#METABOLOMICS WORKBENCH frederico_pinto_20250513_021905 DATATRACK_ID:5908 STUDY_ID:ST003994 ANALYSIS_ID:AN006583 PROJECT_ID:PR002499 VERSION 1 CREATED_ON June 20, 2025, 8:57 am #PROJECT PR:PROJECT_TITLE Optimizing Data-Independent Acquisition (DIA) for Enhanced Untargeted PR:PROJECT_TITLE Metabolomics PR:PROJECT_TYPE Untargeted Metabolomics LC-MS/MS plasma PR:PROJECT_SUMMARY This study developed a nano-sensitive DIA LC-MS/MS method for untargeted PR:PROJECT_SUMMARY metabolomics, systematically optimizing critical mass spectrometric parameters PR:PROJECT_SUMMARY to enhance metabolite coverage. PR:INSTITUTE Federal University of Viçosa PR:DEPARTMENT Campus Rio Paranaíba PR:LABORATORY Applied Analytical Chemistry PR:LAST_NAME Pinto PR:FIRST_NAME Frederico PR:ADDRESS Highway MG 230 Km 7, Rio Paranaíba, Minas Gerais, 38810000, Brazil PR:EMAIL frederico.pinto@ufv.br PR:PHONE na #STUDY ST:STUDY_TITLE Nano-sensitive DIA LC-MS/MS method for untargeted Metabolomics on NIST human ST:STUDY_TITLE plasma to systematically optimize critical mass spectrometric parameters to ST:STUDY_TITLE enhance metabolite coverage ST:STUDY_TYPE Human ST:STUDY_SUMMARY Data-Independent Acquisition (DIA) has emerged as a powerful mass spectrometry ST:STUDY_SUMMARY (MS) strategy for comprehensive metabolomics. This study presents a novel short ST:STUDY_SUMMARY gradient (13min) nano-sensitive analytical method for human plasma analysis ST:STUDY_SUMMARY using DIA LC-MS/MS, focusing on in-depth optimization of MS parameters to ST:STUDY_SUMMARY maximize data quality and metabolite coverage. Key MS parameters, including scan ST:STUDY_SUMMARY speed, isolation window width, resolution, automatic gain control, and collision ST:STUDY_SUMMARY energy, were systematically tuned to balance sensitivity and specificity while ST:STUDY_SUMMARY minimizing interferences. The optimized method, it enabled the detection of 2907 ST:STUDY_SUMMARY features and 675 annotated compounds, leverages recent progress in nano-LC-MS/MS ST:STUDY_SUMMARY for multi-omics applications, showcased the possibility of combining proteomics ST:STUDY_SUMMARY and metabolomics within a single chromatographic system. ST:INSTITUTE Federal University of Viçosa ST:DEPARTMENT Campus Rio Paranaíba ST:LABORATORY Applied Analytical Chemistry ST:LAST_NAME Pinto ST:FIRST_NAME Frederico ST:ADDRESS Highway MG 230 Km 7, Rio Paranaíba, Minas Gerais, 38810000, Brazil ST:EMAIL frederico.pinto@ufv.br ST:PHONE na #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Not applicable #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 Rep_1 NIST_DIA_1 Sample source:Plasma | Factor:DIA RAW_FILE_NAME=AAS_NIST_NIST_DIA SUBJECT_SAMPLE_FACTORS Rep_2 NIST_DIA_2 Sample source:Plasma | Factor:DIA RAW_FILE_NAME=AAS_NIST_NIST_DIA_20241028121157 SUBJECT_SAMPLE_FACTORS Rep_3 NIST_DIA_3 Sample source:Plasma | Factor:DIA RAW_FILE_NAME=AAS_NIST_NIST_DIA_20241028123101 #COLLECTION CO:COLLECTION_SUMMARY Human plasma reference material (SRM 1950) from the National Institute of CO:COLLECTION_SUMMARY Standards and Technology (NIST) CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY Three technical replicates of NIST SRM 1950 reference human plasma were analyzed TR:TREATMENT_SUMMARY by LC-MS/MS using DIA. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from NIST SRM 1950 reference human plasma as SP:SAMPLEPREP_SUMMARY previously described, with minor modifications (Assress et al 2023). An 800 μL SP:SAMPLEPREP_SUMMARY volume of cold methanol was added to 200 μL of plasma in a 1.5 mL Eppendorf SP:SAMPLEPREP_SUMMARY tube. The mixture was vortexed for 10 s and incubated for 15 min at 4 °C on a SP:SAMPLEPREP_SUMMARY ThermoMixer (Eppendorf Inc., Enfield, CT), prior to the centrifugation (18,000g) SP:SAMPLEPREP_SUMMARY at 4 °C for 10 min (Centrifuge 5430R, Eppendorf Inc., Enfield, CT). SP:SAMPLEPREP_SUMMARY Subsequently, 750 μL of the supernatant was collected and dried using a vacuum SP:SAMPLEPREP_SUMMARY concentrator (concentrator RapidVap Vacuum, Labconco, USA). The dried extracts SP:SAMPLEPREP_SUMMARY were reconstituted in 100 μL of a water/methanol solution (95:5) containing SP:SAMPLEPREP_SUMMARY 0.1% formic acid and transferred to a high-performance liquid chromatography SP:SAMPLEPREP_SUMMARY (HPLC) glass vial for analysis. Assress, H. A.; Ferruzzi, M. G.; Lan, R. S. SP:SAMPLEPREP_SUMMARY Optimization of Mass Spectrometric Parameters in Data Dependent Acquisition for SP:SAMPLEPREP_SUMMARY Untargeted Metabolomics on the Basis of Putative Assignments. J. Am. Soc. Mass SP:SAMPLEPREP_SUMMARY Spectrom. 2023, 34 (8), 1621–1631. https://doi.org/10.1021/jasms.3c00084. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Mobile phase A was water acidified with 0.1% of formic acid. Mobile phase B was CH:CHROMATOGRAPHY_SUMMARY 80% acetonitrile-water acidified with 0.1% of formic acid. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME ThermoFisher Scientific EASY-Spray C18 RP capillary (15cm x 75um, 3um) CH:SOLVENT_A 100% water; 0.1% of formic acid CH:SOLVENT_B 80% acetonitrile/20% water; 0.1% of formic acid CH:FLOW_GRADIENT 5-10 % B over 1 min, 10-25 min % B over 2 min, 25-60 % B over 2 min, 60-95 % B CH:FLOW_GRADIENT over 6 min where it was held for 2 min. Total time: 13 min. CH:FLOW_RATE 1000 nL/min CH:COLUMN_TEMPERATURE 25 CH:RETENTION_TIME 13 min CH:SAMPLE_INJECTION 2 uL #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Exploris 480 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS MS spectra were acquired in the range of m/z 50−750. The RF lens was 50%. The MS:MS_COMMENTS ion transfer tube temperature was 275 °C and a spray voltage was set to 1.9 kV MS:MS_COMMENTS in positive polarity. DIA were used for the fragmentation process. MS:MS_RESULTS_FILE ST003994_AN006583_Results.txt UNITS:Intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END