#METABOLOMICS WORKBENCH katherine_heal_20251107_135300 DATATRACK_ID:6638 STUDY_ID:ST004354 ANALYSIS_ID:AN007269 PROJECT_ID:PR002738
VERSION             	1
CREATED_ON             	November 11, 2025, 9:36 am
#PROJECT
PR:PROJECT_TITLE                 	Conserved pathway for homarine catabolism in environmental bacteria
PR:PROJECT_SUMMARY               	Homarine (N-methylpicolinic acid) is a ubiquitous marine metabolite produced by
PR:PROJECT_SUMMARY               	phytoplankton and noted for its bioactivity in marine animals, yet its microbial
PR:PROJECT_SUMMARY               	degradation pathways are uncharacterized. Here, we identify a conserved operon
PR:PROJECT_SUMMARY               	(homABCDER) that mediates homarine catabolism in bacteria using comparative
PR:PROJECT_SUMMARY               	transcriptomics, mutagenesis, and targeted knockouts. Phylogenetic and genomic
PR:PROJECT_SUMMARY               	analyses show this operon distributed across abundant bacterial clades,
PR:PROJECT_SUMMARY               	including coastal copiotrophs (e.g., Rhodobacterales) and open-ocean oligotrophs
PR:PROJECT_SUMMARY               	(e.g., SAR11, SAR116). High-resolution mass spectrometry revealed
PR:PROJECT_SUMMARY               	N-methylglutamic acid and glutamic acid as key metabolic products of homarine in
PR:PROJECT_SUMMARY               	both model and natural systems, with N-methylglutamate dehydrogenase catalyzing
PR:PROJECT_SUMMARY               	their conversion. Metatranscriptomics showed responsive and in situ expression
PR:PROJECT_SUMMARY               	of hom genes aligned with homarine availability. These findings uncover the
PR:PROJECT_SUMMARY               	genetic and metabolic basis of homarine degradation, establish its ecological
PR:PROJECT_SUMMARY               	relevance, and highlight homarine as a versatile growth substrate that feeds
PR:PROJECT_SUMMARY               	into central metabolism via glutamic acid in diverse marine bacteria.
PR:INSTITUTE                     	University of Washington, School of Oceanography
PR:LAST_NAME                     	Heal
PR:FIRST_NAME                    	Katherine
PR:ADDRESS                       	1501 NE Boat Street, Marine Science Building, Room G, Seattle
PR:EMAIL                         	katherine.heal@pnnl.gov
PR:PHONE                         	612-616-4840
PR:PUBLICATIONS                  	DOI 10.21203/rs.3.rs-7359689/v1
#STUDY
ST:STUDY_TITLE                   	Homarine catabolism: Isotope-tracing metabolomics experiments for cruise RC104
ST:STUDY_SUMMARY                 	Stable-isotope probing was performed to track homarine degradation products in
ST:STUDY_SUMMARY                 	natural marine microbial communities from three locations (Figure 2D of DOI
ST:STUDY_SUMMARY                 	10.21203/rs.3.rs-7359689/v1). Seawater incubated with isotopically-labeled
ST:STUDY_SUMMARY                 	13C715N-homarine was analyzed for the compounds enriched in our model organisms
ST:STUDY_SUMMARY                 	(see other studies within this project), with the isotopic labels.
ST:INSTITUTE                     	University of Washington, School of Oceanography
ST:LAST_NAME                     	Heal
ST:FIRST_NAME                    	Katherine
ST:ADDRESS                       	1501 NE Boat Street, Marine Science Building, Room G, Seattle
ST:EMAIL                         	katherine.heal@pnnl.gov
ST:PHONE                         	612-616-4840
ST:STUDY_COMMENTS                	Part of project 6620
ST:PUBLICATIONS                  	DOI 10.21203/rs.3.rs-7359689/v1
#SUBJECT
SU:SUBJECT_TYPE                  	Water sample
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_Con_A_posDDA	Sample source:Filtered Cells | treatment:Control | timepoint:0 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_Con_A_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_Con_B_posDDA	Sample source:Filtered Cells | treatment:Control | timepoint:0 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_Con_B_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_Con_C_posDDA	Sample source:Filtered Cells | treatment:Control | timepoint:0 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_Con_C_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T0_A_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:0 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T0_A_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T0_B_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:0 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T0_B_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T0_C_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:0 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T0_C_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T6_A_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:6 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T6_A_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T6_B_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:6 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T6_B_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T6_C_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:6 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T6_C_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T12_A_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:12 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T12_A_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T12_B_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:12 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T12_B_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T12_C_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:12 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T12_C_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T24_A_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:24 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T24_A_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T24_B_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:24 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T24_B_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T24_C_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:24 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T24_C_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T48_A_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:48 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T48_A_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T48_B_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:48 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T48_B_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T48_C_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:48 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T48_C_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T96_A_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:96 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T96_A_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T96_B_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:96 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T96_B_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T96_C_posDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:96 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T96_C_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_Con_A_negDDA	Sample source:Filtered Cells | treatment:Control | timepoint:0 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_Con_A_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_Con_B_negDDA	Sample source:Filtered Cells | treatment:Control | timepoint:0 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_Con_B_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_Con_C_negDDA	Sample source:Filtered Cells | treatment:Control | timepoint:0 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_Con_C_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T0_A_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:0 