Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA242297

Local Sample ID:	5_post
Subject ID:	SU002509
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

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Subject:

Subject ID:	SU002509
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
5_post	SA242297	FL030347	Growing	classification_based on growth rate
5_post	SA242297	FL030347	High‐risk	classification_based on risks

Collection:

Collection ID:	CO002502
Collection Summary:	Serum samples were thawed on ice and centrifuged at 2000 g for 10 min to remove particulates and debris. Molecular weight cut-off filters (MWCO, 3 kDa, Millipore Amicon Ultra, Burlington, MA) were prerinsed 3 times with optima water at 14000 g for 20 min. Twenty microliter serum supernatant from each sample was diluted in 380 µl water and added to the filter then centrifuged for 20 min at 14000 g. Flowthrough was collected. Then 400 µL water was added to the filter to rinse the sample. An additional rinse step was applied. All the flowthrough (about 1.2 mL) was combined and dried down in Speedvac and stored at −20 °C until labeling. For normalization, a pooled serum sample from screening normal individuals was also prepared. For labeling of serum samples, 40 µL activated DiLeu reagents were mixed with serum flowthrough dissolved in 10 µL 0.5 M TEAB and shaken for 2 h. After the reaction was quenched, 2.5 µL of each reaction mixture with different DiLeu tags were combined at 1: 1: 1: 1 ratio and mixed well. To compare the relative abundance of each set of 4-plex DiLeu labeled metabolites, one channel from each 4-plex combination was selected and combined with 118-DiLeu tag labeled pooled serum samples. Ten µL of the mixture was taken for drying down and desalted using SCX Ziptips. Samples were dissolved in 15 µL 0.1% FA and 3 µL was injected into LC-MS for data acquisition with 3 technical replicates.
Collection Protocol Filename:	methods.txt
Sample Type:	Blood (serum)

Treatment:

Treatment ID:	TR002521
Treatment Summary:	Among the 23 patients carrying adenomas, 15 patients were classified histologically as high-risk and the remaining 8 patients were classified as low-risk cases. In the high-risk cases, 10 were classified as growing by longitudinal CTC analysis, one as static, and 4 as unknown growth status.

Sample Preparation:

Sampleprep ID:	SP002515
Sampleprep Summary:	Serum samples were thawed on ice and centrifuged at 2000 g for 10 min to remove particulates and debris. Molecular weight cut-off filters (MWCO, 3 kDa, Millipore Amicon Ultra, Burlington, MA) were prerinsed 3 times with optima water at 14000 g for 20 min. Twenty microliter serum supernatant from each sample was diluted in 380 µl water and added to the filter then centrifuged for 20 min at 14000 g. Flowthrough was collected. Then 400 µL water was added to the filter to rinse the sample. An additional rinse step was applied. All the flowthrough (about 1.2 mL) was combined and dried down in Speedvac and stored at −20 °C until labeling. For normalization, a pooled serum sample from screening normal individuals was also prepared. For labeling of serum samples, 40 µL activated DiLeu reagents were mixed with serum flowthrough dissolved in 10 µL 0.5 M TEAB and shaken for 2 h. After the reaction was quenched, 2.5 µL of each reaction mixture with different DiLeu tags were combined at 1: 1: 1: 1 ratio and mixed well. To compare the relative abundance of each set of 4-plex DiLeu labeled metabolites, one channel from each 4-plex combination was selected and combined with 118-DiLeu tag labeled pooled serum samples. Ten µL of the mixture was taken for drying down and desalted using SCX Ziptips. Samples were dissolved in 15 µL 0.1% FA and 3 µL was injected into LC-MS for data acquisition with 3 technical replicates.

