Summary

Title

Experiment 03: Macaca mulatta infected with Plasmodium coatneyi Hackeri strain to produce and integrate clinical, hematological, parasitological, and omics measures of acute, recrudescent, and chronic infections.

Description

Malaria-naive male rhesus macaques (Macaca mulatta), approximately four years of age, were inoculated intravenously with salivary gland sporozoites produced and isolated at the Centers for Disease Control and Prevention from multiple Anopheles species (An. dirus, An. gambiae, and An. stephensi) and then profiled for clinical, hematological, parasitological, immunological, functional genomic, lipidomic, proteomic, and metabolomic measurements. The experiment was designed for 100 days, and pre- and post-100 day periods to prepare subjects and administer curative treatments respectively. The anti-malarial drug artemether was subcuratively administered to all subjects at the initial peak of infection, one out of the five macaques received four additional subcurative treatments for subsequent recrudescence peaks. The experimental infection in one subject was ineffective but the macaque was followed-up for the same period of 100 days. The different clinical phases of the infection were clinically determined for each subject. Blood-stage curative doses of artemether were administered to all subjects at the end of the study. Capillary blood samples were collected daily for the measurement of CBCs, reticulocytes, and parasitemias. Capillary blood samples were collected every other day to obtain plasma for metabolomic analysis. Venous blood and bone marrow samples were collected at seven time points for functional genomic, proteomic, lipidomic, and immunological analyses. Within the MaHPIC, this project is known as 'Experiment 03'. This dataset also includes results from MaHPIC Experiment 18 (E18). LC-MS/MS output from E18 were used as a control for analysis. Refer to the E03 and E18 README files for details of these datasets and their production.

Sample Processing Protocol

(1) Allow samples to gently thaw on ice. (2). Label two clean collection tubes and one C18 cartridge (Nest Group, MacroSpin Column – SMM SS18V) per sample. (3) Wash each cartridge with 450ul of 100% methanol. Spin tubes at 500g for 3 minutes and discard flowthrough. (4) Repeat steps 3 and 3a using the following solutions: a. 100% ACN, b. 80% ACN in 0.1% FA, c. 0.1% FA – three times. (5) Add 400ul of thawed sample peptides to their cartridge and disrupt C18 as needed to ensure binding. (6) Spin peptides at 500g for 3 minutes and reapply to cartridge once before discarding flowthrough into biohazardous waste. (7) Repeat steps 5-6 until all peptides have been applied to cartridge twice. (8) Wash cartridge with 450ul of 0.1% FA, spin at 500g for 3 minutes, and discard flowthrough. (9) Repeat step 8 three additional times. (10) Move each cartridge to its’ second collection tube. (11) Add 300ul of 80% ACN in 0.1% FA to cartridge to elute peptides. (11a) Spin tubes at 500g for 3 minutes and transfer peptides to final tube. (12) Repeat steps 11 and 11a once before concentrating peptides in speed vacuum. Refer to the SOP_Prot_MS_Step1.docx document for the protocol described in full detail. See the readme E23M99PRMmCyDaRC_Readme_XXXXXXX_pride.txt for further experimental details.

Data Processing Protocol

Prepare sequence databases according to the protocols described in SOP_Prot_DBCreation_Step2.docx and SOP_Prot_ProValT_DatabaseConfig_Step5.docx. Create Proteome Discoverer Workflow and Byonic Parameters File according to the protocol described in SOP_Prot_ProteinIDParams_Step3.docx. Perform identification of proteins using search engines according to the protocol described in SOP_Prot_ProteinID_Step4.docx. Perform proteome validation using ProValT according to the protocol described in SOP_Prot_ProteomeValidation_Step6.docx. Generate final report as described in SOP_Prot_ProteomeReportGeneration_Step7.docx. The software tools used: ProValT (v. PROVALT_3.1.05_08-31-16 or greater), Proteome Discoverer v2.1, Byonic v2.9, NuSep FASTA Manager 1.2.1. For the full data processing workflow, refer to UGA_Proteomics_Workflow_04-25-17.pptx and the beforementioned SOP files. See the readme E23M99PRMmCyDaRC_Readme_XXXXXXX_pride.txt for further experimental details.

Contact

Jessica Kissinger, Institute of Bioinformatics, University of Georgia
Mary R. Galinski, Emory University ( lab head )

Submission Date

11/03/2019

Publication Date

13/03/2019

Corresponding dataset(s) in other omics resources

http://plasmodb.org/plasmo/mahpic.jsp

Publication

Publication pending