Statement of Work for Identification of Biomarkers for Nerve Agent Exposure Using Peptide Array Analysis of Lysates January 2014 Technical POCs: Milan Mrksich. Objective: The proposed work has the broad goal of identifying sets of biomarkers of threat agent exposure using peptide arrays. Mrksich will prepare peptide libraries and then screen cellular lysates for biomarker screening using SAMDI. Scope: The proposed work will assess the effects of the multiple concentrations of the agents sarin ricin, E. coli shiga toxin, Francisella tularensis, Salmonella enterica, and Yersinia pestis. Following exposure to agents in appropriate cell lines and animal models, screening of lysates and tissue extracts will be carried out using SAMDI mass spectrometry on peptide arrays, representing the entire human proteome, to identify threat-specific protein modifications. We will apply this approach to detect low abundance biomarkers, including products of altered enzymatic activity and other protein modifications, which are indicative of human exposure to all classes of CBW. The work will address the following tasks: Task 1: Design and preparation of peptide arrays. We will first prepare a global array containing all possible tripeptides (203 = 8,000 different peptide substrates). This work includes the chemical synthesis and isolation of the peptides, the preparation of SAMDI array plates and associated peptide arrays and the validation of the integrity of peptide arrays. Array plates will be delivered to Tufts and Battelle for analysis of lysates. We will visit these laboratories to instruct on the use of the peptide arrays for sampling lysates. Section 1.2.2. Profiling global cellular activity by SAMDI array. Team members at Tufts and Battelle will apply lysates from treated cells to the arrays to record proteomic activities, and then clean the arrays and return to Northwestern for analysis. Northwestern will perform SAMDI on the arrays and work with Bagheri to analyze the data. Task 2: Subtask 2.2.Biomarker identification We will prepare peptide arrays for Battelle’s experiments to expose them to sarin. We will then analyze the arrays to identify those peptides that are most reactive with the threat agent. Task 3: Subtask 3.2. Biomarker Identification Design of targeted SAMDI arrays. We will prepare SAMDI arrays having two 400 member peptide sets, which will serve as substrates for the phosphothreoninelyase enzymes. One set will be comprised of peptides having the form (Ac-G-X1-pThr-X2-pTyr-G-G-C-NH2) based on the motif (-T-X-Y) from MAPK that has been previously identified as a substrate for SpvC. The second will be of a more general form (Ac-G-X1– pThr –X2-X3-G-G-C-NH2) that may report on eliminylation targets outside of the MAPK family. The X1, X2, and X3 residues are one of nineteen amino acids (all natural residues excluding cysteine that would interfere with the immobilization reaction). We will also prepare SAMDI arrays to profile serine/threonine acetyltransferase activity. These arrays will comprise peptides having a serine or threonine residue at the central position, flanked on either side by variable residues (Ac-G-X1-Ser/Thr-X2- G-G-C-NH2). The X1 and X2 residues include all 19 amino acids (except for cysteine, see above). Section 3.2.2. Design of arrays to globally profile bacterial effector protein activity: We will analyze bacterial effector proteins from Y. pestis or S. enterica using an array of tripeptides (203 = 8,000 different peptide substrates), as described in Subtask 1.2.
|Effective start/end date||2/20/15 → 2/19/17|
- Tufts University (MFD625//DJF-15-1200-K-0001726)
- Federal Bureau of Investigation (MFD625//DJF-15-1200-K-0001726)
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