Summary of Study ST003302

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 PR002052. The data can be accessed directly via it's Project DOI: 10.21228/M8CG1H 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 IDST003302
Study TitleUntargeted Dialysate Metabolomics Identification and Detection of Novel Neurochemicals via Benzoyl Chloride Derivatization
Study SummaryThis study combines these strategies to obtain in-depth untargeted chemical identification of dialysate. We target the rat dorsal and ventral striatum, given its importance in motivation, movement, and reward processing and identify 489 compounds. Despite the depth of analysis, many well-known neurochemicals, such as all neurotransmitters, were not identified in the untargeted method. These compounds could be detected using BzCl derivation in a targeted method though. By tracking isotopic BzCl feature pair detection and filtering of MS/MS spectra by known benzoyl fragment ions, we found 872 unique features in dialysate suggesting many unknown compounds remain to be identified in dialysate.
Institute
University of Michigan
Last NameAnderson
First NameBrady
Address930 N. University Ann Arbor, MI, 48109, USA
Emailanderbra@umich.edu
Phone734-615-4376
Submit Date2023-05-02
Num Groups1
Total Subjects6
Num Males6
Study CommentsSprague Dawley Rats
Publicationsto be updated later
Raw Data AvailableYes
Raw Data File Type(s)mzML, raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-09-24
Release Version1
Brady Anderson Brady Anderson
https://dx.doi.org/10.21228/M8CG1H
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002052
Project DOI:doi: 10.21228/M8CG1H
Project Title:Untargeted Dialysate Metabolomics Identification and Detection of Novel Neurochemicals via Benzoyl Chloride Derivatization
Project Summary:This study combines these strategies to obtain in-depth untargeted chemical identification of dialysate. We target the rat dorsal and ventral striatum, given its importance in motivation, movement, and reward processing and identify 489 compounds. Despite the depth of analysis, many well-known neurochemicals, such as all neurotransmitters, were not identified in the untargeted method. These compounds could be detected using BzCl derivation in a targeted method though. By tracking isotopic BzCl feature pair detection and filtering of MS/MS spectra by known benzoyl fragment ions, we found 872 unique features in dialysate suggesting many unknown compounds remain to be identified in dialysate.
Institute:University of Michigan
Last Name:Anderson
First Name:Brady
Address:930 N. University Ann Arbor, MI 48109
Email:anderbra@umich.edu
Phone:6519256798
Funding Source:NIH (NINDD,NIEHS)
Publications:to be updated later
Contributors:Brady Anderson, Pavlo Popov, Amanda Cicali, Adana Nwamba, Charles R. Evans, Robert T. Kennedy

Subject:

Subject ID:SU003423
Subject Type:Mammal
Subject Species:Rattus norvegicus
Taxonomy ID:10116
Age Or Age Range:75 days (average)
Weight Or Weight Range:340-375 g
Gender:Male
Animal Animal Supplier:Charles River Laboratory
Animal Housing:Group housed (3 per cage)
Animal Light Cycle:Reversed light cycle (12 h on, 12 h off)
Animal Feed:Lab diet 5LOD rat chow
Species Group:Sprague Dawley

Factors:

Subject type: Mammal; Subject species: Rattus norvegicus (Factor headings shown in green)

mb_sample_id local_sample_id Sample type Dialysate concentration
SA358544RPLC_aCSF_NegaCSF N/A
SA358545RPLC_aCSF_PosaCSF N/A
SA358528RPLC_Blank_Neg_01Blank N/A
SA358529HILIC_Blank_Neg_02Blank N/A
SA358530HILIC_Blank_Neg_01Blank N/A
SA358531HILIC_Blank_Pos_02Blank N/A
SA358532HILIC_Blank_Pos_01Blank N/A
SA358533RPLC_Blank_Neg_02Blank N/A
SA358534RPLC_Blank_Pos_02Blank N/A
SA358535RPLC_Blank_Pos_01Blank N/A
SA358536RPLC_10x_Dialysate_NegDialysate 10x
SA358537RPLC_10x_Dialysate_PosDialysate 10x
SA358538HILIC_10x_Dialysate_PosDialysate 10x
SA358539HILIC_10x_Dialysate_NegDialysate 10x
SA358540HILIC_1x_Dialysate_PosDialysate 1x
SA358541RPLC_1x_Dialysate_NegDialysate 1x
SA358542HILIC_1x_Dialysate_NegDialysate 1x
SA358543RPLC_1x_Dialysate_PosDialysate 1x
Showing results 1 to 18 of 18

