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 |
| 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 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001470 |
| Project DOI: | doi: 10.21228/M8Q99X |
| Project Title: | Quantification of Dissolved Metabolites in Environmental Samples through Cation-Exchange Solid Phase Extraction (CX-SPE) paired with Liquid Chromatography-Mass Spectrometry |
| Project 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 |
| Funding Source: | NSF, Simons Foundation |
| Publications: | Sacks et al., L&O Methods, accepted |
Subject:
| Subject ID: | SU002378 |
| Subject Type: | Water sample |
Factors:
Subject type: Water sample; Subject species: - (Factor headings shown in green)
| mb_sample_id | local_sample_id | Extraction_Approach | Location |
|---|---|---|---|
| SA220266 | LW_C | CX-SPE | Lake Washington |
| SA220267 | LW_A | CX-SPE | Lake Washington |
| SA220268 | LW_B | CX-SPE | Lake Washington |
| SA220269 | Blank3_B | CX-SPE | NA |
| SA220270 | Blank3_A | CX-SPE | NA |
| SA220271 | Blank2_C | CX-SPE | NA |
| SA220272 | Blank2_B | CX-SPE | NA |
| SA220273 | Blank3_C | CX-SPE | NA |
| SA220274 | Blank4_B | CX-SPE | NA |
| SA220275 | Blank4_C | CX-SPE | NA |
| SA220276 | Blank5_B | CX-SPE | NA |
| SA220277 | Blank2_A | CX-SPE | NA |
| SA220278 | Blank5_C | CX-SPE | NA |
| SA220279 | Blank5_A | CX-SPE | NA |
| SA220280 | Blank4_A | CX-SPE | NA |
| SA220281 | Blank1_A | CX-SPE | NA |
| SA220282 | Blank1_B | CX-SPE | NA |
| SA220283 | Blank1_C | CX-SPE | NA |
| SA220284 | NP_A | CX-SPE | North Pacific |
| SA220285 | NP_B | CX-SPE | North Pacific |
| SA220286 | NP_C | CX-SPE | North Pacific |
| SA220287 | PS_A | CX-SPE | Puget Sound |
| SA220288 | PS_B | CX-SPE | Puget Sound |
| SA220289 | PS_C | CX-SPE | Puget Sound |
| SA220290 | Aloha_C | CX-SPE | Station ALOHA |
| SA220291 | Aloha_B | CX-SPE | Station ALOHA |
| SA220292 | Aloha_A | CX-SPE | Station ALOHA |
| SA220293 | TruePoo_Half2 | NA | NA |
| SA220294 | TruePoo_Full1 | NA | NA |
| SA220295 | TruePoo_Full2 | NA | NA |
| SA220296 | TruePoo_Half1 | NA | NA |
| SA220297 | TruePoo_Full3 | NA | NA |
| SA220298 | TruePoo_Half3 | NA | NA |
| SA220299 | PPL_Blank_C | PPL-SPE | NA |
| SA220300 | PPL_Blank_A | PPL-SPE | NA |
| SA220301 | PPL_Blank_B | PPL-SPE | NA |
| SA220302 | PPL_PS_A | PPL-SPE | Puget Sound |
| SA220303 | PPL_PS_C | PPL-SPE | Puget Sound |
| SA220304 | PPL_PS_B | PPL-SPE | Puget Sound |
| SA220305 | PPL_Aloha_C | PPL-SPE | Station ALOHA |
| SA220306 | PPL_Aloha_A | PPL-SPE | Station ALOHA |
| SA220307 | PPL_Aloha_B | PPL-SPE | Station ALOHA |
| Showing results 1 to 42 of 42 |
Collection:
| Collection ID: | CO002371 |
| Collection Summary: | Samples for dissolved metabolites from four unique environmental locations (Lake Washington (LW), Puget Sound (PS), Station ALOHA in the North Pacific Subtropical Gyre (ALOHA), and the North Pacific (NP)) from depths of 15 m for ALOHA and NP samples, 8 m for the PS sample, and 1 m for the LW sample. Samples were processed by filtering the water through 147 mm 0.2 μm PTFE filters using peristaltic pumps (ALOHA, NP, PS samples) and or glass vacuum filtration onto a 47 mm 0.2 μm PTFE filter. The filtrated was collected and stored in 2 L acid-washed polycarbonate bottles (ALOHA, NP, PS samples) or 45 mL acid-washed polypropylene falcon tubes at -20 C for between 1 month and 4 years until analysis. |
| Sample Type: | seawater |
| Storage Conditions: | -20℃ |
Treatment:
| Treatment ID: | TR002390 |
| Treatment Summary: | Not Applicable |
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. |
Combined analysis:
| Analysis ID | AN003744 | AN003745 | AN003746 |
|---|---|---|---|
| Analysis type | MS | MS | MS |
| Chromatography type | HILIC | HILIC | Reversed phase |
| Chromatography system | Waters Acquity I-Class | Waters Acquity I-Class | Waters Acquity I-Class |
| Column | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) | Waters Acquity HSS Cyano (1.8um,2.1 mm x 100 mm) |
| MS Type | ESI | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE |
| Units | Peak Area | Peak Area | Peak Area |
Chromatography:
| Chromatography ID: | CH002774 |
| Chromatography Summary: | See attached summary |
| Methods Filename: | Ingalls_Lab_LC_Methods_2.txt |
| Instrument Name: | Waters Acquity I-Class |
| Column Name: | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 30 |
| Flow Rate: | 0.15 ml/min |
| Solvent A: | 85% acetonitrile/15% water; 10 mM ammonium carbonate |
| Solvent B: | 15% acetonitrile/85% water; 10 mM ammonium carbonate |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH002775 |
| Chromatography Summary: | See attached summary |
| Methods Filename: | Ingalls_Lab_LC_Methods_2.txt |
| Instrument Name: | Waters Acquity I-Class |
| Column Name: | Waters Acquity HSS Cyano (1.8um,2.1 mm x 100 mm) |
| Column Temperature: | 35 |
| Flow Rate: | 0.4 ml/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003491 |
| Analysis ID: | AN003744 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See Protocol |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | CXSPE_Method_Paper_MS_Methods.txt |
| MS ID: | MS003492 |
| Analysis ID: | AN003745 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See Protocol |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | CXSPE_Method_Paper_MS_Methods.txt |
| MS ID: | MS003493 |
| Analysis ID: | AN003746 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See Protocol |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | CXSPE_Method_Paper_MS_Methods.txt |
