DOI: 10.7910/dvn/uzh6bd
title: Targeted Metabolomics in the REGARDS Stroke Case-Cohort Study
title: Amide Column-Based Chromatography for Polar Metabolite Profiling in the REGARDS Stroke Case-Cohort

description: Targeted Metabolomics in the REGARDS Stroke Case-Cohort Study. 
A prospective epidemiological study with a case-cohort design.
Targeted metabolomics was conducted on plasma samples from a nested case-cohort study within the bi-racial REasons for Geographic and Racial Differences in Stroke (REGARDS), a longitudinal prospective cohort. The REGARDS study was designed to investigate different health outcomes with a focus on stroke disparities across the USA. Participants were oversampled from the Southeastern regions of the "stroke belt," where stroke risk and mortality are 2-4 times higher in the African American population. The study recruited 30,239 community-dwelling Black and White participants aged 45 years or older. Plasma samples were collected during the baseline study visit between 2003 and 2007 and stored at -80°C. This dataset includes a total of 2,378 plasma samples representing the stroke case-cohort sub-study. Participants of the random cohort (N=1,056) were selected at the point of enrolment. Stroke cases were adjudicated up to April 2009 (N=1,322).
description: Sample Collection Summary: 
K2EDTA plasma samples were collected at the time of the baseline study visit. All participants were asked to fast at least 8 hours before venous blood drawing in the early morning. Plasma was isolated in a timely manner and stored at -80 °C. Samples went through a freeze-thaw cycle for the purpose of sub-aliquoting and shipping for metabolomics assays. Samples went through a second freeze-thaw cycle immediately before extraction. All procedures requiring manual sample handling were conducted on wet ice.
Sample Type: Blood (plasma)
Storage Conditions: -80℃
description: Sample Preparation Summary:
Samples were extracted over wet ice using protein precipitation. First, 30 μL of K2EDTA plasma was aliquoted onto a 96-well plate. Before quenching, each received 30 µL of internal solution mixture containing stable isotope-labeled proline (13C5, 15N), glutamine (13C5, 15N2), leucine-d10, and phenylalanine-d8 [(15 µM each in methanol: water (50:50)]. Samples were then briefly vortexed for 1 minute at 1400 rpm to ensure homogeneity. Proteins were precipitated with 110 µL of ice-cold acetonitrile: methanol (75:25), and samples were vortexed for a further 5 min at 1400 rpm. Samples were centrifuged at 4000 rpm for 10 min at 4°C, and 100 µL of the clean supernatants were transferred to clean well plates for LC-MS/MS injection.
Processing Storage Conditions: 4℃
Extraction Method: Protein Precipitation (PPE)
description: MS Summary for negative ionization mode:
Instrument Name: Agilent 6495 QQQ
Instrument Type: Triple quadrupole
Ionization source: ESI
MS Comments: Metabolites were measured using an Agilent 6495 triple quadrupole mass spectrometer in dynamic MRM mode. The ESI source settings were as follows: polarity: negative, gas temperature: 230°C, gas flow: 15 L/min, nebulizer pressure: 30 psi, sheath gas temperature: 400°C, sheath gas flow: 12 L/min, capillary voltage: 2500 V, and nozzle voltage: 1000 V. Dynamic MRM transitions with collision energies are detailed in the attached protocol. Data were processed using Agilent MassHunter QQQ Quantitative Analysis utilizing the Agile2 integrator, Quartic/Quintic Savitzky-Golay, and the Root Mean Square (RMS) noise algorithms. Normalized peak areas were calculated by dividing the peak areas of the analytes by the corresponding nearest human pooled plasma (HPP) signal for each analyte. (The provided data included HPP samples; therefore, the values in the data table are pre-normalization).
description: MS Summary for positive ionization mode: 
Instrument Name: Agilent 6495 QQQ
Instrument Type: Triple quadrupole
Ionization source: ESI
MS Comments: Metabolites were measured using an Agilent 6495 triple quadrupole mass spectrometer in dynamic MRM mode. The ESI source settings were as follows: polarity: positive, gas temperature: 230°C, gas flow: 15 L/min, nebulizer pressure: 30 psi, sheath gas temperature: 400°C, sheath gas flow: 12 L/min, capillary voltage: 2000 V, and nozzle voltage: 1000 V. Dynamic MRM transitions with collision energies are detailed in the attached protocol. Data were processed using Agilent MassHunter QQQ Quantitative Analysis utilizing the Agile2 integrator, Quartic/Quintic Savitzky-Golay, and the Root Mean Square (RMS) noise algorithms. Normalized peak areas were calculated by dividing the peak areas of the analytes by the corresponding nearest human pooled plasma (HPP) signal for each analyte. (The provided data included HPP samples; therefore, the values in the data table are pre-normalization).
description: Stata, 17