#METABOLOMICS WORKBENCH Rene_Neuhaus_20250716_073601 DATATRACK_ID:6176 STUDY_ID:ST004080 ANALYSIS_ID:AN006754 PROJECT_ID:PR002561 VERSION 1 CREATED_ON July 16, 2025, 10:25 pm #PROJECT PR:PROJECT_TITLE Revealing metabotypes of IgE-, COX-1 inhibitors- and MRGPRX2 receptor-mediated PR:PROJECT_TITLE drug hypersensitivities PR:PROJECT_TYPE Lipidomics untargeted MS PR:PROJECT_SUMMARY Despite their clinical relevance, the immunological mechanisms underlying PR:PROJECT_SUMMARY immediate drug hypersensitivity reactions (iDHR) are unclear. This study PR:PROJECT_SUMMARY therefore explores the metabolic endotypes (metabotypes) of IgE-, cyclooxygenase PR:PROJECT_SUMMARY (COX)-1 inhibitor-, and Mas-related G-protein coupled receptor X2 PR:PROJECT_SUMMARY (MRGPRX2)-mediated iDHR. Serum samples obtained from 19 patients during the PR:PROJECT_SUMMARY acute and baseline stages of iDHR were analyzed using liquid chromatography PR:PROJECT_SUMMARY coupled to mass spectrometry (LC-MS)-based untargeted metabolomics. Different PR:PROJECT_SUMMARY immunological mechanisms were found to induce distinct metabotypes. The main PR:PROJECT_SUMMARY metabolic classes implicated were amino acids, acyl carnitines, fatty acids, and PR:PROJECT_SUMMARY lysophospholipids, such as lysophosphatidylcholines (LPC) and PR:PROJECT_SUMMARY lysophosphatidylethanolamines (LPE). Most of these compounds decreased during PR:PROJECT_SUMMARY the acute phase of iDHR and exhibited varying trends among the three types of PR:PROJECT_SUMMARY iDHR. PR:INSTITUTE Universidad CEU San Pablo PR:DEPARTMENT Chemistry and Biochemistry PR:LABORATORY CEMBIO PR:LAST_NAME Neuhaus PR:FIRST_NAME René PR:ADDRESS Urb. Montepríncipe, Alcorcón, Madrid, 28925, Spain PR:EMAIL rene.neuhaus@ceu.es PR:PHONE +34611042778 #STUDY ST:STUDY_TITLE Revealing metabotypes of IgE-, COX-1 inhibitors- and MRGPRX2 receptor-mediated ST:STUDY_TITLE drug hypersensitivities ST:STUDY_SUMMARY Despite their clinical relevance, the immunological mechanisms underlying ST:STUDY_SUMMARY immediate drug hypersensitivity reactions (iDHR) are unclear. This study ST:STUDY_SUMMARY therefore explores the metabolic endotypes (metabotypes) of IgE-, cyclooxygenase ST:STUDY_SUMMARY (COX)-1 inhibitor-, and Mas-related G-protein coupled receptor X2 ST:STUDY_SUMMARY (MRGPRX2)-mediated iDHR. Serum samples obtained from 19 patients during the ST:STUDY_SUMMARY acute and baseline stages of iDHR were analyzed using liquid chromatography ST:STUDY_SUMMARY coupled to mass spectrometry (LC-MS)-based untargeted metabolomics. Different ST:STUDY_SUMMARY immunological mechanisms were found to induce distinct metabotypes. The main ST:STUDY_SUMMARY metabolic classes implicated were amino acids, acyl carnitines, fatty acids, and ST:STUDY_SUMMARY lysophospholipids, such as lysophosphatidylcholines (LPC) and ST:STUDY_SUMMARY lysophosphatidylethanolamines (LPE). Most of these compounds decreased during ST:STUDY_SUMMARY the acute phase of iDHR and exhibited varying trends among the three types of ST:STUDY_SUMMARY iDHR. ST:INSTITUTE Universidad CEU San Pablo ST:DEPARTMENT Chemistry and Biochemistry