#METABOLOMICS WORKBENCH jssacks_20220830_132254 DATATRACK_ID:3439 STUDY_ID:ST002292 ANALYSIS_ID:AN003745 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 SeQuant ZIC- pHILIC (150 x 2.1mm, 5um) CH:CHROMATOGRAPHY_TYPE HILIC #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 NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples LW_A LW_B LW_C NP_A NP_B NP_C 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:Lake Washington Extraction_Approach:CX-SPE | Location:Lake Washington Extraction_Approach:CX-SPE | Location:Lake Washington Extraction_Approach:CX-SPE | Location:North Pacific Extraction_Approach:CX-SPE | Location:North Pacific Extraction_Approach:CX-SPE | Location:North Pacific 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 2-Hydroxy-4-(methylthio)butyric_acid 8127787.0708 8718126.9205 11265859.0303 11740865.9099 9226035.6737 11856709.0295 3-Sulfopyruvic Acid 420871.0000 492507.0000 415731.0000 434580.0000 264526.0000 222432.0000 4-Hydroxyisoleucine 1079027.1269 866908.8650 1120315.7052 996959.6520 1007526.7824 1066282.5793 Acetylglutamic acid 5412685.0000 5921881.0000 5456825.0000 5889196.0000 5116136.0000 1956340.0000 Acetylglycine 3515334.0000 4082742.0000 2542140.0000 4221040.0000 4023988.0000 4081362.0000 Aconitic Acid 15787316.0000 12394864.0000 10492650.0000 13997118.0000 11949436.0000 9083870.0000 647364.0000 420542.0000 458857.0000 388217.0000 110242.0000 460964.0000 Allopurinol 5078975.3296 6026991.6049 4831521.6944 5046361.5223 5251868.4262 5779115.8598 Amino Propanesulfonic Acid 2479069.0000 2408201.0000 2494745.0000 2597926.0000 2711006.0000 2525562.0000 AMP 1173652.0000 1106505.0000 1172197.0000 962880.0000 889480.0000 804950.0000 Anserine 2396291.6594 2059540.3976 1958324.9838 1619303.7173 2772169.8396 2053762.4964 Butyrylglycine 5326615.0000 5235258.0000 5258417.0000 5795426.0000 7047344.0000 7437714.0000 cAMP 11019088.0943 9650405.0115 12322848.0620 12174624.1133 10360699.2820 11364935.6539 Carnosine 4192478.0000 4461230.0000 4717396.0000 4192886.0000 4552542.0000 4579276.0000 cGMP 3124706.6693 2994735.3636 2986534.3985 3030638.6978 3244051.1956 3021302.9721 Cyclic diGMP 381848.0000 269121.0000 240445.0000 Cysteic Acid 667787.0000 579676.0000 1678581.0000 1695498.0000 1623742.0000 1594063.0000 1468908.0000 1430752.0000 1602316.0000 Cysteinesulfinic acid 447185.0000 403914.0000 478253.0000 316414.0000 249482.0000 440274.0000 DHPS 17277308.0000 15551170.0000 16930946.0000 17789808.0000 18601168.0000 18797114.0000 Ethanesulfonic Acid 14116526.0000 14375641.0000 13908760.0000 14420304.0000 14742160.0000 15638154.0000 Ethyl Dihydroxybenzoate 3981318.0000 4092031.0000 4130384.0000 3817708.0000 3852158.0000 4393132.0000 Fructose 6 phosphate 1065201.0000 1112343.0000 924515.0000 1255712.0000 288018.0000 833576.0000 Glucose 1 phosphate 1065201.0000 1112343.0000 924515.0000 1255712.0000 288018.0000 833576.0000 Glucose 6 phosphate 659123.0000 657069.0000 484939.0000 525778.0000 486280.0000 272848.0000 Glutamylphenylalanine 8604247.6612 7830922.0647 9224601.5043 8816331.4665 7970870.8811 9101189.2161 glycerol 3 phosphate 836791.0000 717869.0000 599993.0000 730468.0000 730826.0000 439968.0000 GMP 384115.0000 359631.0000 42096.0000 161372.0000 164496.0000 Hypoxanthine 10305944.9622 9524107.7892 11294213.6595 12225207.8621 13925425.4262 13541010.7236 Imidazoleacrylic Acid 2002307.0000 3396132.0000 2850845.0000 2353998.0000 3970765.0000 2442636.0000 8807004.0000 7602981.0000 6581910.0000 9473536.0000 10291132.0000 7178012.0000 Indole 3 carbinol 456141.0000 450255.0000 741887.0000 Inosine 2850600.0000 3185111.0000 3387419.0000 2727778.0000 3075672.0000 3322714.0000 Isethionic Acid 1667081.0000 1520085.0000 2964617.0000 17631598.0000 17022848.0000 18432030.0000 17029816.0000 19341790.0000 18957888.0000 Ketoglutaric Acid 2594344.0000 2561578.0000 2417437.0000 660240.0000 480383.0000 1037340.0000 1050235.0000 643978.0000 959396.0000 Keto-?