

Peptide array technology is an integrated platform of unique proteomics and bioinformatics that is used to identify disease biomarkers and drug targets. Ultimately this work fosters signal transduction research and collaboration between scientists.

Peptides are analyzed in solution or on membranes. Changing the peptide sequence by synthesizing a peptide allows an investigator to change an amino acid in the sequence and enable profiling of the proteins phosphosites. The synthesized peptide can interrogate the specificity of a reagent, for example a drug that targets the phosphosite. Applications for peptide array technology are epitope mapping, substrate optimization, and drug targeting. A synthetic peptide can be very specific, even identifying phosphomimic sites.

Once a peptide is synthesized, it is used to immunize rabbits and produce desired antibodies. The antibodies are purified and analyzed for reactivity by immunoblot. Useful antibodies that pass quality control are printed on a chip forming an array of multiple proteins of interest.

Generally, protein targets are low abundance proteins in a mix of proteins making them hard to detect with accuracy. Most phosphorylation sites in a cell are also time dependent and the vast majority of time, cells need to be stimulated to show the phosphorylation site. The printed array contains almost one thousand phosphosite-specific antibodies that detect changes in the phosphorylation sites in a sample on one slide. When coupled with pan-specific phosphoproteins antibodies there are over 2000 proteins being examined, on one array, at one time.

An advantage of using the microarray for analyzing proteins from lysate sample preparations is a reduction of protein-protein interactions that can hide epitopes or small proteins that yield a weak signals. This new technology stabilizes post-translational modifications and reduces internal antibody competition for target proteins and then normalizes protein signals to similar fragment sizes. Proteins are labile and once a cell is disrupted phosphosites are dephosphorylated within minutes. Even phosphatase inhibitors don’t completely stop this issue of rapid loss of phosphosite detection. An approach to overcome this issue is by chemical cleavage of the protein. Generally, trypsin is used to cleave proteins into fragments.

Cysteine cleavage cuts the protein at cysteine residues generating larger protein fragments in the lysate over, for example, typical trypsin cleavage. A cysteine residue is carefully placed in the synthetic peptide to avoid a target on the protein. A dye-labelled anti-biotin antibody is used to bind the target protein in an indirect method. The biotinylating protocol increases the sensitivity of detection while lowering any background signals. The reactions are then confirmed by Western Blots.

The array uses small amounts of material, as small as 20 micrograms of protein, to analyze 2000 proteins, in duplicate. This technology significantly increases the data that can be obtained over mass spectrometry analysis. While it is true mass spec has high sensitivity and accuracy, to achieve the analysis for 2000 proteins the starting material needed would be milligram amounts.

Validation of the microarray results involves two dimensional immunoblotting, called multiblots. The multiblot results are compared back to microarray results to confirm the reactivity of a protein. Additionally, the technology can detect the size of a protein by its cross-reactivity and then identify the protein by size using immunoprecipitation. This information tracks a target protein and can identify the protein and the specific reactive site on the protein. Because the microarray tracks multiple proteins at the same time it is possible to follow pathways and observe the changes in a protein relative to another protein or phosphosite.

The results can be used to track or map cell signaling. Cell signaling is highly interconnected. Changes are associated with each other directly or even steps along the way. Like a jigsaw puzzle, when 2000 proteins are observed, typically 10% change at one time, showing relationships. Pathway related proteins are changing together. A Kinetscape map looks at the interconnectivity between the proteins.

Underlying interconnectivity shown in Kinetscape mapping was derived from 20,000 kinase substrate relationships. What is known is a kinase, its substrate, and the phosphorylation sites on the protein. Sometimes the effect of the phosphorylation is known. The map shows empirical changes between proteins as increased, decreased, or unchanged. Lines between the proteins show an effect that can be an increase, decrease or no change in phosphorylation sites as they are altered and tracked on the target proteins. The lines are indicating activation as a plus, inhibitory actions are shown as a negative. Most importantly, the mapping shows the dynamic relationships between proteins. There are databases with individual proteins providing known relationships available in open access websites.

Microarray is faster and less expensive than mass spectrometry for a similar analysis. A microarray of 78 protein kinases, 24 protein phosphatases, and 31 phosphoproteins from brain and spinal cord tissue from the SOD1 mouse model and thoracic spinal cord fractions from humans with ALS were analyzed by western blot. Extensive protein kinase expression was obtained revealing some overlap between the mouse and human tissue. However, data showed that genetic mouse model SOD1 doesn’t recapitulate human sALS and may indicate that the genetic mouse model doesn’t represent the disease processes in the sporadic ALS cases in people.

Application of the array technology may be useful in early detection of ALS. It is possible to target and reduce pathology thereby slowing disease processes if the cell signaling markers and pathways were known early in the disease process.

The hypothesis going forward is mutated proteins that are associated with fALS may also be important in sALS. Abnormal phosphorylation of disease-associated proteins, and their associated regulatory proteins, may be targeted in all ALS. These proteins are conserved and may be interconnected in signaling networks involving regulatory protein kinases. The objective is to identify markers of ALS for diagnostic purposes, uncover the molecular mechanisms that underlie the common pathology of ALS and identify potential drug targets for specific treatment of ALS.

Studies will identify proteins that have been linked with ALS to identify phosphosites on these proteins that are functional, highly conserved (and likely to be functional in disease) and detectable. The sites can be found on the PhosphoNET and PhosphoSitePlus websites as well as literature analyses.

Over 97 new phosphosite specific rabbit antibodies generated against synthetic peptides patterned after these known phosphosites has been accomplished. This milestone adds to the 160 phosphosites in 62 target proteins in the Kinexus bank and made available to all ALS researchers. It is hoped that this will provide a cornerstone for ALS research.

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