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MTBLS6:  Development and validation of the PyMS Mass Spectrometry software

 Authors: David De Souza , Sean O'Callaghan

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  Submitted: 29-Mar-2012 , Release date: 29-Mar-2012 , Update date: 03-Dec-2015

 Submitted by:  Sean O'Callaghan  |   Study status: Public

Study Description

To provide useful data for development and validation of the PyMS Mass Spectrometry software, a test dataset was run at Metabolomics Australia. A biologically complex mix of a biological background material (foetal calf serum), spiked with 2-fold increasing amounts of a mix of metabolite standards. In addition, a single sample consisting of a simple mix of 45 metabolites representing a variety of chemical classes (sufars, organic acids, amino acids, sugar phosphates), was run through a standard Metabolomics Australia GC-MS analysis. The resulting data is a valuable tool in testing GC-MS data analysis software.

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  Organism(s)

Bos taurus

reference compound

  Study Design Description

PyMS

CHMO:gas chromatography-mass spectrometry

MS:data processing software

NCIT:Validation

 Publications
[1]  Development and validation of the PyMS Mass Spectrometry software
  Experimental Factors

Sample type

Spike in volume

Gap Penalty

Retention Time Modulation

Threshold

Min Number Ions

Relative Intensity

Skewing

Window

Protocol Description
Sample collection Two types of samples were prepared. A mixture of 45 metabolites, representing a variety of chemical classes (sugars, organic acids, amino acids, sugar phosphates), was prepared for GC-MS analysis. 10 µl of the mixture was transferred to a GC-MS vial insert and evaporated to dryness in vacuo. The samples were resuspended in 20 mg/ml methoxyamine in pyridine (20 µl, 16 hr, 25 °C) with continuous shaking, followed by derivatization with BSTFA + 1% TMCS (Pierce; 20 µl, 1 hr, 25 °C) using a Gerstel MPS2 autosampler robot. Second group a Foetal calf serum was spiked with 2-fold increasing amounts of a mix of metabolite standards.
Extraction The Foetal calf serum samples (120 µl) were extracted by the addition of 200 µl of 3:2 chloroform:methanol, followed by vigorous mixing and incubation on ice for 10 min. Samples were centrifuged at 4 °C for 10 mins at 16,000 rpm to enable biphasic sparation. 15 µl of the upper aqueous phase was transferred to GC-MS vial inserts and dried in vacuo. The samples were resuspended in 20 mg/ml methoxyamine in pyridine (20 µl, 16 hr, 25 °C) with continuous shaking, followed by derivatization with BSTFA + 1% TMCS (Pierce; 20 µl, 1 hr, 25 °C) using a Gerstel MPS2 autosampler robot.
Chromatography Gas chromatography was performed using a 30 m VF5-MS column with 0.25 mm inner diameter and 0.25 mm film thickness (Varian Inc.). Agilent 7890A gas chromatograph interfaced with a 5975C mass selective detector.
Mass spectrometry The injection temperature was 250 °C, the interface set at 280 °C, and the ionsource adjusted to 250 °C. The carrier gas was helium (flow rate 1 ml/min). The temperature program was 1 min isothermal heating at 70 °C, followed by a 1 °C/min oven temperature ramp to 76 °C, then 5 °C/min to 325 °C and held for 10 min. Mass spectra were recorded at 2.66 scans/s (m/z 50–600).
Data transformation Data Analysis was performed using the PyMS software package (code.google.com/p/pyms). There are two steps, deconvolution (peak picking), and alignment. Each step produces output – for each sample a peak list was created. Deconvolution was carried out using PyMS with the following parameters: Threshold: 6000, Window: 13 scan, Num Ions per Peak: 3, Relative Intensity: 2 and Spectral Skewing parameter: 2 for the Foetal Calf Serum and Threshold: 3000, and Window: 5 scans difference for the Metabolite mix sample. Alignment was carried out using PyMS, with gap penalty 0.3 and retention time modulation parameter 2.5.
Metabolite identification No metabolites were identified.
Source Name Organism Organism part Protocol REF Sample Name Sample type Spike in volume Unit Gap Penalty Retention Time Modulation Threshold Window Min Number Ions Relative Intensity Skewing
