#METABOLOMICS WORKBENCH jssacks_20220830_132254 DATATRACK_ID:3439 STUDY_ID:ST002292 ANALYSIS_ID:AN003746 VERSION 1 CREATED_ON 09-28-2022 #PROJECT PR:PROJECT_TITLE Quantification of Dissolved Metabolites in Environmental Samples through PR:PROJECT_TITLE Cation-Exchange Solid Phase Extraction (CX-SPE) paired with Liquid PR:PROJECT_TITLE Chromatography-Mass Spectrometry PR:PROJECT_SUMMARY Small, biologically produced, organic molecules called metabolites play key PR:PROJECT_SUMMARY roles in microbial systems where they directly mediate exchanges of nutrients, PR:PROJECT_SUMMARY energy, and information. However, the study of dissolved polar metabolites in PR:PROJECT_SUMMARY seawater and other environmental matrices has been hampered by analytical PR:PROJECT_SUMMARY challenges including high inorganic ion concentrations, low analyte PR:PROJECT_SUMMARY concentrations, and high chemical diversity. Here we show that a cation-exchange PR:PROJECT_SUMMARY solid phase extraction (CX-SPE) sample preparation approach separates positively PR:PROJECT_SUMMARY charged and zwitterionic metabolites from seawater and freshwater samples, PR:PROJECT_SUMMARY allowing their analysis by liquid chromatography-mass spectrometry (LC-MS). We PR:PROJECT_SUMMARY successfully extracted 69 known compounds from an in-house compound collection PR:PROJECT_SUMMARY and evaluated the performance of the method by establishing extraction PR:PROJECT_SUMMARY efficiencies and limits of detection (pM to low nM range) for these compounds. PR:PROJECT_SUMMARY CX-SPE extracted a range of compounds including amino acids and compatible PR:PROJECT_SUMMARY solutes, resulted in very low matrix effects, and performed robustly across PR:PROJECT_SUMMARY large variations in salinity and dissolved organic matter (DOM) concentration. PR:PROJECT_SUMMARY We compared CX-SPE to an established solid phase extraction procedure (PPL-SPE) PR:PROJECT_SUMMARY and demonstrate that these two methods extract fundamentally different fractions PR:PROJECT_SUMMARY of the dissolved metabolite pool with CX-SPE extracting compounds that are on PR:PROJECT_SUMMARY average smaller and more polar. We use CX-SPE to analyze four environmental PR:PROJECT_SUMMARY samples from distinct aquatic biomes, producing some of the first CX-SPE PR:PROJECT_SUMMARY dissolved metabolomes. Quantified compounds ranged in concentration from 0.0093 PR:PROJECT_SUMMARY nM to 49 nM and were composed primarily of amino acids (0.15 – 16 nM) and PR:PROJECT_SUMMARY compatible solutes such as TMAO (0.89 – 49 nM) and glycine betaine (2.8 – PR:PROJECT_SUMMARY 5.2 nM). PR:INSTITUTE University of Washington PR:DEPARTMENT Oceanography PR:LABORATORY Ingalls Lab PR:LAST_NAME Sacks PR:FIRST_NAME Joshua PR:ADDRESS Ocean Sciences Building, 1492 NE Boat St. Seattle, WA 98105 PR:EMAIL jssacks@uw.edu PR:PHONE 4074090052 PR:FUNDING_SOURCE NSF, Simons Foundation PR:PUBLICATIONS Sacks et al., L&O Methods, accepted PR:DOI http://dx.doi.org/10.21228/M8Q99X #STUDY ST:STUDY_TITLE Quantification of Dissolved Metabolites in Environmental Samples through ST:STUDY_TITLE Cation-Exchange Solid Phase Extraction (CX-SPE) paired with Liquid ST:STUDY_TITLE Chromatography-Mass Spectrometry ST:STUDY_TYPE Method Development for Dissolved Metabolomics in Seawater ST:STUDY_SUMMARY Small, biologically produced, organic molecules called metabolites play key ST:STUDY_SUMMARY roles in microbial systems where they directly mediate exchanges of nutrients, ST:STUDY_SUMMARY energy, and