Malaria Host-Pathogen Interaction Center (MaHPIC) http://plasmodb.org/plasmo/mahpic.jsp Experiment 03 Lipidomics Dataset ********** ***** This Readme file was generated by the MaHPIC Informatics Core to document: ***** 0. Project and Experiment description ***** 1. Dataset description ***** 2. Known Issues and Important Notes ***** 3. File Organization and Description ***** 4. Experimental Results Column Header Definitions ********** Please contact mahpic-infodepo@uga.edu with any questions / concerns / suggestions ********** ********** ********** 0. Project and Experiment Description: MaHPIC Project Description: The Malaria Host-Pathogen Interaction Center (MaHPIC) is a transdisciplinary malaria systems biology research program supported by an NIH/NIAID contract (# HHSN272201200031C; see http://www.systemsbiology.emory.edu). The MaHPIC generates many data types (e.g., clinical, hematological, parasitological, metabolomics, functional genomics, lipidomics, proteomics, immune response) and mathematical models, to iteratively test and develop hypotheses related to the complex host-parasite dynamics in the course of malaria in non-human primates, and metabolomics data via collaborations with investigators conducting clinical studies in malaria endemic countries, with the overarching goal of better understanding human disease, pathogenesis, and immunity. Curation and maintenance of all data and metadata are the responsibility of the MaHPIC. MaHPIC Experiment Title: MaHPIC Experiment 03: Lipidomics from Macaca mulatta infected with Plasmodium coatneyi Hackeri strain to produce and integrate clinical, hematological, parasitological, and omics measures of acute, recrudescent, and chronic infections. MaHPIC Experiment 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 was produced by: The MaHPIC Consortium, Monica Cabrera, Jeremy D. DeBarry, Mary G. Galinski, Jay Humphrey, Ebru Karpuzoglu, Jessica C. Kissinger, Regina Joice, Esmeralda Meyer, Alberto Moreno, Vishal Nayak, Mustafa Nural, Dan Ory, Suman Pakala, and Xuntian Jiang. The experimental design and protocols for this study were approved by the Emory University Institutional Animal Care and Use Committee (IACUC). 1. Dataset Description: This dataset consists of relative quantification results of targeted lipid species, raw and intermediate data, related metadata, and supporting documents from the MaHPIC Lipidomics Team at Washington University in St. Louis for Experiment 03. Experiment duration: 101 days plus 5 pre-inoculation and 34 post-experiment days Experiment start date: 03/06/2014 (Day -5) Inoculation date: 03/11/2014 (Day 0) Experiment end date: 06/19/2014 (Day 100) Post-experiment end date: 07/23/2014 (Day 134) Identifiers of Non-Human Primate (NHP) subjects: RCs13 RWr13 RUn13 RZe13 RTi13 Time points days were determined by infection stages, and are defined as follows: Time point 1 = Baseline/pre-infection (inoculation day minus 5) Time point 2 = Peak of Infection Time point 3 = Post-peak Time point 4 = Recrudescence peak Time point 5 = Post-peak Time point 6 = Recrudescence peak Time point 7 = Post-peak LC-MS/MS was used to relatively quantify the following lipids: In Whole Blood: phosphaditylcholine (PC), sphingomyelin (SM), Ceramide (Cer), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylethanolamine (PE), glucosylceramide (GC), lactosylceramide (LC), sulfatide (ST), ganglioside (GM3) In Bone Marrow: PC, SM, PI, PA, PS, Cer, diacyl glyceride (DG), PE, GC, ST, GM3 Lipid nomenclature, Internal standards and quality control (QC) sample used, sample preparation, lipid classes investigated in different runs, LC-MS/MS conditions for each run, process employed for