Metabolomics Australia.Group-1.Subject-1.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA1_1 Spiked 50 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-1.Subject-2.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA1_2 Spiked 50 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-1.Subject-3.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA1_3 Spiked 50 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-2.Subject-1.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA2_1 Spiked 25 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-2.Subject-2.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA2_2 Spiked 25 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-2.Subject-3.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA2_3 Spiked 25 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-3.Subject-1.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA3_1 Spiked 12 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-3.Subject-2.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA3_2 Spiked 12 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-3.Subject-3.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA3_3 Spiked 12 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-4.Subject-1.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA4_1 Spiked 6 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-4.Subject-2.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA4_2 Spiked 6 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-4.Subject-3.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA4_3 Spiked 6 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-5.Subject-1.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA5_1 Spiked 3 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-5.Subject-2.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA5_2 Spiked 3 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-5.Subject-3.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA5_3 Spiked 3 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-6.Subject-1.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA6_1 Spiked 0 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-6.Subject-2.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA6_2 Spiked 0 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-6.Subject-3.bovine serum albumin-grown cell Bos taurus fetal serum Sample collection APC_BSTFA6_3 Spiked 0 UO:microliter 0.3 2.5 6000 13 3 2 2
Metabolomics Australia.Group-1.Subject-1.Metabolite Mix reference compound mixture Sample collection GC01_0812_066 mixture 0 UO:microliter 0.3 2.5 2000 5 3 2 2
Validations marked with (*) have been allowed by the MetaboLights Curators.
Click here for the detailed description of Validations.
Condition Status Description Requirement Group Message
PASSES Study Title MANDATORY STUDY OK
PASSES Study Description MANDATORY STUDY OK
PASSES Study text successfully parsed OPTIONAL STUDY OK
PASSES Study Contact(s) have listed email MANDATORY CONTACT OK
PASSES Sample(s) MANDATORY SAMPLES OK
PASSES Sample Name consistency check MANDATORY ASSAYS OK
* PASSES Publication Author list is missing MANDATORY PUBLICATION Please provide Author List
* PASSES Publication PubMed ID and Publication DOI are missing OPTIONAL PUBLICATION Please provide Pubmed ID and/or DOI
PASSES Minimal Experimental protocol MANDATORY PROTOCOLS OK
PASSES Comprehensive Experimental protocol OPTIONAL PROTOCOLS OK
PASSES Sample Collection protocol MANDATORY PROTOCOLS OK
PASSES Protocols text successfully parsed OPTIONAL PROTOCOLS OK
PASSES Organism name MANDATORY ORGANISM OK
PASSES Organism part MANDATORY ORGANISM OK
PASSES Study Factors MANDATORY FACTORS OK
PASSES Assay platform information OPTIONAL ASSAYS OK
PASSES Assay has raw files referenced MANDATORY FILES OK
PASSES Assay referenced raw files detection in filesystem MANDATORY FILES OK
PASSES Raw files in the Assay(s) have the correct format MANDATORY FILES OK
PASSES Assay(s) MANDATORY ASSAYS OK
* PASSES At least one assay has Metabolite Assignment Files (MAFs) referenced MANDATORY FILES Metabolite identification protocol is described but no Metabolite Assignment File (MAF) is referenced in the Assay table
PASSES ISA-Tab investigation file check MANDATORY ISATAB OK

Assay 

Assay file name: a_live10_mtbl_metabolite profiling_mass spectrometry.txt
Measurement: metabolite profiling
Technology: mass spectrometry
Platform: Agilent 5975E GC/MSD (Agilent)