Collection:

Collection ID:CO003416
Collection Summary:For this study, animal treatment was approved by the University Committee on Use and Care of Animals (UCUCA) at the University of Michigan, the National Institute of Health (NIH) Guidelines for the Care and Use of Laboratory Animals. For dialysate sample collection, we used six male Sprague-Dawley rats (Charles River Laboratories; Wilmington, MA), approximately 75 days old and weighing 340 to 375 g. Rats were group-housed before and after stereotaxic surgery in a reverse light cycle vivarium (12 h on/12 h off; lights off 6 AM) with ad libitum access to food and water. Before dialysate collection, a single microdialysis cannula was implanted using stereotaxic surgery targeting the striatum with coordinates from bregma: +1.8 AP, ±1.8 ML, -4.0 DV. Animals recovered for 36 to 48 h before microdialysis probe placement. On the day of sample collection (at the beginning of the rat dark cycle), a microdialysis probe was inserted under isoflurane anesthesia. CMA 12 Elite microdialysis probes with a 4 mm long membrane (0.5 mm O.D.) and 20,000-dalton molecular weight cutoff were used (Harvard Apparatus; Holliston, MA). After insertion, the microdialysis probe membrane spanned the rostral areas of the dorsomedial striatum, the nucleus accumbens core, and the lateral nucleus accumbens shell.Before sample collection, aCSF solution was perfused at a flow rate of 2 uL/min for 45 min, followed by 30 min at 1 uL/min. After probe conditioning, all samples were collected at a 1 uL/min perfusion rate and 30-min fractions for 12 continuous hours. The collected dialysate fractions were kept in a -20 ºC freezer during the 12-h sampling period and stored in a -80 ºC freezer after.
Sample Type:Brain
Collection Method:Microdialysate
Collection Location:Dorsal and ventral striatum
Collection Frequency:Continuous
Collection Duration:12 h
Volumeoramount Collected:4 mL
Storage Conditions:-80℃
Collection Tube Temp:0 C

Treatment:

Treatment ID:TR003432
Treatment Summary:No treatment was administered. Rats were freely moving with access to food and water.

Sample Preparation:

Sampleprep ID:SP003430
Sampleprep Summary:For underivatized experiments, aliquots of pooled dialysate were transferred to tapered glass HPLC vials (Thermo Fisher Scientific; Waltham, MA) and dried in an EZ-2 vacuum centrifuge (GeneVac; Ipswich, United Kingdom) for three hours. Samples were then preconcentrated 10-fold by volume in 9:1 water:methanol or 85:15 acetonitrile:water for RPLC and HILIC analyses.
Processing Storage Conditions:On ice
Extract Storage:-80℃

Combined analysis:

Analysis ID AN005409 AN005410 AN005411 AN005412
Analysis type MS MS MS MS
Chromatography type Reversed phase Reversed phase HILIC HILIC
Chromatography system Thermo Vanquish Thermo Vanquish Thermo Vanquish Thermo Vanquish
Column Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um)
MS Type ESI ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Orbitrap ID-X Tribrid Thermo Orbitrap ID-X Tribrid Thermo Orbitrap ID-X Tribrid Thermo Orbitrap ID-X Tribrid
Ion Mode POSITIVE NEGATIVE POSITIVE NEGATIVE
Units Peak area Peak area Peak area Peak area

Chromatography:

Chromatography ID:CH004102
Chromatography Summary:Reversed phase liquid chromatography (RPLC)
Instrument Name:Thermo Vanquish
Column Name:Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um)
Column Temperature:55 C
Flow Gradient:0 min, 0%B; 0-10, 0-99%B; 10-17, 99%B; 17-17.1, 99-0%B; 17.1-20, 0%B
Flow Rate:0.450 mL/min
Sample Injection:5 uL
Solvent A:100% water; 0.1% formic acid
Solvent B:100% Methanol; 0.025% formic acid
Washing Buffer:9:1 water:methanol
Target Sample Temperature:4 C
Chromatography Type:Reversed phase
  