information. However, the study of dissolved polar metabolites in ST:STUDY_SUMMARY seawater and other environmental matrices has been hampered by analytical ST:STUDY_SUMMARY challenges including high inorganic ion concentrations, low analyte ST:STUDY_SUMMARY concentrations, and high chemical diversity. Here we show that a cation-exchange ST:STUDY_SUMMARY solid phase extraction (CX-SPE) sample preparation approach separates positively ST:STUDY_SUMMARY charged and zwitterionic metabolites from seawater and freshwater samples, ST:STUDY_SUMMARY allowing their analysis by liquid chromatography-mass spectrometry (LC-MS). We ST:STUDY_SUMMARY successfully extracted 69 known compounds from an in-house compound collection ST:STUDY_SUMMARY and evaluated the performance of the method by establishing extraction ST:STUDY_SUMMARY efficiencies and limits of detection (pM to low nM range) for these compounds. ST:STUDY_SUMMARY CX-SPE extracted a range of compounds including amino acids and compatible ST:STUDY_SUMMARY solutes, resulted in very low matrix effects, and performed robustly across ST:STUDY_SUMMARY large variations in salinity and dissolved organic matter (DOM) concentration. ST:STUDY_SUMMARY We compared CX-SPE to an established solid phase extraction procedure (PPL-SPE) ST:STUDY_SUMMARY and demonstrate that these two methods extract fundamentally different fractions ST:STUDY_SUMMARY of the dissolved metabolite pool with CX-SPE extracting compounds that are on ST:STUDY_SUMMARY average smaller and more polar. We use CX-SPE to analyze four environmental ST:STUDY_SUMMARY samples from distinct aquatic biomes, producing some of the first CX-SPE ST:STUDY_SUMMARY dissolved metabolomes. Quantified compounds ranged in concentration from 0.0093 ST:STUDY_SUMMARY nM to 49 nM and were composed primarily of amino acids (0.15 – 16 nM) and ST:STUDY_SUMMARY compatible solutes such as TMAO (0.89 – 49 nM) and glycine betaine (2.8 – ST:STUDY_SUMMARY 5.2 nM). ST:INSTITUTE University of Washington ST:DEPARTMENT Oceanography ST:LABORATORY Ingalls Lab ST:LAST_NAME Sacks ST:FIRST_NAME Joshua ST:ADDRESS Ocean Sciences Building, 1492 NE Boat St. Seattle, WA 98105 ST:EMAIL jssacks@uw.edu ST:PHONE 4074090052 ST:SUBMIT_DATE 2022-08-30 #SUBJECT SU:SUBJECT_TYPE Water sample #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - LW_A Extraction_Approach:CX-SPE | Location:Lake Washington Replicate=A; Type=Smp; Depth_m=1; Vol_mL=40; Latitude=47.633; Longitude=-122.3; Date=2020-06-20; RAW_FILE_NAME=210322_Smp_LW_A; RAW_FILE_NAME=210322_Smp_LW_A; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - LW_B Extraction_Approach:CX-SPE | Location:Lake Washington Replicate=B; Type=Smp; Depth_m=1; Vol_mL=40; Latitude=47.633; Longitude=-122.3; Date=2020-06-20; RAW_FILE_NAME=210322_Smp_LW_B; RAW_FILE_NAME=210322_Smp_LW_B; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - LW_C Extraction_Approach:CX-SPE | Location:Lake Washington Replicate=C; Type=Smp; Depth_m=1; Vol_mL=40; Latitude=47.633; Longitude=-122.3; Date=2020-06-20; RAW_FILE_NAME=210322_Smp_LW_C; RAW_FILE_NAME=210322_Smp_LW_C; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - Blank1_A Extraction_Approach:CX-SPE | Location:NA Replicate=A; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_Blank1_A; RAW_FILE_NAME=210322_Blk_Blank1_A; RAW_FILE_NAME=210329_Blk_Blank1_A SUBJECT_SAMPLE_FACTORS - Blank1_B Extraction_Approach:CX-SPE | Location:NA Replicate=B; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_Blank1_B; RAW_FILE_NAME=210322_Blk_Blank1_B; RAW_FILE_NAME=210329_Blk_Blank1_B