Data

Sample Name Protocol REF Post Extraction Derivatization Extract Name Protocol REF Chromatography Instrument Column model Column type Labeled Extract Name Label Protocol REF Scan polarity Scan m/z range Instrument Ion source Mass analyzer MS Assay Name Raw Spectral Data File Protocol REF Normalization Name Derived Spectral Data File Protocol REF Data Transformation Name Metabolite Assignment File
APC_BSTFA1_1 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA1_1.CDF Data transformation APC_BSTFA1_1_peaks.csv Metabolite identification
APC_BSTFA1_2 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA1_2.CDF Data transformation APC_BSTFA1_2_peaks.csv Metabolite identification
APC_BSTFA1_3 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA1_3.CDF Data transformation APC_BSTFA1_3_peaks.csv Metabolite identification
APC_BSTFA2_1 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA2_1.CDF Data transformation APC_BSTFA2_1_peaks.csv Metabolite identification
APC_BSTFA2_2 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA2_2.CDF Data transformation APC_BSTFA2_2_peaks.csv Metabolite identification
APC_BSTFA2_3 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA2_3.CDF Data transformation APC_BSTFA2_3_peaks.csv Metabolite identification
APC_BSTFA3_1 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA3_1.CDF Data transformation APC_BSTFA3_1_peaks.csv Metabolite identification
APC_BSTFA3_2 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA3_2.CDF Data transformation APC_BSTFA3_2_peaks.csv Metabolite identification
APC_BSTFA3_3 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA3_3.CDF Data transformation APC_BSTFA3_3_peaks.csv Metabolite identification
APC_BSTFA4_1 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA4_1.CDF Data transformation APC_BSTFA4_1_peaks.csv Metabolite identification
APC_BSTFA4_2 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA4_2.CDF Data transformation APC_BSTFA4_2_peaks.csv Metabolite identification
APC_BSTFA4_3 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA4_3.CDF Data transformation APC_BSTFA4_3_peaks.csv Metabolite identification
APC_BSTFA5_1 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA5_1.CDF Data transformation APC_BSTFA5_1_peaks.csv Metabolite identification
APC_BSTFA5_2 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA5_2.CDF Data transformation APC_BSTFA5_2_peaks.csv Metabolite identification
APC_BSTFA5_3 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA5_3.CDF Data transformation APC_BSTFA5_3_peaks.csv Metabolite identification
APC_BSTFA6_1 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA6_1.CDF Data transformation APC_BSTFA6_1_peaks.csv Metabolite identification
APC_BSTFA6_2 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA6_2.CDF Data transformation APC_BSTFA6_2_peaks.csv Metabolite identification
APC_BSTFA6_3 Extraction 20 mg/ml methoxyamine in pyridine sylilation spiked-foetal-calf-serum-sample1rep1-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole APC_BSTFA6_3.CDF Data transformation APC_BSTFA6_3_peaks.csv Metabolite identification
GC01_0812_066 Extraction None sylilation Metabolomics Australia.Group-1.Subject-1.Metabolite Mix-extract Chromatography Agilent 7890A GC VF-5ms GC (0.25 µm, 0.25 mm x 30 m; Varian) low polarity Mass spectrometry positive 50-600 Agilent 5975C MSD electron ionization MS:quadrupole gc01_0812_066.cdf Data transformation GC01_0812_066_peaks.csv Metabolite identification

Pathways - Assay 



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)
  Download whole study (SLOW)  |  Download study (FTP)  |    Download metadata    

Aspera Download Details:

List of study files   Subset

File
audit
metexplore_mapping.json
APC_BSTFA1_1.CDF
APC_BSTFA1_1_peaks.csv
APC_BSTFA1_2.CDF
APC_BSTFA1_2_peaks.csv
APC_BSTFA1_3.CDF
APC_BSTFA1_3_peaks.csv
APC_BSTFA2_1.CDF
APC_BSTFA2_1_peaks.csv
APC_BSTFA2_2.CDF
APC_BSTFA2_2_peaks.csv
APC_BSTFA2_3.CDF
APC_BSTFA2_3_peaks.csv
APC_BSTFA3_1.CDF
APC_BSTFA3_1_peaks.csv
APC_BSTFA3_2.CDF
APC_BSTFA3_2_peaks.csv
APC_BSTFA3_3.CDF
APC_BSTFA3_3_peaks.csv
APC_BSTFA4_1.CDF
APC_BSTFA4_1_peaks.csv
APC_BSTFA4_2.CDF
APC_BSTFA4_2_peaks.csv
APC_BSTFA4_3.CDF
APC_BSTFA4_3_peaks.csv
APC_BSTFA5_1.CDF
APC_BSTFA5_1_peaks.csv
APC_BSTFA5_2.CDF
APC_BSTFA5_2_peaks.csv
APC_BSTFA5_3.CDF
APC_BSTFA5_3_peaks.csv
APC_BSTFA6_1.CDF
APC_BSTFA6_1_peaks.csv
APC_BSTFA6_2.CDF
APC_BSTFA6_2_peaks.csv
APC_BSTFA6_3.CDF
APC_BSTFA6_3_peaks.csv
GC01_0812_066_peaks.csv
a_live10_mtbl_metabolite profiling_mass spectrometry.txt
aligned_areas.csv
aligned_rts.csv
gc01_0812_066.cdf
i_Investigation.txt
s_live10_mtbl.txt

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