Chromatography ID:CH004103
Chromatography Summary:Hydrophilic interaction liquid chromatography (HILIC)
Instrument Name:Thermo Vanquish
Column Name:Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um)
Column Temperature:55 C
Flow Gradient:0-0.5 min, 100%B; 0.5-7, 100-85%B; 7-9, 85%B; 9-16, 85-50%B; 16-16.1, 50-100%B; 16.1-20, 100%B
Flow Rate:0.300 mL/min
Sample Injection:2 uL
Solvent A:95% water/5% acetonitrile; 0.125% formic acid; 10 mM ammonium formate
Solvent B:95% acetonitrile/5% water; 0.125% formic acid; 10 mM ammonium formate
Washing Buffer:85:15 acetonitrile:water
Target Sample Temperature:4 C
Chromatography Type:HILIC

MS:

MS ID:MS005136
Analysis ID:AN005409
Instrument Name:Thermo Orbitrap ID-X Tribrid
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Mass spectrometer settings for all full scan (MS1) methods were set as follows: sheath gas, 40; aux gas, 10; sweep gas, 1; ion transfer tube temp, 325 ºC; vaporizer temp, 300 ºC; orbitrap resolution. 120000; scan range, 70-800 m/z; RF lens, 45%; normalized AGC target, 25%; maximum injection time, auto; microscans, 1; data type, profile; internal mass calibration, EASY-ICTM. Spray voltages were set to 3200 V and -3200 V for positive and negative ionization modes. For MS/MS methods, the instrument settings above were maintained except for full scan orbitrap resolution, which was lowered to maximize MS/MS spectra collection. The data-dependent acquisition methods utilized the following settings: full scan orbitrap resolution, 60000; intensity threshold, 1.0x104; dynamic exclusion properties; exclusion duration 3 seconds (exclude after one time with +/- 5 ppm); isolation mode, quadrupole; isolation window, 1.2 m/z; activation type, HCD; collision energy mode, assisted; collision energies, 20, 40, and 80%; detector type, orbitrap; orbitrap resolution, 30000; normalized AGC target, 20%; maximum injection time, 54 ms; microscans, 1; data type, centroid; cycle time, 1.2 s. Five iterative injections (i.e., rolling precursor ion exclusion) were performed for underivatized samples to better collect MS/MS spectra of lower abundance metabolites.
Ion Mode:POSITIVE
Capillary Temperature:325 C
Dry Gas Flow:Sheath 40, Auxiliary 10, Sweep 1
Ion Source Temperature:300 C
Ion Spray Voltage:+3200
Ionization Energy:positive
Mass Accuracy:120,000 resolution
Automatic Gain Control:45%
  
MS ID:MS005137
Analysis ID:AN005410
Instrument Name:Thermo Orbitrap ID-X Tribrid
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Mass spectrometer settings for all full scan (MS1) methods were set as follows: sheath gas, 40; aux gas, 10; sweep gas, 1; ion transfer tube temp, 325 ºC; vaporizer temp, 300 ºC; orbitrap resolution. 120000; scan range, 70-800 m/z; RF lens, 45%; normalized AGC target, 25%; maximum injection time, auto; microscans, 1; data type, profile; internal mass calibration, EASY-ICTM. Spray voltages were set to 3200 V and -3200 V for positive and negative ionization modes. For MS/MS methods, the instrument settings above were maintained except for full scan orbitrap resolution, which was lowered to maximize MS/MS spectra collection. The data-dependent acquisition methods utilized the following settings: full scan orbitrap resolution, 60000; intensity threshold, 1.0x104; dynamic exclusion properties; exclusion duration 3 seconds (exclude after one time with +/- 5 ppm); isolation mode, quadrupole; isolation window, 1.2 m/z; activation type, HCD; collision energy mode, assisted; collision energies, 20, 40, and 80%; detector type, orbitrap; orbitrap resolution, 30000; normalized AGC target, 20%; maximum injection time, 54 ms; microscans, 1; data type, centroid; cycle time, 1.2 s. Five iterative injections (i.e., rolling precursor ion exclusion) were performed for underivatized samples to better collect MS/MS spectra of lower abundance metabolites.
Ion Mode:NEGATIVE
Capillary Temperature:325 C
Dry Gas Flow:Sheath 40, Auxiliary 10, Sweep 1
Ion Source Temperature:300 C
Ion Spray Voltage:-3200
Ionization Energy:negative
Mass Accuracy:120,000 resolution
Automatic Gain Control:45%
  