-(methylthio)butyric acid 1549294.7861 1671066.1180 1694600.7122 1559708.5426 1619552.8114 1569145.4772 Lipoic Acid 772536.0000 854574.0000 809245.0000 708588.0000 866594.0000 896368.0000 L-Pyroglutamic acid 22743982.0000 21517126.0000 21845260.0000 23372584.0000 22398254.0000 22077564.0000 6543074.0000 4351397.0000 8017369.0000 21668774.0000 21540382.0000 21622328.0000 Malic Acid 82324104.0000 82361872.0000 82619648.0000 MESNA (Sodium 2-mercaptoethanesulfonate) 7862480.0000 9878884.0000 65435656.0000 7686955.0000 7756097.0000 7047229.0000 8726156.0000 8636362.0000 8444854.0000 N-Acetylmuramic acid 3329782.0000 3835350.0000 3729039.0000 3050168.0000 3384950.0000 2966350.0000 NAD 177508.0000 151548.0000 75378.0000 Propanesulfonate 14627002.0000 14062148.0000 2503125.0000 13995480.0000 13977872.0000 13236262.0000 Propionylglycine 7502026.0000 7398067.0000 7278883.0000 7647096.0000 7748456.0000 8110154.0000 Resorcylic Acid 5627308.0000 3210771.0000 1926205.0000 8167926.0000 6549928.0000 6434494.0000 240010.0000 176803.0000 374452.0000 Riboflavin Monophosphate 318337.0000 149386.0000 55511.0000 291782.0000 106816.0000 Ribose 5 phosphate 163248.0000 71958.0000 Salicylic Acid 17596654.0000 16729924.0000 20090206.0000 19470084.0000 18253244.0000 22051814.0000 20939104.0000 19385680.0000 22137278.0000 704441.0000 643855.0000 936888.0000 641018.0000 647852.0000 670245.0000 Succinic semialdehyde 844038886.9361 939194819.9275 1004615386.3912 1057861336.6599 956606321.7842 1078392040.5434 Sulfanilic Acid 3988129.7676 3201418.8017 3870391.1641 4053699.3521 3939898.3006 4335864.9740 Sulfoacetic Acid 97611008.0000 92024408.0000 182221920.0000 104317176.0000 103158864.0000 180597008.0000 Sulfolactic Acid 1115412.0000 1135785.0000 2742509.0000 Taurine 59587.0000 Threonic acid 13276418.0000 11515300.0000 10194328.0000 15162984.0000 13221462.0000 11654642.0000 Thymidine 453682.0000 422003.0000 600927.0000 1113504.0000 421524.0000 1096356.0000 Thymine 14406773.0000 8324188.0000 13742464.0000 9988274.0000 23247168.0000 16057880.0000 UDP-glucosamine 587060.0000 618446.0000 573766.0000 493324.0000 474732.0000 348822.0000 UDP-glucose 660481.0000 491349.0000 507135.0000 496132.0000 533386.0000 371574.0000 Uracil 6516748.1742 5358868.8459 7563974.5288 7612915.0940 8874055.7573 6920269.9346 Uridine 1386485.0000 1585495.0000 1300808.0000 1187054.0000 1445226.0000 890454.0000 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 2-Hydroxy-4-(methylthio)butyric_acid NA 3-Sulfopyruvic Acid cpd:C05528 4-Hydroxyisoleucine cpd:C20802 Acetylglutamic acid cpd:C00624 Acetylglycine NA Aconitic Acid cpd:C00417 Allopurinol NA Amino Propanesulfonic Acid NA AMP cpd:C00020 Anserine cpd:C01262 Butyrylglycine NA cAMP cpd:C00575 Carnosine cpd:C00386 cGMP cpd:C00942 Cyclic diGMP cpd:C16463 Cysteic Acid cpd:C00506 Cysteinesulfinic acid cpd:C00606 DHPS cpd:C19675 Ethanesulfonic Acid NA Ethyl Dihydroxybenzoate NA Fructose 6 phosphate cpd:C00085 Glucose 1 phosphate cpd:C00103 Glucose 6 phosphate cpd:C00092 Glutamylphenylalanine NA glycerol 3 phosphate cpd:C00093 GMP cpd:C00144 Hypoxanthine cpd:C00262 Imidazoleacrylic Acid cpd:C00785 Indole 3 carbinol NA Inosine cpd:C00294 Isethionic Acid cpd:C05123 Ketoglutaric Acid cpd:C00026 Keto-?-(methylthio)butyric acid NA Lipoic Acid cpd:C00725 L-Pyroglutamic acid cpd:C01879 Malic Acid cpd:C00149 MESNA (Sodium 2-mercaptoethanesulfonate) NA N-Acetylmuramic acid cpd:C02713 NAD cpd:C00003 Propanesulfonate NA Propionylglycine NA Resorcylic Acid cpd:C00628 Riboflavin Monophosphate cpd:C00061 Ribose 5 phosphate cpd:C00117 Salicylic Acid cpd:C00805 Succinic semialdehyde cpd:C00232 Sulfanilic Acid cpd:C06335 Sulfoacetic Acid cpd:C14179 Sulfolactic Acid cpd:C11537 Taurine cpd:C00245 Threonic acid NA Thymidine cpd:C00214 Thymine cpd:C00178 UDP-glucosamine cpd:C00043 UDP-glucose cpd:C00029 Uracil cpd:C00106 Uridine cpd:C00299 METABOLITES_END #END