SUBJECT_SAMPLE_FACTORS - Blank1_C Extraction_Approach:CX-SPE | Location:NA Replicate=C; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_Blank1_C; RAW_FILE_NAME=210322_Blk_Blank1_C; RAW_FILE_NAME=210329_Blk_Blank1_C SUBJECT_SAMPLE_FACTORS - Blank2_A Extraction_Approach:CX-SPE | Location:NA Replicate=A; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_Blank2_A; RAW_FILE_NAME=210322_Blk_Blank2_A; RAW_FILE_NAME=210329_Blk_Blank2_A SUBJECT_SAMPLE_FACTORS - Blank2_B Extraction_Approach:CX-SPE | Location:NA Replicate=B; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_Blank2_B; RAW_FILE_NAME=210322_Blk_Blank2_B; RAW_FILE_NAME=210329_Blk_Blank2_B SUBJECT_SAMPLE_FACTORS - Blank2_C Extraction_Approach:CX-SPE | Location:NA Replicate=C; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_Blank2_C; RAW_FILE_NAME=210322_Blk_Blank2_C; RAW_FILE_NAME=210329_Blk_Blank2_C SUBJECT_SAMPLE_FACTORS - Blank3_A Extraction_Approach:CX-SPE | Location:NA Replicate=A; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_Blank3_A; RAW_FILE_NAME=210322_Blk_Blank3_A; RAW_FILE_NAME=210329_Blk_Blank3_A SUBJECT_SAMPLE_FACTORS - Blank3_B Extraction_Approach:CX-SPE | Location:NA Replicate=B; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_Blank3_B; RAW_FILE_NAME=210322_Blk_Blank3_B; RAW_FILE_NAME=210329_Blk_Blank3_B SUBJECT_SAMPLE_FACTORS - Blank3_C Extraction_Approach:CX-SPE | Location:NA Replicate=C; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_Blank3_C; RAW_FILE_NAME=210322_Blk_Blank3_C; RAW_FILE_NAME=210329_Blk_Blank3_C SUBJECT_SAMPLE_FACTORS - Blank4_A Extraction_Approach:CX-SPE | Location:NA Replicate=A; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_Blank4_A; RAW_FILE_NAME=210322_Blk_Blank4_A; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - Blank4_B Extraction_Approach:CX-SPE | Location:NA Replicate=B; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_Blank4_B; RAW_FILE_NAME=210322_Blk_Blank4_B; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - Blank4_C Extraction_Approach:CX-SPE | Location:NA Replicate=C; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_Blank4_C; RAW_FILE_NAME=210322_Blk_Blank4_C; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - Blank5_A Extraction_Approach:CX-SPE | Location:NA Replicate=A; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_Blank5_A; RAW_FILE_NAME=210322_Blk_Blank5_A; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - Blank5_B Extraction_Approach:CX-SPE | Location:NA Replicate=B; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_Blank5_B; RAW_FILE_NAME=210322_Blk_Blank5_B; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - Blank5_C Extraction_Approach:CX-SPE | Location:NA Replicate=C; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_Blank5_C; RAW_FILE_NAME=210322_Blk_Blank5_C; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - NP_A Extraction_Approach:CX-SPE | Location:North Pacific Replicate=A; Type=Smp; Depth_m=15; Vol_mL=40; Latitude=41.4; Longitude=-158; Date=2016-04-26; RAW_FILE_NAME=210322_Smp_NP_A; RAW_FILE_NAME=210322_Smp_NP_A; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - NP_B Extraction_Approach:CX-SPE | Location:North Pacific Replicate=B; Type=Smp; Depth_m=15; Vol_mL=40; Latitude=41.4; Longitude=-158; Date=2016-04-26; RAW_FILE_NAME=210322_Smp_NP_B; RAW_FILE_NAME=210322_Smp_NP_B; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - NP_C Extraction_Approach:CX-SPE | Location:North Pacific Replicate=C; Type=Smp; Depth_m=15; Vol_mL=40; Latitude=41.4; Longitude=-158; Date=2016-04-26; RAW_FILE_NAME=210322_Smp_NP_C; RAW_FILE_NAME=210322_Smp_NP_C; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - PS_A Extraction_Approach:CX-SPE | Location:Puget Sound Replicate=A; Type=Smp; Depth_m=8; Vol_mL=40; Latitude=47.683; Longitude=-122.416; Date=2017-05-07; RAW_FILE_NAME=210322_Smp_PS_A; RAW_FILE_NAME=210322_Smp_PS_A; RAW_FILE_NAME=210329_Smp_PS_A SUBJECT_SAMPLE_FACTORS - PS_B Extraction_Approach:CX-SPE | Location:Puget Sound Replicate=B; Type=Smp; Depth_m=8; Vol_mL=40; Latitude=47.683; Longitude=-122.416; Date=2017-05-07; RAW_FILE_NAME=210322_Smp_PS_B; RAW_FILE_NAME=210322_Smp_PS_B; RAW_FILE_NAME=210329_Smp_PS_B SUBJECT_SAMPLE_FACTORS - PS_C Extraction_Approach:CX-SPE | Location:Puget Sound Replicate=C; Type=Smp; Depth_m=8; Vol_mL=40; Latitude=47.683; Longitude=-122.416; Date=2017-05-07; RAW_FILE_NAME=210322_Smp_PS_C; RAW_FILE_NAME=210322_Smp_PS_C; RAW_FILE_NAME=210329_Smp_PS_C SUBJECT_SAMPLE_FACTORS - Aloha_A Extraction_Approach:CX-SPE | Location:Station ALOHA Replicate=A; Type=Smp; Depth_m=15; Vol_mL=40; Latitude=22.75; Longitude=-158; Date=2016-04-20; RAW_FILE_NAME=210322_Smp_Aloha_A; RAW_FILE_NAME=210322_Smp_Aloha_A; RAW_FILE_NAME=210329_Smp_Aloha_A SUBJECT_SAMPLE_FACTORS - Aloha_B Extraction_Approach:CX-SPE | Location:Station ALOHA Replicate=B; Type=Smp; Depth_m=15; Vol_mL=40; Latitude=22.75; Longitude=-158; Date=2016-04-20; RAW_FILE_NAME=210322_Smp_Aloha_B; RAW_FILE_NAME=210322_Smp_Aloha_B; RAW_FILE_NAME=210329_Smp_Aloha_B SUBJECT_SAMPLE_FACTORS - Aloha_C Extraction_Approach:CX-SPE | Location:Station ALOHA Replicate=C; Type=Smp; Depth_m=15; Vol_mL=40; Latitude=22.75; Longitude=-158; Date=2016-04-20; RAW_FILE_NAME=210322_Smp_Aloha_C; RAW_FILE_NAME=210322_Smp_Aloha_C; RAW_FILE_NAME=210329_Smp_Aloha_C SUBJECT_SAMPLE_FACTORS - TruePoo_Full1 Extraction_Approach:NA | Location:NA Replicate=1; Type=Poo; Depth_m=NA; Vol_mL=NA; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Poo_TruePoo_Full1; RAW_FILE_NAME=210322_Poo_TruePoo_Full1; RAW_FILE_NAME=210329_Poo_TruePoo_Full1 SUBJECT_SAMPLE_FACTORS - TruePoo_Full2 Extraction_Approach:NA | Location:NA Replicate=2; Type=Poo; Depth_m=NA; Vol_mL=NA; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Poo_TruePoo_Full2; RAW_FILE_NAME=210322_Poo_TruePoo_Full2; RAW_FILE_NAME=210329_Poo_TruePoo_Full2 SUBJECT_SAMPLE_FACTORS - TruePoo_Full3 Extraction_Approach:NA | Location:NA Replicate=3; Type=Poo; Depth_m=NA; Vol_mL=NA; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Poo_TruePoo_Full3; RAW_FILE_NAME=210322_Poo_TruePoo_Full3; RAW_FILE_NAME=210329_Poo_TruePoo_Full3 SUBJECT_SAMPLE_FACTORS - TruePoo_Half1 Extraction_Approach:NA | Location:NA Replicate=1; Type=Poo; Depth_m=NA; Vol_mL=NA; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Poo_TruePoo_Half1; RAW_FILE_NAME=210322_Poo_TruePoo_Half1; RAW_FILE_NAME=210329_Poo_TruePoo_Half1 SUBJECT_SAMPLE_FACTORS - TruePoo_Half2 Extraction_Approach:NA | Location:NA Replicate=2; Type=Poo; Depth_m=NA; Vol_mL=NA; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Poo_TruePoo_Half2; RAW_FILE_NAME=210322_Poo_TruePoo_Half2; RAW_FILE_NAME=210329_Poo_TruePoo_Half2 SUBJECT_SAMPLE_FACTORS - TruePoo_Half3 Extraction_Approach:NA | Location:NA Replicate=3; Type=Poo; Depth_m=NA; Vol_mL=NA; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Poo_TruePoo_Half3; RAW_FILE_NAME=210322_Poo_TruePoo_Half3; RAW_FILE_NAME=210329_Poo_TruePoo_Half3 