Summary of Study ST002292

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 PR001470. The data can be accessed directly via it's Project DOI: 10.21228/M8Q99X 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	ST002292
Study Title	Quantification of Dissolved Metabolites in Environmental Samples through Cation-Exchange Solid Phase Extraction (CX-SPE) paired with Liquid Chromatography-Mass Spectrometry
Study Type	Method Development for Dissolved Metabolomics in Seawater
Study Summary	Small, biologically produced, organic molecules called metabolites play key roles in microbial systems where they directly mediate exchanges of nutrients, energy, and information. However, the study of dissolved polar metabolites in seawater and other environmental matrices has been hampered by analytical challenges including high inorganic ion concentrations, low analyte concentrations, and high chemical diversity. Here we show that a cation-exchange solid phase extraction (CX-SPE) sample preparation approach separates positively charged and zwitterionic metabolites from seawater and freshwater samples, allowing their analysis by liquid chromatography-mass spectrometry (LC-MS). We successfully extracted 69 known compounds from an in-house compound collection and evaluated the performance of the method by establishing extraction efficiencies and limits of detection (pM to low nM range) for these compounds. CX-SPE extracted a range of compounds including amino acids and compatible solutes, resulted in very low matrix effects, and performed robustly across large variations in salinity and dissolved organic matter (DOM) concentration. We compared CX-SPE to an established solid phase extraction procedure (PPL-SPE) and demonstrate that these two methods extract fundamentally different fractions of the dissolved metabolite pool with CX-SPE extracting compounds that are on average smaller and more polar. We use CX-SPE to analyze four environmental samples from distinct aquatic biomes, producing some of the first CX-SPE dissolved metabolomes. Quantified compounds ranged in concentration from 0.0093 nM to 49 nM and were composed primarily of amino acids (0.15 – 16 nM) and compatible solutes such as TMAO (0.89 – 49 nM) and glycine betaine (2.8 – 5.2 nM).
Institute	University of Washington
Department	Oceanography
Laboratory	Ingalls Lab
Last Name	Sacks
First Name	Joshua
Address	Ocean Sciences Building, 1492 NE Boat St. Seattle, WA 98105
Email	jssacks@uw.edu
Phone	4074090052
Submit Date	2022-08-30
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2022-10-19
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP002384
Sampleprep Summary:	Cation-Exchange Solid Phase Extraction (CX-SPE) Sample Volume: 40 mLs Solid Phase Resin/Column: Strong cation-exchange resin (Dowex 50WX8; H+ form, 100-200 mesh, Sigma-Aldrich, Vienna, Austria) Solvents/Reagents: 1 M NH3, 3 M HNO3 Brief Procedure: 35 g of resin was added to a glass chromatography column with a fritted disk and a PTFE stopcock. The resin was equilibrated with 50 mL water, 100 mL of 1M NH3, 50 mL water, 100 mL 3M HNO3, and 50 mL H2O. The samples were acidified with HNO3 to pH 2 and heavy isotope-labeled internal standards added. The sample was loaded onto column, allowed to stand for 5 minutes, and then drained from the column. The column was then rinsed with 50 mL water. Approximately 200 mL of 1M NH3 was added to column. Ammonia eluted from column in 10 mL fractions. The pH of each fraction checked by dabbing a small drop of sample onto a pH strip with a combusted glass Pasteur pipette. The alkaline front (the 10 mL fraction where the pH increases from approximately 2-4 to 9-11), the fraction before, and the two fractions after were collected, combined, and dried down under N2 gas in a water bath of approximately 32 degrees C. Dried fractions were redissolved in 380 uL of water. 20 uL of isotope-labeled injection standards in water were added to the fractions. Columns were regenerated for reuse through the addition of 50 mL of water, 100 mL 3M HNO3, and 50 mL of water. When not in use, columns were stored completely filled with 0.01 M HNO3. Water was extracted alongside samples as methodological blanks. PPL-Solid Phase Extraction (PPL-SPE) Sample Volume: 40 mLs Solid Phase Resin/Column: Agilent Bond Elut PPL cartridge, 1 g bed mass, 6 mL volume Solvents/Reagents: methanol, 0.01 M HCl, Brief Procedure: Sample acidified with HCl to pH 2 and heavy isotope-labeled internal standards added. Column prepped by adding 1 cartridge volumes of each of methanol followed by 0.01 M HCl. Sample loaded onto columns using a peristaltic pump at a flow rate of 10 mL/min Column rinsed with 2 cartridge volumes 0.01 M HCl. Sample eluted with 1 cartridge volume methanol and dried down under N2 gas. Dried fractions were redissolved in 380 uL of water. 20 uL of isotope-labeled injection standards in water were added to the fractions. Water was extracted alongside samples as methodological blanks.

Sampleprep ID:

SP002384

Sampleprep Summary:

Cation-Exchange Solid Phase Extraction (CX-SPE) Sample Volume: 40 mLs Solid Phase Resin/Column: Strong cation-exchange resin (Dowex 50WX8; H+ form, 100-200 mesh, Sigma-Aldrich, Vienna, Austria) Solvents/Reagents: 1 M NH3, 3 M HNO3 Brief Procedure: 35 g of resin was added to a glass chromatography column with a fritted disk and a PTFE stopcock. The resin was equilibrated with 50 mL water, 100 mL of 1M NH3, 50 mL water, 100 mL 3M HNO3, and 50 mL H2O. The samples were acidified with HNO3 to pH 2 and heavy isotope-labeled internal standards added. The sample was loaded onto column, allowed to stand for 5 minutes, and then drained from the column. The column was then rinsed with 50 mL water. Approximately 200 mL of 1M NH3 was added to column. Ammonia eluted from column in 10 mL fractions. The pH of each fraction checked by dabbing a small drop of sample onto a pH strip with a combusted glass Pasteur pipette. The alkaline front (the 10 mL fraction where the pH increases from approximately 2-4 to 9-11), the fraction before, and the two fractions after were collected, combined, and dried down under N2 gas in a water bath of approximately 32 degrees C. Dried fractions were redissolved in 380 uL of water. 20 uL of isotope-labeled injection standards in water were added to the fractions. Columns were regenerated for reuse through the addition of 50 mL of water, 100 mL 3M HNO3, and 50 mL of water. When not in use, columns were stored completely filled with 0.01 M HNO3. Water was extracted alongside samples as methodological blanks. PPL-Solid Phase Extraction (PPL-SPE) Sample Volume: 40 mLs Solid Phase Resin/Column: Agilent Bond Elut PPL cartridge, 1 g bed mass, 6 mL volume Solvents/Reagents: methanol, 0.01 M HCl, Brief Procedure: Sample acidified with HCl to pH 2 and heavy isotope-labeled internal standards added. Column prepped by adding 1 cartridge volumes of each of methanol followed by 0.01 M HCl. Sample loaded onto columns using a peristaltic pump at a flow rate of 10 mL/min Column rinsed with 2 cartridge volumes 0.01 M HCl. Sample eluted with 1 cartridge volume methanol and dried down under N2 gas. Dried fractions were redissolved in 380 uL of water. 20 uL of isotope-labeled injection standards in water were added to the fractions. Water was extracted alongside samples as methodological blanks.