Untargeted Metabolomics Dataset from Rheumatic Heart Disease, Degenerative Aortic Stenosis, and Healthy Controls

Study SummaryThis dataset contains untargeted LC–MS/MS metabolomics profiles of serum samples collected from patients with rheumatic heart disease (RHD, n = 33), degenerative aortic stenosis (AS, n = 9), and healthy controls (n = 15). The study aimed to characterize metabolic alterations associated with valvular heart diseases relative to healthy individuals.
Serum samples were analyzed using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC–QTOF–MS) operated in both positive and negative electrospray ionization (ESI⁺/ESI⁻) modes. The dataset includes processed peak tables, and metadata describing the clinical samples.
Study DesignStudy groups:Rheumatic Heart Disease (RHD): n = 33Degenerative Aortic Stenosis (AS): n = 9Healthy Controls: n = 15Quality control and blanks:Pooled QC: prepared by combining equal aliquots of all serum samples (RHD, AS, and controls). Multiple aliquots were stored to avoid repeated freeze–thaw cycles.Long-term QC: NIST SRM 1950 plasma reference standard (NIST, USA) was included in each batch as a long-term reference.Blanks: extraction blanks were processed alongside study samples to monitor background contamination.Sample CollectionVenous blood was collected from all participants after written informed consent. For RHD and AS patients, samples were obtained prior to general anaesthesia. Blood was drawn into VACUETTE® CAT serum clot activator tubes (Greiner Bio-One, Austria), allowed to clot for 30 minutes at room temperature, and centrifuged at 2,000 × g for 15 minutes at 4 °C. Serum aliquots were stored at −80 °C until analysis (within 1–2 years of collection).
Sample Preparation and ExtractionFrozen serum samples were thawed on ice. Metabolites were extracted from 100 µL of each sample (including QC and reference materials) by adding 400 µL of ice-cold acetonitrile:methanol (9:1, v/v) containing 1% formic acid (for ESI⁺) or 1 mM acetic acid (for ESI⁻). The mixture was vortexed for 2 minutes and centrifuged at 14,000 × g for 15 minutes at 4 °C. Supernatants were dried under nitrogen gas, reconstituted in 100 µL of deionized water with 0.1% formic acid (ESI⁺) or 1 mM acetic acid (ESI⁻), and centrifuged again before LC–MS analysis.
Chromatographic SeparationChromatographic separation was performed on an ExionLC™ AD UPLC system (SCIEX, USA) using an Omega Polar C18 column (1.6 µm, 100 Å, 3 × 100 mm; Phenomenex, USA) maintained at 40 °C.
Mobile phase A: HPLC-grade waterMobile phase B: acetonitrile:methanol (1:1, v/v)Modifiers: 2 mM ammonium formate and 0.1% formic acid (ESI⁺) or 1 mM acetic acid (ESI⁻)Gradient: 60 minutes at 0.4 mL/minInjection volume: 5 µLAutosampler temperature: 15 °CNeedle rinsing: water:methanol:isopropanol (6:3:1, v/v/v) between injectionsMass SpectrometryMass spectrometric analysis was performed on a SCIEX X500R Q-TOF equipped with a dual ESI source and operated in data-dependent acquisition (DDA) mode using SCIEX OS 1.4.
Source parameters:
Ion source gas 1: 40 psiIon source gas 2: 65 psiCurtain gas: 25 psiSource temperature: 500 °CIon spray voltage: +5000 V (ESI⁺)Collision gas (CAD): 7 psiAcquisition settings:
Mass range: m/z 50–1200TOF–MS accumulation time: 200 msTOF–MS/MS accumulation time: 60 msMaximum candidate ions per cycle: 7Intensity threshold: 50 cpsCollision energy: 35 ± 15 VQ1 resolution: unitMS1 rate: 4 scans/sMS2 rate: 8 scans/sData ProcessingRaw LC–MS/MS data were converted to .mzML using MSConvert (ProteoWizard 3.0.1908) and processed in MS-DIAL 4.38.
Peak detection and alignment: performed using MS-DIAL.Missing value filtering: features with >30% missing values overall or within any study group were removed.Drift correction: QC-based LOESS normalization using pooled QC samples injected every five samples, with additional QCs at the start and end of each batch.Background filtering: features retained if sample/blank intensity ratio > 5.Features annotations: Features were annotated by MS and MS/MS spectral matching to HMDB, KEGG, and PubChem (similarity >70%), yielding putative identifications consistent with MSI Level 2.Output files: Putative annotated and aligned peak tables as obtained from MSDIAL for both ESI⁺ and ESI⁻ modes.Quality ControlPooled QC: used to assess analytical reproducibility and applied for normalization.NIST SRM 1950: served as a long-term performance reference across analytical batches.Blank samples: used to identify and filter out background or contaminant signals.EthicsAll participants provided written informed consent. The study was approved by the Faculty of Health Sciences Human Research Ethics Committee, University of Cape Town (HREC Ref: 574/2018 and 061/2018).
Data AvailabilityProcessed data: MS-DIAL aligned and annotated peak tables (.csv) positive and negative mode.Metadata: includes sample information.