SUBJECT_SAMPLE_FACTORS - PPL_Blank_A Extraction_Approach:PPL-SPE | Location:NA Replicate=A; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_PPL_Blank_A; RAW_FILE_NAME=210322_Blk_PPL_Blank_A; RAW_FILE_NAME=210329_Blk_PPL_Blank_A SUBJECT_SAMPLE_FACTORS - PPL_Blank_B Extraction_Approach:PPL-SPE | Location:NA Replicate=B; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_PPL_Blank_B; RAW_FILE_NAME=210322_Blk_PPL_Blank_B; RAW_FILE_NAME=210329_Blk_PPL_Blank_B SUBJECT_SAMPLE_FACTORS - PPL_Blank_C Extraction_Approach:PPL-SPE | Location:NA Replicate=C; Type=Blk; Depth_m=NA; Vol_mL=40; Latitude=NA; Longitude=NA; Date=NA; RAW_FILE_NAME=210322_Blk_PPL_Blank_C; RAW_FILE_NAME=210322_Blk_PPL_Blank_C; RAW_FILE_NAME=210329_Blk_PPL_Blank_C SUBJECT_SAMPLE_FACTORS - PPL_PS_A Extraction_Approach:PPL-SPE | Location:Puget Sound Replicate=A; Type=Smp; Depth_m=8; Vol_mL=40; Latitude=47.683; Longitude=-122.416; Date=2017-05-07; RAW_FILE_NAME=210322_Smp_PPL_PS_A; RAW_FILE_NAME=210322_Smp_PPL_PS_A; RAW_FILE_NAME=210329_Smp_PPL_PS_A SUBJECT_SAMPLE_FACTORS - PPL_PS_B Extraction_Approach:PPL-SPE | Location:Puget Sound Replicate=B; Type=Smp; Depth_m=8; Vol_mL=40; Latitude=47.683; Longitude=-122.416; Date=2017-05-07; RAW_FILE_NAME=210322_Smp_PPL_PS_B; RAW_FILE_NAME=210322_Smp_PPL_PS_B; RAW_FILE_NAME=210329_Smp_PPL_PS_B SUBJECT_SAMPLE_FACTORS - PPL_PS_C Extraction_Approach:PPL-SPE | Location:Puget Sound Replicate=C; Type=Smp; Depth_m=8; Vol_mL=40; Latitude=47.683; Longitude=-122.416; Date=2017-05-07; RAW_FILE_NAME=210322_Smp_PPL_PS_C; RAW_FILE_NAME=210322_Smp_PPL_PS_C; RAW_FILE_NAME=210329_Smp_PPL_PS_C SUBJECT_SAMPLE_FACTORS - PPL_Aloha_A Extraction_Approach:PPL-SPE | Location:Station ALOHA Replicate=A; Type=Smp; Depth_m=15; Vol_mL=40; Latitude=22.75; Longitude=-158; Date=2016-04-20; RAW_FILE_NAME=210322_Smp_PPL_Aloha_A; RAW_FILE_NAME=210322_Smp_PPL_Aloha_A; RAW_FILE_NAME=210329_Smp_PPL_Aloha_A SUBJECT_SAMPLE_FACTORS - PPL_Aloha_B Extraction_Approach:PPL-SPE | Location:Station ALOHA Replicate=B; Type=Smp; Depth_m=15; Vol_mL=40; Latitude=22.75; Longitude=-158; Date=2016-04-20; RAW_FILE_NAME=210322_Smp_PPL_Aloha_B; RAW_FILE_NAME=210322_Smp_PPL_Aloha_B; RAW_FILE_NAME=210329_Smp_PPL_Aloha_B SUBJECT_SAMPLE_FACTORS - PPL_Aloha_C Extraction_Approach:PPL-SPE | Location:Station ALOHA Replicate=C; Type=Smp; Depth_m=15; Vol_mL=40; Latitude=22.75; Longitude=-158; Date=2016-04-20; RAW_FILE_NAME=210322_Smp_PPL_Aloha_C; RAW_FILE_NAME=210322_Smp_PPL_Aloha_C; RAW_FILE_NAME=210329_Smp_PPL_Aloha_C #COLLECTION CO:COLLECTION_SUMMARY Samples for dissolved metabolites from four unique environmental locations (Lake CO:COLLECTION_SUMMARY Washington (LW), Puget Sound (PS), Station ALOHA in the North Pacific CO:COLLECTION_SUMMARY Subtropical Gyre (ALOHA), and the North Pacific (NP)) from depths of 15 m for CO:COLLECTION_SUMMARY ALOHA and NP samples, 8 m for the PS sample, and 1 m for the LW sample. Samples CO:COLLECTION_SUMMARY were processed by filtering the water through 147 mm 0.2 μm PTFE filters using CO:COLLECTION_SUMMARY peristaltic pumps (ALOHA, NP, PS samples) and or glass vacuum filtration onto a CO:COLLECTION_SUMMARY 47 mm 0.2 μm PTFE filter. The filtrated was collected and stored in 2 L CO:COLLECTION_SUMMARY acid-washed polycarbonate bottles (ALOHA, NP, PS samples) or 45 mL acid-washed CO:COLLECTION_SUMMARY polypropylene falcon tubes at -20 C for between 1 month and 4 years until CO:COLLECTION_SUMMARY analysis. CO:SAMPLE_TYPE seawater CO:STORAGE_CONDITIONS -20℃ #TREATMENT