relative quantification of each lipid species in a lipid class, normalization by QC, are all described in the accompanying SOP - E03_Lipidomics_SOP_WB_and_BM_09202016_v2.docx 1.c. Number of samples: Two samples were extracted from each of the 5 NHPs on each time point day. Total number of barcoded samples = 2 * 5 * 7 = 70 1.d. List of barcoded samples, subject IDs, and time points: A separate excel file is made available with this information: E03M99LIMmCoDaZZ_Barcodes-Info_MULTIPL.xlsx ********** ********** ********** 2. Notes and Known Issues: 2.a. Blood transfusion events - RTi13 was given a blood transfusion on 4/4/14 (second day after time point 2). 100ml of blood was given from donor NHP GC9B. From an email from Dr. Kissinger: “The change in monkey blood will affect many events for up to 100 or 120 days depending on amount of life left in all of the cell populations.” USE THE DATA FROM THAT DAY AND BEYOND, FOR THIS NHP, WITH CAUTION. 2.b. Two instruments were used. API4000 and TSQ Sulfatide and Ganglioside are two classes of sphingolipids that are low abundant, and TSQ cannot be used for those. 2.c. There were 4 runs. First 3 runs were for the abundant sphingolipids and phospholipids, and were run on TSQ. Fourth run was for the low abundant sphingolipids, and API4000 was used. 2.d. Lipid Classes: Glycerophospholipids Phosphatidic acid (PA) Phosphatidyl coline (PC) Phosphatidyl serine (PS) Phosphatidyl ethanolamine (PE) Phosphatidyl inositol (PI) Sphingolipids Ceramide (Cer) Sphingomyelin (SM) Glucosyl ceramide (GC) Lactosyl ceramide (LC) Sulfatide (ST) Ganglioside GM3 (GM3) Glycerolipids Diacylglycerol (DG) BM from API4000: GM3 and ST BM from TSQ: Cer, DG, GC, PA, PC, PE, PI, PS, SM WB from API4000: GM3 and ST WB from TSQ: Cer, GC, LC, PA, PC, PE, PI, PS, SM 2.e. Use QC normalized data for analyses - All users should only use the files which contain QC based normalization results. E03M99LIMmCoDaBM_09202016-QC-Normalized-Results_MULTIPL.xlsx E03M99LIMmCoDaWB_09202016-QC-Normalized-Results_MULTIPL.xlsx Other supporting files are also provided, but, if there is a need to use any data from those files, please contact the MaHIPC Informatics Core first (mahpic-infodepo@uga.edu). This is to ensure that all MaHPIC analyses are done on the same underlying data, and that users are informed about the context of normalization. 2.f. Data should be further normalized by RBC and/or WBC counts, as needed, before any analyses are performed. The RBC and WBC counts data are available in a separate file: E03M99LIMmCoDaZZ_09202016-WBC-and-RBC-for-Normalization_MULTIPL.xlsx 2.g. Samples were randomized by the MaHPIC Lipidomics Team before the LC-MS/MS runs. The order of injection of the samples after the randomization is available on the second sheet of the analytical metadata files 2.h. Samples were not run in replicates as was done in some of the earlier MaHPIC experiments ********** ********** ********** 3. File Organization and Description: Files listed below are named according to the naming document Supplementary/MaHPIC_File_Naming_Standards_1Sep2016.docx RESULTS: E03M99LIMmCoDaBM_09202016-QC-Normalized-Results_MULTIPL.xlsx E03M99LIMmCoDaWB_09202016-QC-Normalized-Results_MULTIPL.xlsx E03M99LIMmCoDaZZ_09202016-WBC-and-RBC-for-Normalization_MULTIPL.xlsx - Excel files containing final results - These are the files that all MAHPIC investigators should use by default. The clinical data (RBC and WBC counts) are also placed in this directory though they are not directly results from the MaHPIC Lipidomics Team. RAW DATA: E03M99LIMmCoDaZZ_09202016-API4000-Files_MULTIPL.zip E03M99LIMmCoDaZZ_09202016-TSQ-Files_MULTIPL.zip - These are the raw