ST:LABORATORY CEMBIO ST:LAST_NAME Neuhaus ST:FIRST_NAME René ST:ADDRESS Urb. Montepríncipe, Alcorcón, Madrid, 28925, Spain ST:EMAIL rene.neuhaus@ceu.es ST:PHONE +34611042778 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS P_01 1_1 Sample source:Serum | Metabotype:IgE | Timepoint:Acute stage RAW_FILE_NAME=1_1_P.mzML; RAW_FILE_NAME=1_1_N.mzML SUBJECT_SAMPLE_FACTORS P_02 2_1 Sample source:Serum | Metabotype:IgE | Timepoint:Acute stage RAW_FILE_NAME=2_1_P.mzML; RAW_FILE_NAME=2_1_N.mzML SUBJECT_SAMPLE_FACTORS P_03 3_1 Sample source:Serum | Metabotype:IgE | Timepoint:Acute stage RAW_FILE_NAME=3_1_P.mzML; RAW_FILE_NAME=3_1_N.mzML SUBJECT_SAMPLE_FACTORS P_04 4_1 Sample source:Serum | Metabotype:IgE | Timepoint:Acute stage RAW_FILE_NAME=4_1_P.mzML; RAW_FILE_NAME=4_1_N.mzML SUBJECT_SAMPLE_FACTORS P_05 5_1 Sample source:Serum | Metabotype:IgE | Timepoint:Acute stage RAW_FILE_NAME=5_1_P.mzML; RAW_FILE_NAME=5_1_N.mzML SUBJECT_SAMPLE_FACTORS P_06 6_1 Sample source:Serum | Metabotype:IgE | Timepoint:Acute stage RAW_FILE_NAME=6_1_P.mzML; RAW_FILE_NAME=6_1_N.mzML SUBJECT_SAMPLE_FACTORS P_07 7_1 Sample source:Serum | Metabotype:IgE | Timepoint:Acute stage RAW_FILE_NAME=7_1_P.mzML; RAW_FILE_NAME=7_1_N.mzML SUBJECT_SAMPLE_FACTORS P_08 8_1 Sample source:Serum | Metabotype:IgE | Timepoint:Acute stage RAW_FILE_NAME=8_1_P.mzML; RAW_FILE_NAME=8_1_N.mzML SUBJECT_SAMPLE_FACTORS P_18 18_1 Sample source:Serum | Metabotype:COX-1 inhibitors | Timepoint:Acute stage RAW_FILE_NAME=18_1_P.mzML; RAW_FILE_NAME=18_1_N.mzML SUBJECT_SAMPLE_FACTORS P_19 19_1 Sample source:Serum | Metabotype:COX-1 inhibitors | Timepoint:Acute stage RAW_FILE_NAME=19_1_P.mzML; RAW_FILE_NAME=19_1_N.mzML SUBJECT_SAMPLE_FACTORS P_20 20_1 Sample source:Serum | Metabotype:COX-1 inhibitors | Timepoint:Acute stage RAW_FILE_NAME=20_1_P.mzML; RAW_FILE_NAME=20_1_N.mzML SUBJECT_SAMPLE_FACTORS P_21 21_1 Sample source:Serum | Metabotype:COX-1 inhibitors | Timepoint:Acute stage RAW_FILE_NAME=21_1_P.mzML; RAW_FILE_NAME=21_1_N.mzML SUBJECT_SAMPLE_FACTORS P_22 22_1 Sample source:Serum | Metabotype:COX-1 inhibitors | Timepoint:Acute stage RAW_FILE_NAME=22_1_P.mzML; RAW_FILE_NAME=22_1_N.mzML SUBJECT_SAMPLE_FACTORS P_23 23_1 Sample source:Serum | Metabotype:COX-1 inhibitors | Timepoint:Acute stage RAW_FILE_NAME=23_1_P.mzML; RAW_FILE_NAME=23_1_N.mzML SUBJECT_SAMPLE_FACTORS P_36 36_1 Sample source:Serum | Metabotype:MRGPRX2 | Timepoint:Acute stage RAW_FILE_NAME=36_1_P.mzML; RAW_FILE_NAME=36_1_N.mzML SUBJECT_SAMPLE_FACTORS P_37 37_1 Sample source:Serum | Metabotype:MRGPRX2 | Timepoint:Acute stage RAW_FILE_NAME=37_1_P.mzML; RAW_FILE_NAME=37_1_N.mzML SUBJECT_SAMPLE_FACTORS P_38 38_1 Sample source:Serum | Metabotype:MRGPRX2 | Timepoint:Acute stage RAW_FILE_NAME=38_1_P.mzML; RAW_FILE_NAME=38_1_N.mzML SUBJECT_SAMPLE_FACTORS P_39 39_1 Sample source:Serum | Metabotype:MRGPRX2 | Timepoint:Acute stage RAW_FILE_NAME=39_1_P.mzML; RAW_FILE_NAME=39_1_N.mzML SUBJECT_SAMPLE_FACTORS P_40 40_1 Sample source:Serum | Metabotype:MRGPRX2 | Timepoint:Acute stage RAW_FILE_NAME=40_1_P.mzML; RAW_FILE_NAME=40_1_N.mzML SUBJECT_SAMPLE_FACTORS P_01 1_3 Sample source:Serum | Metabotype:IgE | Timepoint:Baseline stage RAW_FILE_NAME=1_3_P.mzML; RAW_FILE_NAME=1_3_N.mzML SUBJECT_SAMPLE_FACTORS P_02 2_3 Sample source:Serum | Metabotype:IgE | Timepoint:Baseline stage RAW_FILE_NAME=2_3_P.mzML; RAW_FILE_NAME=2_3_N.mzML SUBJECT_SAMPLE_FACTORS P_03 3_3 Sample source:Serum | Metabotype:IgE | Timepoint:Baseline stage RAW_FILE_NAME=3_3_P.mzML; RAW_FILE_NAME=3_3_N.mzML SUBJECT_SAMPLE_FACTORS P_04 4_3 Sample source:Serum | Metabotype:IgE | Timepoint:Baseline stage RAW_FILE_NAME=4_3_P.mzML; RAW_FILE_NAME=4_3_N.mzML SUBJECT_SAMPLE_FACTORS P_05 5_3 Sample source:Serum | Metabotype:IgE | Timepoint:Baseline stage RAW_FILE_NAME=5_3_P.mzML; RAW_FILE_NAME=5_3_N.mzML SUBJECT_SAMPLE_FACTORS P_06 6_3 Sample source:Serum | Metabotype:IgE | Timepoint:Baseline stage RAW_FILE_NAME=6_3_P.mzML; RAW_FILE_NAME=6_3_N.mzML SUBJECT_SAMPLE_FACTORS P_07 7_3 Sample source:Serum | Metabotype:IgE | Timepoint:Baseline stage RAW_FILE_NAME=7_3_P.mzML; RAW_FILE_NAME=7_3_N.mzML SUBJECT_SAMPLE_FACTORS P_08 8_3 Sample source:Serum | Metabotype:IgE | Timepoint:Baseline stage RAW_FILE_NAME=8_3_P.mzML; RAW_FILE_NAME=8_3_N.mzML SUBJECT_SAMPLE_FACTORS P_18 18_3 Sample source:Serum | Metabotype:COX-1 inhibitors | Timepoint:Baseline stage RAW_FILE_NAME=18_3_P.mzML; RAW_FILE_NAME=18_3_N.mzML SUBJECT_SAMPLE_FACTORS P_19 19_3 Sample source:Serum | Metabotype:COX-1 inhibitors | Timepoint:Baseline stage RAW_FILE_NAME=19_3_P.mzML; RAW_FILE_NAME=19_3_N.mzML SUBJECT_SAMPLE_FACTORS P_20 20_3 Sample source:Serum | Metabotype:COX-1 inhibitors | Timepoint:Baseline stage RAW_FILE_NAME=20_3_P.mzML; RAW_FILE_NAME=20_3_N.mzML SUBJECT_SAMPLE_FACTORS P_21 21_3 Sample source:Serum | Metabotype:COX-1 inhibitors | Timepoint:Baseline stage RAW_FILE_NAME=21_3_P.mzML; RAW_FILE_NAME=21_3_N.mzML SUBJECT_SAMPLE_FACTORS P_22 22_3 Sample source:Serum | Metabotype:COX-1 inhibitors | Timepoint:Baseline stage RAW_FILE_NAME=22_3_P.mzML; RAW_FILE_NAME=22_3_N.mzML SUBJECT_SAMPLE_FACTORS P_23 23_3 Sample source:Serum | Metabotype:COX-1 inhibitors | Timepoint:Baseline stage RAW_FILE_NAME=23_3_P.mzML; RAW_FILE_NAME=23_3_N.mzML SUBJECT_SAMPLE_FACTORS P_36 36_3 Sample source:Serum | Metabotype:MRGPRX2 | Timepoint:Baseline stage RAW_FILE_NAME=36_3_P.mzML; RAW_FILE_NAME=36_3_N.mzML SUBJECT_SAMPLE_FACTORS P_37 37_3 Sample source:Serum | Metabotype:MRGPRX2 | Timepoint:Baseline stage RAW_FILE_NAME=37_3_P.mzML; RAW_FILE_NAME=37_3_N.mzML SUBJECT_SAMPLE_FACTORS P_38 38_3 Sample source:Serum | Metabotype:MRGPRX2 | Timepoint:Baseline stage RAW_FILE_NAME=38_3_P.mzML; RAW_FILE_NAME=38_3_N.mzML SUBJECT_SAMPLE_FACTORS P_39 39_3 Sample source:Serum | Metabotype:MRGPRX2 | Timepoint:Baseline stage RAW_FILE_NAME=39_3_P.mzML; RAW_FILE_NAME=39_3_N.mzML SUBJECT_SAMPLE_FACTORS P_40 40_3 Sample source:Serum | Metabotype:MRGPRX2 | Timepoint:Baseline stage RAW_FILE_NAME=40_3_P.mzML; RAW_FILE_NAME=40_3_N.mzML SUBJECT_SAMPLE_FACTORS QC QC_1 Sample source:Serum | Metabotype:QC | Timepoint:QC RAW_FILE_NAME=QC_1_P.mzML; RAW_FILE_NAME=QC_1_N.mzML SUBJECT_SAMPLE_FACTORS QC QC_2 Sample source:Serum | Metabotype:QC | Timepoint:QC RAW_FILE_NAME=QC_2_P.mzML; RAW_FILE_NAME=QC_2_N.mzML