Sampleprep ID:

SP002515

Sampleprep Summary:

Serum samples were thawed on ice and centrifuged at 2000 g for 10 min to remove particulates and debris. Molecular weight cut-off filters (MWCO, 3 kDa, Millipore Amicon Ultra, Burlington, MA) were prerinsed 3 times with optima water at 14000 g for 20 min. Twenty microliter serum supernatant from each sample was diluted in 380 µl water and added to the filter then centrifuged for 20 min at 14000 g. Flowthrough was collected. Then 400 µL water was added to the filter to rinse the sample. An additional rinse step was applied. All the flowthrough (about 1.2 mL) was combined and dried down in Speedvac and stored at −20 °C until labeling. For normalization, a pooled serum sample from screening normal individuals was also prepared. For labeling of serum samples, 40 µL activated DiLeu reagents were mixed with serum flowthrough dissolved in 10 µL 0.5 M TEAB and shaken for 2 h. After the reaction was quenched, 2.5 µL of each reaction mixture with different DiLeu tags were combined at 1: 1: 1: 1 ratio and mixed well. To compare the relative abundance of each set of 4-plex DiLeu labeled metabolites, one channel from each 4-plex combination was selected and combined with 118-DiLeu tag labeled pooled serum samples. Ten µL of the mixture was taken for drying down and desalted using SCX Ziptips. Samples were dissolved in 15 µL 0.1% FA and 3 µL was injected into LC-MS for data acquisition with 3 technical replicates.

Combined analysis:

Analysis ID	AN003941
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters NanoAcquity
Column	Waters ACQUITY UPLC BEH C18 (150 mm x 75 μm, 1.7 μm, 150 Å)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE
Units	1

Chromatography:

Chromatography ID:	CH002918
Chromatography Summary:	Four-plex pooled samples were reconstituted in 0.1% formic acid before injection. The HPLC-MS/MS analysis was conducted using a Waters nanoACQUITY UPLC coupled with Thermo Q Exactive Orbitrap MS. The separation column was in-house made with an emitter tip and dimensions of 75 μm inner diameter × 15 cm length. The column was packed with 1.7 μm, 150 Å, ethylene-bridged-hybrid (BEH) C18 material (Waters, Milford, MA). Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid. The flow rate was set as 0.3 μL/min, and the LC gradient was 55 min and set as follows: 0−10 min, 3%-30% solvent B; 10−30 min, 30−80% B; 30-30.5 min, 80%-95% B; 30.5− 40.5 min, 95% B; 40.5−41 min, 95%-3% B; 41-55 min, 3% B. Positive ionization mode was used and full MS scans were acquired from m/z 180 to 800 at a resolution of 60 k, automatic gain control (AGC) was set as 5x 10^5, and a maximum injection time as set as 30 ms. The top 20 precursors were selected for normalized collision energy (NCE) dissociation (NCE = 30) with an isolation window of m/z 1, fixed first m/z 110, dynamic exclusion of 5 seconds, charge exclusion of >2, and a resolution of 35 k.
Instrument Name:	Waters NanoAcquity
Column Name:	Waters ACQUITY UPLC BEH C18 (150 mm x 75 μm, 1.7 μm, 150 Å)
Column Temperature:	Room temperature
Flow Gradient:	LC gradient was 55 min and set as follows: 0−10 min, 3%-30% solvent B; 10−30 min, 30−80% B; 30-30.5 min, 80%-95% B; 30.5− 40.5 min, 95% B; 40.5−41 min, 95%-3% B; 41-55 min, 3% B.
Flow Rate:	0.3 μL/min
Solvent A:	100% water; 0.01% formic acid
Solvent B:	100% acetonitrile; 0.01% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003677
Analysis ID:	AN003941
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Positive ionization mode was used and full MS scans were acquired from m/z 180 to 800 at a resolution of 60 k, automatic gain control (AGC) was set as 5x 10^5, and a maximum injection time as set as 30 ms. The top 20 precursors were selected for normalized collision energy (NCE) dissociation (NCE = 30) with an isolation window of m/z 1, fixed first m/z 110, dynamic exclusion of 5 seconds, charge exclusion of >2, and a resolution of 35 k.
Ion Mode:	POSITIVE