TR:TREATMENT_SUMMARY Not Applicable #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cation-Exchange Solid Phase Extraction (CX-SPE) Sample Volume: 40 mLs Solid SP:SAMPLEPREP_SUMMARY Phase Resin/Column: Strong cation-exchange resin (Dowex 50WX8; H+ form, 100-200 SP:SAMPLEPREP_SUMMARY mesh, Sigma-Aldrich, Vienna, Austria) Solvents/Reagents: 1 M NH3, 3 M HNO3 Brief SP:SAMPLEPREP_SUMMARY Procedure: 35 g of resin was added to a glass chromatography column with a SP:SAMPLEPREP_SUMMARY fritted disk and a PTFE stopcock. The resin was equilibrated with 50 mL water, SP:SAMPLEPREP_SUMMARY 100 mL of 1M NH3, 50 mL water, 100 mL 3M HNO3, and 50 mL H2O. The samples were SP:SAMPLEPREP_SUMMARY acidified with HNO3 to pH 2 and heavy isotope-labeled internal standards added. SP:SAMPLEPREP_SUMMARY The sample was loaded onto column, allowed to stand for 5 minutes, and then SP:SAMPLEPREP_SUMMARY drained from the column. The column was then rinsed with 50 mL water. SP:SAMPLEPREP_SUMMARY Approximately 200 mL of 1M NH3 was added to column. Ammonia eluted from column SP:SAMPLEPREP_SUMMARY in 10 mL fractions. The pH of each fraction checked by dabbing a small drop of SP:SAMPLEPREP_SUMMARY sample onto a pH strip with a combusted glass Pasteur pipette. The alkaline SP:SAMPLEPREP_SUMMARY front (the 10 mL fraction where the pH increases from approximately 2-4 to SP:SAMPLEPREP_SUMMARY 9-11), the fraction before, and the two fractions after were collected, SP:SAMPLEPREP_SUMMARY combined, and dried down under N2 gas in a water bath of approximately 32 SP:SAMPLEPREP_SUMMARY degrees C. Dried fractions were redissolved in 380 uL of water. 20 uL of SP:SAMPLEPREP_SUMMARY isotope-labeled injection standards in water were added to the fractions. SP:SAMPLEPREP_SUMMARY Columns were regenerated for reuse through the addition of 50 mL of water, 100 SP:SAMPLEPREP_SUMMARY mL 3M HNO3, and 50 mL of water. When not in use, columns were stored completely SP:SAMPLEPREP_SUMMARY filled with 0.01 M HNO3. Water was extracted alongside samples as methodological SP:SAMPLEPREP_SUMMARY blanks. PPL-Solid Phase Extraction (PPL-SPE) Sample Volume: 40 mLs Solid Phase SP:SAMPLEPREP_SUMMARY Resin/Column: Agilent Bond Elut PPL cartridge, 1 g bed mass, 6 mL volume SP:SAMPLEPREP_SUMMARY Solvents/Reagents: methanol, 0.01 M HCl, Brief Procedure: Sample acidified with SP:SAMPLEPREP_SUMMARY HCl to pH 2 and heavy isotope-labeled internal standards added. Column prepped SP:SAMPLEPREP_SUMMARY by adding 1 cartridge volumes of each of methanol followed by 0.01 M HCl. Sample SP:SAMPLEPREP_SUMMARY loaded onto columns using a peristaltic pump at a flow rate of 10 mL/min Column SP:SAMPLEPREP_SUMMARY rinsed with 2 cartridge volumes 0.01 M HCl. Sample eluted with 1 cartridge SP:SAMPLEPREP_SUMMARY volume methanol and dried down under N2 gas. Dried fractions were redissolved in SP:SAMPLEPREP_SUMMARY 380 uL of water. 20 uL of isotope-labeled injection standards in water were SP:SAMPLEPREP_SUMMARY added to the fractions. Water was extracted alongside samples as methodological SP:SAMPLEPREP_SUMMARY blanks. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY See attached summary CH:METHODS_FILENAME Ingalls_Lab_LC_Methods_2.txt CH:INSTRUMENT_NAME Waters Acquity I-Class CH:COLUMN_NAME Waters Acquity HSS Cyano column (1.8 µm particle size, 2.1 mm x 100 mm) CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE CXSPE_Method_Paper_MS_Methods.txt #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS See Protocol MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples PS_A PS_B PS_C Aloha_A Aloha_B Aloha_C TruePoo_Full1 TruePoo_Full2 TruePoo_Full3 TruePoo_Half1 TruePoo_Half2 TruePoo_Half3 PPL_PS_A PPL_PS_B PPL_PS_C PPL_Aloha_A PPL_Aloha_B PPL_Aloha_C Factors Extraction_Approach:CX-SPE | Location:Puget Sound Extraction_Approach:CX-SPE | Location:Puget Sound Extraction_Approach:CX-SPE | Location:Puget Sound Extraction_Approach:CX-SPE | Location:Station ALOHA Extraction_Approach:CX-SPE | Location:Station ALOHA Extraction_Approach:CX-SPE | Location:Station ALOHA Extraction_Approach:NA | Location:NA Extraction_Approach:NA | Location:NA Extraction_Approach:NA | Location:NA Extraction_Approach:NA | Location:NA Extraction_Approach:NA | Location:NA Extraction_Approach:NA | Location:NA Extraction_Approach:PPL-SPE | Location:Puget Sound Extraction_Approach:PPL-SPE | Location:Puget Sound Extraction_Approach:PPL-SPE | Location:Puget Sound Extraction_Approach:PPL-SPE | Location:Station ALOHA Extraction_Approach:PPL-SPE | Location:Station ALOHA Extraction_Approach:PPL-SPE | Location:Station ALOHA 3-Indoleacetonitrile 36750.0000 39722.0000 17035.0000 3 Indolepropionic Acid 269709.6535 265272.5392 240480.6683 226834.0225 214513.4587 211206.9730 3OC10-HSL 45690.0000 37865.0000 33722.0000 50408.0000 27332.0000 29422.0000 Acetyl-L-carnitine 571867.4010 650901.2850 524674.9603 1901598.3419 1504125.3238 1466791.5588 6408043.3380 5934756.0843 7484130.3909 6385103.3502 7501292.9404 8471481.5676 479898.5188 316919.7123 247094.0871 131696.2035 246419.4902 54841.1528 Adenosyl Homocysteine 4504905.0000 4876843.0000 4192125.0000 4468368.0000 4696082.0000 4171764.0000 52586.7333 33455.3333 50680.6000 35932.4000 63629.2667 44805.6667 Adenosyl Methionine 12316.0000 13322.0000 29549.0000 11202.0000 6670.0000 11773.0000 1648172.0000 1845729.0000 1418581.0000 1496502.0000 1333402.0000 1108868.0000 Betaine 126795088.1190 170433075.8783 146004057.2062 107830170.5251 167113953.7785 73829792.8638 157578296.5943 153620016.2703 149616563.7749 156351693.1504 164036993.8758 148258099.5463 Butyryl-L-carnitine 262881296.0000 290417056.0000 236483072.0000 270618592.0000 300761376.0000 249060384.0000 C4-HSL 83999.0000 91736.0000 76652.0000 Caffeine 246922.0000 270325.0000 397238.0000 Camalexin 23512.0000 25170.0000 39676.0000 Choline 394003.9740 394904.5117 670119.9748 103261768.4522 100187568.0905 97811298.7423 93426247.7762 93431882.2993 89952501.5858 Decarboxylated S-Adenosylmethionine 88145.0000 116885.0000 90081.0000 131713.0000 114166.0000 179119.0000 9102.4667 29090.6000 14502.0000 28727.9333 35886.1333 45350.6667 Desthiobiotin 137166.0000 166256.0000 61586.0000 122504.0000 139903.0000 118876.0000 105866.0000 119434.0000 103176.0000 Dimethyl glycine 18776343.9649 17980923.0810 18550250.2055 19563606.6694 18800600.1305 18727304.7344 Domoic Acid 37302.5963 333018.3127 103144.6384 63553.7894 44786.1664 60181.6797 Glutathione 458374.2667 422023.9121 326652.1207 638149.4396 481749.6088 394266.1885 97032.6741 23853.2078 19091.9987 26302.3794 29922.0679 28315.8126 Glutathione Disulfide 283507.0000 352049.0000 258019.0000 256722.0000 265488.0000 312214.0000 Indole 3 Lactic Acid 628832.1615 693780.2486 713079.1699 722083.7889 657516.6249 715583.1582 Indole 3 methyl acetate 269709.6535 265272.5392 240480.6683 226834.0225 214513.4587 211206.9730 Isobutyryl-L-carnitine 262881296.0000 290417056.0000 236483072.0000 270618592.0000 300761376.0000 249060384.0000 Kynurenine 280944.3684 315186.0878 