Summary of Study ST003500

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002146. The data can be accessed directly via it's Project DOI: 10.21228/M8783Z This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST003500
Study TitleA UHPLC-MS/MS Method for Profiling of Urinary Mercapturic Acids using Positive Ion Mode
Study SummaryWe report the first application of a UHPLC-MS/MS method using positive ion mode detection for the unbiased characterization of mercapturic acids. The proposed method utilizes a neutral loss monitoring paradigm to monitor for two diagnostic fragmentation pathways for this class of compound. Using a cohort of 20 nonsmokers and 20 smokers, we detected 180 putative mercapturic acid signatures that exhibited a high degree of reproducibility from the complex urine metabolome background. Following a combination of multivariate and univariate statistics, we found 33 putative mercapturic acids associated with smoking status.
Institute
University of Minnesota
Last NameMurray
First NameKevin
Address2-210 CCRB, 2231 6th St SE, Minneapolis, MN 55455
Emailmurra668@umn.edu
Phone612-626-2182
Submit Date2024-08-01
Num Groups2
Total Subjects20
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2025-02-03
Release Version1
Kevin Murray Kevin Murray
https://dx.doi.org/10.21228/M8783Z
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:


Project:

Project ID:PR002146
Project DOI:doi: 10.21228/M8783Z
Project Title:A UHPLC-MS/MS Method for Profiling of Urinary Mercapturic Acids using Positive Ion Mode
Project Summary:We describe an analytical global profiling approach with machine learning predicted structural annotations for the characterization of mercapturic acids, a detoxification product of chemical environmental exposure.
Institute:University of Minnesota
Department:School of Public Health, Division of Environmental Health Sciences
Laboratory:Balbo Research Group
Last Name:Murray
First Name:Kevin
Address:2-210 CCRB, 2231 6th St SE, Minneapolis, MN 55455
Email:murra668@umn.edu
Phone:612-626-2182
Funding Source:National Cancer Institute (5R01CA222005-05)

Subject:

Subject ID:SU003629
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:21-61
Gender:Male and female
Human Smoking Status:Smoking and Nonsmoking
Species Group:Mammals

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Smoking Status
SA385691N149Urine Non-Smoker
SA385692N082Urine Non-Smoker
SA385693N215Urine Non-Smoker
SA385694N207Urine Non-Smoker
SA385695N194Urine Non-Smoker
SA385696N189Urine Non-Smoker
SA385697N167Urine Non-Smoker
SA385698N162Urine Non-Smoker
SA385699N161Urine Non-Smoker
SA385700N079Urine Non-Smoker
SA385701N142Urine Non-Smoker
SA385702N087Urine Non-Smoker
SA385703N140Urine Non-Smoker
SA385704N134Urine Non-Smoker
SA385705N133Urine Non-Smoker
SA385706N128Urine Non-Smoker
SA385707N096Urine Non-Smoker
SA385708N094Urine Non-Smoker
SA385709N088Urine Non-Smoker
SA385710N141Urine Non-Smoker
SA385711pool_10Urine QC
SA385712pool_09Urine QC
SA385713pool_08Urine QC
SA385714pool_07Urine QC
SA385715pool_06Urine QC
SA385716pool_05Urine QC
SA385717pool_03Urine QC
SA385718pool_02Urine QC
SA385719pool_01Urine QC
SA385720pool_04Urine QC
SA385721S147Urine Smoker
SA385722S190Urine Smoker
SA385723S171Urine Smoker
SA385724S163Urine Smoker
SA385725S157Urine Smoker
SA385726S156Urine Smoker
SA385727S152Urine Smoker
SA385728S151Urine Smoker
SA385729S150Urine Smoker
SA385730S144Urine Smoker
SA385731S146Urine Smoker
SA385732S145Urine Smoker
SA385733S138Urine Smoker
SA385734S136Urine Smoker
SA385735S135Urine Smoker
SA385736S132Urine Smoker
SA385737S131Urine Smoker
SA385738S117Urine Smoker
SA385739S102Urine Smoker
SA385740S110Urine Smoker
Showing results 1 to 50 of 50

Collection:

Collection ID:CO003622
Collection Summary:Adult participants were recruited as part of an ongoing study conducted at the University of Minnesota. Demographic information including gender, race, age, and tobacco usage was obtained through a questionnaire. Urine samples were collected throughout a 24-hour period and stored at -80 °C until the LC-MS/MS analysis. Urine samples were normalized by total 24-hour collection volume prior to sample preparation. Urinary creatinine levels measured using the RayBio Creatinine Assay kit (RayBiotech, Norcross, GA). This study was approved by the University of Minnesota Institutional Review Board.
Sample Type:Urine
Storage Conditions:-80℃

Treatment:

Treatment ID:TR003638
Treatment Summary:Urine samples were collected from non smokers and smokers. Urine samples were collected from patients over a 24-hour period. Sample were snap frozen after collection. No additional sample treatment was applied.