MS ID:MS005138
Analysis ID:AN005411
Instrument Name:Thermo Orbitrap ID-X Tribrid
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Mass spectrometer settings for all full scan (MS1) methods were set as follows: sheath gas, 40; aux gas, 10; sweep gas, 1; ion transfer tube temp, 325 ºC; vaporizer temp, 300 ºC; orbitrap resolution. 120000; scan range, 70-800 m/z; RF lens, 45%; normalized AGC target, 25%; maximum injection time, auto; microscans, 1; data type, profile; internal mass calibration, EASY-ICTM. Spray voltages were set to 3200 V and -3200 V for positive and negative ionization modes. For MS/MS methods, the instrument settings above were maintained except for full scan orbitrap resolution, which was lowered to maximize MS/MS spectra collection. The data-dependent acquisition methods utilized the following settings: full scan orbitrap resolution, 60000; intensity threshold, 1.0x104; dynamic exclusion properties; exclusion duration 3 seconds (exclude after one time with +/- 5 ppm); isolation mode, quadrupole; isolation window, 1.2 m/z; activation type, HCD; collision energy mode, assisted; collision energies, 20, 40, and 80%; detector type, orbitrap; orbitrap resolution, 30000; normalized AGC target, 20%; maximum injection time, 54 ms; microscans, 1; data type, centroid; cycle time, 1.2 s. Five iterative injections (i.e., rolling precursor ion exclusion) were performed for underivatized samples to better collect MS/MS spectra of lower abundance metabolites.
Ion Mode:POSITIVE
Capillary Temperature:325 C
Dry Gas Flow:Sheath 40, Auxiliary 10, Sweep 1
Ion Source Temperature:300 C
Ion Spray Voltage:+3200
Ionization Energy:positive
Mass Accuracy:120,000 resolution
Automatic Gain Control:45%
  
MS ID:MS005139
Analysis ID:AN005412
Instrument Name:Thermo Orbitrap ID-X Tribrid
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Mass spectrometer settings for all full scan (MS1) methods were set as follows: sheath gas, 40; aux gas, 10; sweep gas, 1; ion transfer tube temp, 325 ºC; vaporizer temp, 300 ºC; orbitrap resolution. 120000; scan range, 70-800 m/z; RF lens, 45%; normalized AGC target, 25%; maximum injection time, auto; microscans, 1; data type, profile; internal mass calibration, EASY-ICTM. Spray voltages were set to 3200 V and -3200 V for positive and negative ionization modes. For MS/MS methods, the instrument settings above were maintained except for full scan orbitrap resolution, which was lowered to maximize MS/MS spectra collection. The data-dependent acquisition methods utilized the following settings: full scan orbitrap resolution, 60000; intensity threshold, 1.0x104; dynamic exclusion properties; exclusion duration 3 seconds (exclude after one time with +/- 5 ppm); isolation mode, quadrupole; isolation window, 1.2 m/z; activation type, HCD; collision energy mode, assisted; collision energies, 20, 40, and 80%; detector type, orbitrap; orbitrap resolution, 30000; normalized AGC target, 20%; maximum injection time, 54 ms; microscans, 1; data type, centroid; cycle time, 1.2 s. Five iterative injections (i.e., rolling precursor ion exclusion) were performed for underivatized samples to better collect MS/MS spectra of lower abundance metabolites.
Ion Mode:NEGATIVE
Capillary Temperature:325 C
Dry Gas Flow:Sheath 40, Auxiliary 10, Sweep 1
Ion Source Temperature:300 C
Ion Spray Voltage:-3200
Ionization Energy:negative
Mass Accuracy:120,000 resolution
Automatic Gain Control:45%
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