262098.4058 255728.3315 245643.5506 275199.3291 175775.0349 233222.3217 564043.3995 248275.9223 74237.8945 28040.5587 Methylthioadenosine 41632330.3253 40124763.3434 40345162.3092 38336870.7980 36453042.2738 35950630.3300 24003.9590 43065.5948 108469.9681 3611.6746 4052.8472 13210.9953 Phenylalanine 7268751.3954 7129721.4760 8010883.5905 10254584.5435 8377802.4407 6464228.5364 27122991.3746 26675020.3824 26854247.7383 26810146.5515 27262436.1170 26777909.2666 18716120.4183 21961499.3867 21443290.9162 19883060.6770 19958550.8297 21838604.3288 Propionyl-L-carnitine 71548840.0000 77596240.0000 65170520.0000 70008880.0000 77959368.0000 65867568.0000 12097.1333 25620.0667 26654.0667 34805.1333 40463.8667 38875.8667 Pyridoxal Phosphate 3265312.6508 3143915.0128 2842675.6179 3080730.2030 3108906.7691 2748040.9717 Pyridoxamine 332482.8619 420512.8519 355564.2892 409118.8207 442431.2676 384877.7262 Sulfurol 35894.9876 56853.3401 33158.9822 23259.8858 17255.0315 11214.7346 1069954.6861 960490.4762 930380.6203 1127871.4599 1019974.2009 981153.9915 337173.1747 300462.2136 358753.9394 153682.8747 150262.8549 157100.7111 Syringaldehyde 171685.0000 176476.0000 209591.0000 Thiamine monophosphate 2019872.0000 2065956.0000 1658522.0000 2035768.0000 1920878.0000 1687246.0000 8146.7333 12563.8000 10050.3333 7619.0000 9766.2000 9791.0667 Tryptophan 11635080.4582 11146117.7217 11059861.2527 10824520.8470 11006203.9755 10856493.9845 122235.3496 204159.0889 139416.6580 223982.4010 85005.7542 157395.3765 Tyrosine 1726345.3343 1435449.0916 1896631.8965 3449761.7086 2755290.8245 2220886.4900 11841929.2855 11652300.6740 11596810.1687 11786910.9250 11901871.5263 12153063.9025 1770052.4237 1725513.9045 1646074.5589 4145795.6106 4469574.1807 4155622.0815 Vanillin 1510425.5478 1458211.5631 1603634.6724 1444009.7668 1588599.1580 1522508.9970 Vitamin B3 325441.0356 115773.9996 275009.0354 255988.3046 115139.6371 89435.0197 Vitamin B5 1013399.0000 361802.0000 46857.0000 904476.0000 298597.0000 6301.0000 118786.0000 154486.0000 44661.0000 120804.0000 187446.0000 21642.0000 94379.8000 70183.6000 82904.6667 21669.9333 25354.2000 25796.7333 Vitamin B6 784933.8086 672802.3781 665268.1156 341986.9537 418369.0941 349968.3776 447783.9204 393146.4617 422763.1699 Vitamin B9 7147.0680 5628.1602 14736.7884 6855.1739 5483.5888 5948.1295 Xanthine 192200.0000 165348.0000 202288.0000 154885.0000 172203.0000 115760.0000 4636.9333 691.9333 4945.0000 259.6667 1078.9333 1148.8667 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant 3-Indoleacetonitrile cpd:C02938 3 Indolepropionic Acid NA 3OC10-HSL NA Acetyl-L-carnitine cpd:C02571 Adenosyl Homocysteine cpd:C00021 Adenosyl Methionine cpd:C00019 Betaine cpd:C00719 Butyryl-L-carnitine NA C4-HSL cpd:C11837 Caffeine cpd:C07481 Camalexin NA Choline cpd:C00114 Decarboxylated S-Adenosylmethionine NA Desthiobiotin cpd:C01909 Dimethyl glycine cpd:C01026 Domoic Acid cpd:C13732 Glutathione cpd:C00051 Glutathione Disulfide cpd:C00127 Indole 3 Lactic Acid cpd:C02043 Indole 3 methyl acetate cpd:C20635 Isobutyryl-L-carnitine NA Kynurenine cpd:C00328 Methylthioadenosine cpd:C00170 Phenylalanine cpd:C00079 Propionyl-L-carnitine cpd:C03017 Pyridoxal Phosphate cpd:C00018 Pyridoxamine cpd:C00534 Sulfurol cpd:C04294 Syringaldehyde NA Thiamine monophosphate cpd:C01081 Tryptophan cpd:C00078 Tyrosine cpd:C00082 Vanillin cpd:C00755 Vitamin B3 cpd:C00253 Vitamin B5 cpd:C00864 Vitamin B6 cpd:C00314 Vitamin B9 cpd:C00504 Xanthine cpd:C00385 METABOLITES_END #END