Sample Preparation:

Sampleprep ID:SP003636
Sampleprep Summary:Urine samples (200 μL) were acidified using 40 μL of a 30% aqueous HCl solution and vortexed gently. A mixture of 10 ng D5-PhMA, 20 ng D3-2CaEMA, 20 ng D3-2CyEMA, 10 ng D3-2HPMA, 50 ng D3-3HPMA, and 50 ng D3-3HMPMA was added to each sample. Oasis MAX mixed mode reverse phase anion exchange solid phase extraction cartridges (60 mg, 60 u, 2 mL reservoir size) were obtained from Waters Corp. (Milford, MA, USA). Before sample introduction, the plate was preconditioned with MeOH, water, and 2% aqueous NH4OH solutions. The samples were applied and washed with 0.7 mL 2% aqueous NH4OH and 0.7 mL MeOH, then dried with nitrogen gas for 20 min. The plate was washed with 0.7 mL 2% aqueous formic acid before sample collection. Finally, the unfractionated urine extracts were collected using 0.7 mL 90% MeOH in 2% formic acid wash. The samples were transferred from the 96-well collection plate to fresh 1.2 mL silanized vials and dried to completeness in a SpeedVac without heat. The dried samples were stored at -80 °C until ready for LC-MS/MS analysis.
Processing Storage Conditions:Room temperature
Extract Storage:-80℃

Combined analysis:

Analysis ID AN005745
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Dionex Ultimate 3000 RS
Column Self-packed C18 column Dr. Maisch GmbH ReproSil-PUR (450 mm x 100 µm, 1.9 µm, 120 Å C18aq)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Orbitrap Fusion Lumos Tribrid
Ion Mode POSITIVE
Units Peak heights

Chromatography:

Chromatography ID:CH004360
Methods Filename:MA_18786_hcdOT_30K_CNL-MS3-50CE_77min_20230510
Instrument Name:Thermo Dionex Ultimate 3000 RS
Column Name:Self-packed C18 column Dr. Maisch GmbH ReproSil-PUR (450 mm x 100 µm, 1.9 µm, 120 Å C18aq)
Column Temperature:55 °C
Flow Gradient:Chromatographic separation was performed using a linear gradient starting at 0% B and increased to 50% B at 40 min, 90% B at 60 min, and held for 8 min followed by a return to starting conditions.
Flow Rate:325 nL/min
Sample Injection:1% sample equivalent load in 1 μL reconstituted using a load solvent mixture of 0.1% aqueous formic acid.
Solvent A:99.9% water; 0.1% formic acid
Solvent B:99.9% acetonitrile; 0.1% formic acid
Analytical Time:68 min
Sample Loop Size:10 µL injection loop
Chromatography Type:Reversed phase

MS:

MS ID:MS005468
Analysis ID:AN005745
Instrument Name:Thermo Orbitrap Fusion Lumos Tribrid
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Discovery DDA-CNL-MS3 analyses of experimental samples were performed using full-scan detection followed by data-dependent MS2 acquisition and a conditional neutral loss-triggered MS3 acquisition. All analyses were performed in positive ion mode. Full-scan detection was performed using Orbitrap detection at a resolution of 120,000, AGC targeted setting of 4 × 105, a maximum ion injection time of 50 ms, and an S-Lens RF setting of 40%. Scan ranges of 170 m/z – 600 m/z were used for full-scan detection. MS2 spectra were collected using a DDA design with a 2 sec cycle time in centroid mode with a 15 sec dynamic exclusion list. Fragment spectra were acquired with quadrupole isolation of 0.8 m/z, Orbitrap detection at a resolution of 30,000, an AGC setting of 1 × 105, and a 100 ms maximum injection time. The analysis utilized HCD fragmentation at a fixed collision energy of 30%. MS3 spectra were collected using a conditional neutral loss design that acquired an additional fragmentation scan on ions exhibiting a neutral loss of 131.0582 Da or 105.0426 Da from the selected precursor. MS3 spectra were acquired with a full-scan MS isolation of 1.5 m/z and MS2 isolation of 2.0 m/z, Orbitrap detection at a resolution of 30,000, AGC setting of 2 × 105, and a 200 ms maximum injection time. The MS3 analysis utilized HCD fragmentation at a fixed collision energy of 50%. Data processing: Fragmentation filtering was performed using the DFBuilder module incorporated in the open-source software MZmine to monitor diagnostic neutral loss and fragment ions characteristic of mercapturic acid conjugates. Putative mercapturic acid detection required the observation of a neutral loss of 131.0582 Da (C5H9NO3) or 105.0426 Da (C3H7NO3) in acquired MS/MS spectrum. A complete list of the processing parameters is summarized in Table S2. Fragmentation filtering using the DFBuilder module was performed for the specified neutral loss using a 5 ppm mass tolerance and minimum fragment ion intensity of 1 x 104 with a 1% base peak threshold. A targeted chromatogram was drawn around detected precursor masses using a 1.0 min retention time window. Masses of interest were detected using the centroid detector and noise threshold of 1 x 105. Chromatogram traces were computed using the ADAP chromatogram builder module with a mass tolerance of 5 ppm. Chromatographic peaks were detected using the Local Minimum search algorithm using a 30% chromatographic threshold, 0.1 min retention time minimum search range, a minimum peak height of 1 x 105, a minimum peak top/edge ratio of 10, and minimum peak width tolerance of 0.1 min. Duplicate peaks were removed using a 0.1 min retention time tolerance. Feature lists were deisotoped using the 13C isotope filter module. All samples and pooled replicate feature lists were aligned using the Join Aligner module with a mass tolerance of 5 ppm and retention time tolerance of 0.4 min. Commonly observed in-source fragments, adducts, and multimer complexes were detected and removed from consideration. Missing peak annotations were replaced using the Gap-Filling algorithm. Acquired MS/MS spectra were matched against the NIST mass spectral library (version 2020) to remove unrelated metabolic signatures. Finally, the remaining features were exported as CSV files for downstream analysis.
Ion Mode:POSITIVE
Collision Energy:NCE 30%
Fragmentation Method:HCD
Ion Spray Voltage:2.1 kV
Ionization:ESI
Mass Accuracy:5 ppm
Precursor Type:[M+H]+
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