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

<h2><b>Study Summary</b></h2><p dir="ltr">This 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.</p><p dir="ltr">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.</p><h2><b>Study Design</b></h2><ul><li><b>Study groups:</b></li><li><ul><li>Rheumatic Heart Disease (RHD): n = 33</li><li>Degenerative Aortic Stenosis (AS): n = 9</li><li>Healthy Controls: n = 15</li></ul></li><li><b>Quality control and blanks:</b></li><li><ul><li><i>Pooled QC:</i> prepared by combining equal aliquots of all serum samples (RHD, AS, and controls). Multiple aliquots were stored to avoid repeated freeze–thaw cycles.</li><li><i>Long-term QC:</i> NIST SRM 1950 plasma reference standard (NIST, USA) was included in each batch as a long-term reference.</li><li><i>Blanks:</i> extraction blanks were processed alongside study samples to monitor background contamination.</li></ul></li></ul><h2><b>Sample Collection</b></h2><p dir="ltr">Venous 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).</p><h2><b>Sample Preparation and Extraction</b></h2><p dir="ltr">Frozen 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.</p><h2><b>Chromatographic Separation</b></h2><p dir="ltr">Chromatographic 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.</p><ul><li><b>Mobile phase A:</b> HPLC-grade water</li><li><b>Mobile phase B:</b> acetonitrile:methanol (1:1, v/v)</li><li><b>Modifiers:</b> 2 mM ammonium formate and 0.1% formic acid (ESI⁺) or 1 mM acetic acid (ESI⁻)</li><li><b>Gradient:</b> 60 minutes at 0.4 mL/min</li><li><b>Injection volume:</b> 5 µL</li><li><b>Autosampler temperature:</b> 15 °C</li><li><b>Needle rinsing:</b> water:methanol:isopropanol (6:3:1, v/v/v) between injections</li></ul><h2><b>Mass Spectrometry</b></h2><p dir="ltr">Mass 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.</p><p dir="ltr"><b>Source parameters:</b></p><ul><li>Ion source gas 1: 40 psi</li><li>Ion source gas 2: 65 psi</li><li>Curtain gas: 25 psi</li><li>Source temperature: 500 °C</li><li>Ion spray voltage: +5000 V (ESI⁺)</li><li>Collision gas (CAD): 7 psi</li></ul><p dir="ltr"><b>Acquisition settings:</b></p><ul><li>Mass range: m/z 50–1200</li><li>TOF–MS accumulation time: 200 ms</li><li>TOF–MS/MS accumulation time: 60 ms</li><li>Maximum candidate ions per cycle: 7</li><li>Intensity threshold: 50 cps</li><li>Collision energy: 35 ± 15 V</li><li>Q1 resolution: unit</li><li>MS1 rate: 4 scans/s</li><li>MS2 rate: 8 scans/s</li></ul><h2><b>Data Processing</b></h2><p dir="ltr">Raw LC–MS/MS data were converted to <code>.mzML</code> using MSConvert (ProteoWizard 3.0.1908) and processed in MS-DIAL 4.38.</p><ul><li><b>Peak detection and alignment:</b> performed using MS-DIAL.</li><li><b>Missing value filtering:</b> features with >30% missing values overall or within any study group were removed.</li><li><b>Drift correction:</b> QC-based LOESS normalization using pooled QC samples injected every five samples, with additional QCs at the start and end of each batch.</li><li><b>Background filtering:</b> features retained if sample/blank intensity ratio > 5.</li><li><b>Features annotations:</b> Features were annotated by MS and MS/MS spectral matching to HMDB, KEGG, and PubChem (similarity >70%), yielding <i>putative identifications</i> consistent with MSI Level 2.</li><li><b>Output files:</b> Putative annotated and aligned peak tables as obtained from MSDIAL for both ESI⁺ and ESI⁻ modes.</li></ul><h2><b>Quality Control</b></h2><ul><li><b>Pooled QC:</b> used to assess analytical reproducibility and applied for normalization.</li><li><b>NIST SRM 1950:</b> served as a long-term performance reference across analytical batches.</li><li><b>Blank samples:</b> used to identify and filter out background or contaminant signals.</li></ul><h2><b>Ethics</b></h2><p dir="ltr">All 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).</p><h2><b>Data Availability</b></h2><ul><li><b>Processed data:</b> MS-DIAL aligned and annotated peak tables (<code>.csv</code>) positive and negative mode.</li><li><b>Metadata:</b> includes sample information.</li></ul><p></p>