Summary of Study ST002420

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 PR001558. The data can be accessed directly via it's Project DOI: 10.21228/M89M65 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.

Study ID	ST002420
Study Title	Colorectal cancer isobaric labeling for metabolite quantification
Study Summary	A major challenge in reducing the death rate of colorectal cancer is to screen patients using low-invasive testing. Blood test shows a high compliance rate with reduced invasiveness. In this work, a multiplex isobaric tag labeling strategy coupled with mass spectrometry is adopted to relatively quantify primary and secondary amine-containing metabolites in serum for the discovery of metabolite biomarkers of colorectal cancer. Serum samples from patients at different risk statuses and colorectal cancer growth statuses are studied. Metabolite identification is based on accurate mass matching and/or retention time of labeled metabolite standards. We quantify 40 metabolites across all the serum samples, including 18 metabolites validated with standards. We find significantly decreased levels of threonine and asparagine in the patients with growing adenomas or high-risk adenomas (p < 0.05). Glutamine levels decrease in patients with adenomas of unknown growth status or high-risk adenomas. In contrast, arginine levels are elevated in patients with low-risk adenoma. Receiver operating characteristic analysis shows high sensitivity and specificity of these metabolites for detecting growing adenomas. Based on these results, we conclude that potential metabolite biomarkers identified here contribute to distinguishing colorectal patients with growing adenomas from normal individuals and patients with unknown growth status of adenomas.
Institute	University of Wisconsin-Madison
Last Name	Liu
First Name	Yuan
Address	777 Highland ave, Madison, Wisconsin, 53705, USA
Email	liu788@wisc.edu
Phone	6089606939
Submit Date	2022-12-28
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2023-03-31
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001558
Project DOI:	doi: 10.21228/M89M65
Project Title:	Isobaric labeling metabolites CRC studies
Project Type:	MS relative quantitative analysis
Project Summary:	Isobaric labeling-based relative quantification for the discovery of serum metabolite biomarkers associated with growing early colorectal adenomas
Institute:	University of Wisconsin - Madison
Last Name:	Liu
First Name:	Yuan
Address:	777 Highland ave, Madison, Wisconsin, 53705, USA
Email:	liu788@wisc.edu
Phone:	6089606939

Subject:

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

Factors:

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

mb_sample_id	local_sample_id	classification_based on growth rate	classification_based on risks
SA242323	N14	control	control
SA242324	N13	control	control
SA242325	N11	control	control
SA242326	N15	control	control
SA242327	N12	control	control
SA242328	N17	control	control
SA242329	N10	control	control
SA242330	N19	control	control
SA242331	N18	control	control
SA242332	N20	control	control
SA242333	N16	control	control
SA242334	N5	control	control
SA242335	N9	control	control
SA242336	N2	control	control
SA242337	N1	control	control
SA242338	N4	control	control
SA242339	N3	control	control
SA242340	N6	control	control
SA242341	N8	control	control
SA242342	N7	control	control
SA242281	23_pre	Growing	High‐risk
SA242282	23_post	Growing	High‐risk
SA242283	19_post	Growing	High‐risk
SA242284	19_pre	Growing	High‐risk
SA242285	15_post	Growing	High‐risk
SA242286	24_pre	Growing	High‐risk
SA242287	25_post	Growing	High‐risk
SA242288	33_pre	Growing	High‐risk
SA242289	33_post	Growing	High‐risk
SA242290	28_post	Growing	High‐risk
SA242291	28_pre	Growing	High‐risk
SA242292	15_pre	Growing	High‐risk
SA242293	25_pre	Growing	High‐risk
SA242294	24_post	Growing	High‐risk
SA242295	5_pre	Growing	High‐risk
SA242296	8_post	Growing	High‐risk
SA242297	5_post	Growing	High‐risk
SA242298	8_pre	Growing	High‐risk
SA242299	7_post	Growing	High‐risk
SA242300	7_pre	Growing	High‐risk
SA242343	2_post	growing	Low‐Risk
SA242344	2_pre	growing	Low‐Risk
SA242301	9_pre	Growing	Low‐Risk
SA242302	18_post	Growing	Low‐Risk
SA242303	32_post	Growing	Low‐Risk
SA242304	32_pre	Growing	Low‐Risk
SA242305	9_post	Growing	Low‐Risk
SA242306	18_pre	Growing	Low‐Risk
SA242345	20_post	static	High‐risk
SA242346	20_pre	static	High‐risk
SA242307	1_post	Static	Low‐Risk
SA242308	3_post	Static	Low‐Risk
SA242309	3_pre	Static	Low‐Risk
SA242310	1_pre	Static	Low‐Risk
SA242311	10_post	Unknown	High‐risk
SA242312	29_post	Unknown	High‐risk
SA242313	29_pre	Unknown	High‐risk
SA242314	12_post	Unknown	High‐risk
SA242315	12_pre	Unknown	High‐risk
SA242316	21_post	Unknown	High‐risk
SA242317	21_pre	Unknown	High‐risk
SA242318	10_pre	Unknown	High‐risk
SA242319	11_post	Unknown	Low‐Risk
SA242320	26_pre	Unknown	Low‐Risk
SA242321	26_post	Unknown	Low‐Risk
SA242322	11_pre	Unknown	Low‐Risk

Showing results 1 to 66 of 66

Showing results 1 to 66 of 66

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