SUBJECT_SAMPLE_FACTORS QC QC_3 Sample source:Serum | Metabotype:QC | Timepoint:QC RAW_FILE_NAME=QC_3_P.mzML; RAW_FILE_NAME=QC_3_N.mzML SUBJECT_SAMPLE_FACTORS QC QC_4 Sample source:Serum | Metabotype:QC | Timepoint:QC RAW_FILE_NAME=QC_4_P.mzML; RAW_FILE_NAME=QC_4_N.mzML SUBJECT_SAMPLE_FACTORS QC QC_5 Sample source:Serum | Metabotype:QC | Timepoint:QC RAW_FILE_NAME=QC_5_P.mzML; RAW_FILE_NAME=QC_5_N.mzML SUBJECT_SAMPLE_FACTORS QC QC_6 Sample source:Serum | Metabotype:QC | Timepoint:QC RAW_FILE_NAME=QC_6_P.mzML; RAW_FILE_NAME=QC_6_N.mzML SUBJECT_SAMPLE_FACTORS QC QC_7 Sample source:Serum | Metabotype:QC | Timepoint:QC RAW_FILE_NAME=QC_7_P.mzML; RAW_FILE_NAME=QC_7_N.mzML SUBJECT_SAMPLE_FACTORS QC QC_8 Sample source:Serum | Metabotype:QC | Timepoint:QC RAW_FILE_NAME=QC_8_P.mzML; RAW_FILE_NAME=QC_8_N.mzML SUBJECT_SAMPLE_FACTORS QC QC_9 Sample source:Serum | Metabotype:QC | Timepoint:QC RAW_FILE_NAME=QC_9_P.mzML; RAW_FILE_NAME=QC_9_N.mzML #COLLECTION CO:COLLECTION_SUMMARY Two peripheral blood samples were collected from each patient at different time CO:COLLECTION_SUMMARY points using BD Vacutainer® SST™ II Advance tubes: one at the acute stage CO:COLLECTION_SUMMARY (immediately after a drug provocation test with the elicitor drug) and one at CO:COLLECTION_SUMMARY the baseline stage (at least 14 days after the drug provocation test). CO:SAMPLE_TYPE Blood (serum) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Serum samples were obtained by centrifuging blood samples (1200 x g, 10 min, TR:TREATMENT_SUMMARY 4°C) and stored at -80°C until analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 100 μL of the acute and baseline serum samples (nIgE = 8, nCOX = 6, nMRG = 5) SP:SAMPLEPREP_SUMMARY were deproteinized by adding 300 μL of a cold solution consisting of MeOH:EtOH SP:SAMPLEPREP_SUMMARY (1:1, v/v, -20⁰C). Samples were then vortex-mixed, incubated on ice (20 min, SP:SAMPLEPREP_SUMMARY 0°C) and centrifuged (16100 x g, 20 min, 4°C). Resulting supernatants were SP:SAMPLEPREP_SUMMARY transferred to LC vials, centrifuged (2000 x g, 5 min, 15 °C) and then injected SP:SAMPLEPREP_SUMMARY into the LC-MS system. Blank samples were prepared using the same protocol, SP:SAMPLEPREP_SUMMARY except that serum was replaced with water. In addition, 25 μL of each serum SP:SAMPLEPREP_SUMMARY sample were pooled to prepare the quality control (QC) samples. QC samples were SP:SAMPLEPREP_SUMMARY processed using the same steps and injected every 3-4 samples throughout the SP:SAMPLEPREP_SUMMARY analysis to monitor the reproducibility of the sample preparation protocol and SP:SAMPLEPREP_SUMMARY the performance and stability of the analytical system. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Serum samples were analyzed in an untargeted approach using an Agilent 1290 CH:CHROMATOGRAPHY_SUMMARY Infinity II ultra-high performance liquid chromatography (UHPLC) system coupled CH:CHROMATOGRAPHY_SUMMARY to an Agilent 6545 quadrupole-time-of-flight mass spectrometer (QTOF-MS) CH:CHROMATOGRAPHY_SUMMARY (Agilent Technologies, Santa Clara, CA, USA) used in both positive and negative CH:CHROMATOGRAPHY_SUMMARY electrospray ionization modes (ESI(+) and ESI(-), respectively) with the CH:CHROMATOGRAPHY_SUMMARY following characteristics. For ESI(+) and ESI(-), 0.5 μL and 1.0 μL of the CH:CHROMATOGRAPHY_SUMMARY extracted samples, respectively, were injected into a Discovery® HS C18 CH:CHROMATOGRAPHY_SUMMARY reversed-phase column (2.1 mm × 150 mm, 3.0 μm) coupled to a compatible CH:CHROMATOGRAPHY_SUMMARY Discovery® HS C18 guard column (2.1 mm × 20 mm, 3.0 μm) (Supelco, Sigma CH:CHROMATOGRAPHY_SUMMARY Aldrich, Darmstadt, Germany), maintained at 60°C. Of note, the multisampler CH:CHROMATOGRAPHY_SUMMARY temperature was maintained at 4°C throughout the analysis to ensure compound CH:CHROMATOGRAPHY_SUMMARY stability. The mobile phases used for both ionization modes were (A) 0.1% FA in CH:CHROMATOGRAPHY_SUMMARY water and (B) 0.1% FA in ACN. The flow rate was set at 0.6 mL/min and the CH:CHROMATOGRAPHY_SUMMARY chromatographic gradient consisted of: 5% of B at 0.0 – 1.0, from 5% to 80% of CH:CHROMATOGRAPHY_SUMMARY B at 1.0 – 7.0, from 80% to 100% of B at 7.0 – 11.5, from 100% to 5% of B at CH:CHROMATOGRAPHY_SUMMARY 11.5 – 12.0. The initial conditions were restored by min 12.0 and were CH:CHROMATOGRAPHY_SUMMARY followed by a 3.0 min re-equilibration period, which led to a total running time CH:CHROMATOGRAPHY_SUMMARY of 15.0 min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Discovery HS C18 (150 x 2.1mm, 3.0um) with a Discovery HS C18 guard column (20 x CH:COLUMN_NAME 2.1mm, 3.0um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0.0 – 1.0 min: 5% B; 1.0 – 7.0 min: linear increase until 80% B; 7.0 – CH:FLOW_GRADIENT 11.5 min: linear increase until 100% B. Then the equipment returned to the CH:FLOW_GRADIENT initial conditions in 0.5 min, which were held for 3.0 min for column CH:FLOW_GRADIENT reconditioning. CH:FLOW_RATE 0.6 mL/min CH:COLUMN_TEMPERATURE 60 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The QTOF mass spectrometer, equipped with a dual atmospheric jet stream MS:MS_COMMENTS electrospray ionization (ESI) ion source, was configured with the following MS:MS_COMMENTS parameters: 175 V fragmentor, 65 V skimmer, 4000 V capillary voltage, 750 V MS:MS_COMMENTS octopole radio frequency voltage, 12 L/min nebulizer gas flow, 250 °C gas MS:MS_COMMENTS temperature, 52 psi nebulizer gas pressure, 11 L/min sheath gas flow, and 370 MS:MS_COMMENTS °C sheath gas temperature. In full scan mode, operated from 100 to 1.700 m/z MS:MS_COMMENTS with a scan rate of 1.0 spectra/s, data were collected for both ionization MS:MS_COMMENTS modes. A solution of two reference mass compounds was infused throughout the MS:MS_COMMENTS analysis using an Agilent 1260 Iso Pump at a flow rate of 1.5 mL/min: purine MS:MS_COMMENTS (detected as [C5H4N4-H]−) at m/z 119.0363 and HP-0921 (detected as MS:MS_COMMENTS [C18H18O6N3P3F24+HCOO]−) at m/z 966.0007. These masses were continuously MS:MS_COMMENTS infused into the system to provide constant mass correction. MS:MS_RESULTS_FILE ST004080_AN006754_Results.txt UNITS:Peak area Has m/z:Neutral masses Has RT:Yes RT units:Minutes #END