data from the instruments, supporting information, as well as the intermediate files produced from the two instruments used. The files are zipped and only the zipped file is renamed according to MaHPIC file naming standard. For detailed description of the files contained within these zipped files, refer to the documentation in the SOP - sections 8.1 (Files generated on TSQ Quantum Ultra triple quadrupole mass spectrometer) and 8.2 (Files generated on API4000 mass spectrometer) METADATA: E03M99LIMmCyDaBM_09202016-BM-AnalyticalMetadata_MULTIPL.xlsx E03M99LIMmCyDaWB_09202016-WB-AnalyticalMetadata_MULTIPL.xlsx - Excel files containing Analytical Metadata and sheets documenting the run order of the samples and other instrumentation information SUPPLEMENTARY: E03M99LIMmCoDaZZ_Barcodes-Info_MULTIPL.xlsx - List of barcoded samples, subject IDs, and time points E03_Lipidomics_SOP_WB_and_BM_09202016_v2.docx - SOP describing Lipid nomenclature, Internal standards and quality control (QC) sample used, sample preparation, lipid classes investigated in different runs, LC-MS/MS conditions for each run, process employed for relative quantification of each lipid species in a lipid class, and normalization by QC. E03M99LIMmCoDaBM_Ceramides-Results_MULTIPL.xls E03M99LIMmCoDaBM_DiacylGlycerides-Results_MULTIPL.xls E03M99LIMmCoDaBM_GlucosylCeramides-Results_MULTIPL.xls E03M99LIMmCoDaBM_PhosphatidicAcid-Results_MULTIPL.xls E03M99LIMmCoDaBM_PhosphatidylCholine-Results_MULTIPL.xls E03M99LIMmCoDaBM_PhosphatidylEthanolamine-Results_MULTIPL.xls E03M99LIMmCoDaBM_PhosphatidylSerine-Results_MULTIPL.xls E03M99LIMmCoDaBM_Phosphatidylinositol-Results_MULTIPL.xls E03M99LIMmCoDaBM_Sphingomyelin-Results_MULTIPL.xls E03M99LIMmCyDaBM_Gangliosides-Sulfatides-Results_MULTIPL.xlsx E03M99LIMmCoDaWB_Ceramides-Results_MULTIPL.xls E03M99LIMmCoDaWB_GlucosylCeramides-Results_MULTIPL.xls E03M99LIMmCoDaWB_LactosylCeramide-Results_MULTIPL.xls E03M99LIMmCoDaWB_PhosphatidicAcid-Results_MULTIPL.xls E03M99LIMmCoDaWB_PhosphatidylCholine-Results_MULTIPL.xls E03M99LIMmCoDaWB_PhosphatidylEthanolamine-Results_MULTIPL.xls E03M99LIMmCoDaWB_PhosphatidylSerine-Results_MULTIPL.xls E03M99LIMmCoDaWB_Phosphatidylinositol-Results_MULTIPL.xls E03M99LIMmCoDaWB_Sphingomyelin-Results_MULTIPL.xls E03M99LIMmCyDaWB_Gangliosides-Sulfatides-Results_MULTIPL.xlsx - Intermediate result data for each lipid species are available as separate files. They contain the peak area and other data. Please note that these data are not QC-normalized and cannot be used directly. ********** ********** ********** 4. Experimental Results Column Header Definitions In the final result files (listed below), the column headers are the lipid names. The numbers within parentheses next to the species abbreviation refer to the total number of carbons and double bonds of all the acyl chains. The alkyl ether linkage in PC is represented by the "A" prefix, e.g. PC(A32:0) and the (1Z)-alkenyl ether (neutral Plasmalogen) species by the "P" prefix, e.g. PC(P34:2). PE(18:0-18:1)/PE(16:0-20:1) means there are 2 PE isomers: one has 18:0 and 18:1 acyl groups, and another has 16:0 and 20:1 acyl groups. Their molecular weights are same PAF C16 is platelet-activating factor C-16 E03M99LIMmCoDaBM_09202016-QC-Normalized-Results_MULTIPL.xlsx E03M99LIMmCoDaWB_09202016-QC-Normalized-Results_MULTIPL.xlsx The first column contains the LIMS barcode of the sample. For column header definitions/descriptions of the intermediate result files, refer to the documentation in the SOP - sections 8.1 (Files generated on TSQ Quantum Ultra triple quadrupole mass spectrometer) and 8.2 (Files generated on API4000 mass spectrometer) ********** ********** **********