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T0_A_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T0_B_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:0 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T0_B_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T0_C_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:0 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T0_C_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T6_A_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:6 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T6_A_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T6_B_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:6 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T6_B_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T6_C_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:6 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T6_C_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T12_A_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:12 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T12_A_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T12_B_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:12 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T12_B_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T12_C_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:12 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T12_C_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T24_A_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:24 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T24_A_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T24_B_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:24 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T24_B_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T24_C_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:24 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T24_C_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T48_A_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:48 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T48_A_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T48_B_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:48 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T48_B_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T48_C_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:48 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T48_C_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T96_A_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:96 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T96_A_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T96_B_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:96 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T96_B_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Smp_RC104_HomFate_T96_C_negDDA	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:96 | lat:48.15104 | lon:-123.00699	date=20230901; RAW_FILE_NAME(raw_file_name)=240926_Smp_RC104_HomFate_T96_C_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Blk_RC104_HomFate_T6_Blk_A_negDDA	Sample source:Blank | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Blk_RC104_HomFate_T6_Blk_A_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Blk_RC104_HomFate_T6_Blk_B_negDDA	Sample source:Blank | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Blk_RC104_HomFate_T6_Blk_B_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Blk_RC104_HomFate_T6_Blk_C_negDDA	Sample source:Blank | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Blk_RC104_HomFate_T6_Blk_C_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Poo_RC104_HomFate_Full1_negDDA	Sample source:QC | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Poo_RC104_HomFate_Full1_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Poo_RC104_HomFate_Full2_negDDA	Sample source:QC | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Poo_RC104_HomFate_Full2_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Poo_RC104_HomFate_Full3_negDDA	Sample source:QC | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Poo_RC104_HomFate_Full3_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Poo_RC104_HomFate_Full4_negDDA	Sample source:QC | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Poo_RC104_HomFate_Full4_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Poo_RC104_HomFate_Full5_negDDA	Sample source:QC | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Poo_RC104_HomFate_Full5_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Std_4uMStdsMix1InH2O-1_negDDA	Sample source:Standards | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Std_4uMStdsMix1InH2O-1_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Std_4uMStdsMix1InH2O-2_negDDA	Sample source:Standards | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Std_4uMStdsMix1InH2O-2_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Std_4uMStdsMix2InH2O-1_negDDA	Sample source:Standards | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Std_4uMStdsMix2InH2O-1_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Std_4uMStdsMix2InH2O-2_negDDA	Sample source:Standards | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Std_4uMStdsMix2InH2O-2_negDDA.mzML; ms_type=negative
SUBJECT_SAMPLE_FACTORS           	-	240926_Blk_RC104_HomFate_T6_Blk_A_posDDA	Sample source:Blank | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Blk_RC104_HomFate_T6_Blk_A_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Blk_RC104_HomFate_T6_Blk_B_posDDA	Sample source:Blank | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Blk_RC104_HomFate_T6_Blk_B_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Blk_RC104_HomFate_T6_Blk_C_posDDA	Sample source:Blank | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Blk_RC104_HomFate_T6_Blk_C_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Poo_RC104_HomFate_Full1_posDDA	Sample source:QC | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Poo_RC104_HomFate_Full1_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Poo_RC104_HomFate_Full2_posDDA	Sample source:QC | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Poo_RC104_HomFate_Full2_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Poo_RC104_HomFate_Full3_posDDA	Sample source:QC | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Poo_RC104_HomFate_Full3_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Poo_RC104_HomFate_Full4_posDDA	Sample source:QC | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Poo_RC104_HomFate_Full4_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Poo_RC104_HomFate_Full5_posDDA	Sample source:QC | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Poo_RC104_HomFate_Full5_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Std_4uMStdsMix1InH2O-1_posDDA	Sample source:Standards | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Std_4uMStdsMix1InH2O-1_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Std_4uMStdsMix1InH2O-2_posDDA	Sample source:Standards | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Std_4uMStdsMix1InH2O-2_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Std_4uMStdsMix2InH2O-1_posDDA	Sample source:Standards | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Std_4uMStdsMix2InH2O-1_posDDA.mzML; ms_type=positive
SUBJECT_SAMPLE_FACTORS           	-	240926_Std_4uMStdsMix2InH2O-2_posDDA	Sample source:Standards | treatment:N/A | timepoint:N/A | lat:N/A | lon:N/A	date=N/A; RAW_FILE_NAME(raw_file_name)=240926_Std_4uMStdsMix2InH2O-2_posDDA.mzML; ms_type=positive
#COLLECTION
CO:COLLECTION_SUMMARY            	Experiments using 13C7,15N-homarine-homarine were performed on research cruise
CO:COLLECTION_SUMMARY            	RC104 in the Summer of 2023 at two different stations in Puget Sound (described
CO:COLLECTION_SUMMARY            	in Table S8 and displayed in Figure 2 of 10.21203/rs.3.rs-7359689/v1). The
CO:COLLECTION_SUMMARY            	preparation of the 13C7-15N-labeled homarine is described in detail in
CO:COLLECTION_SUMMARY            	10.21203/rs.3.rs-7359689/v1. Seawater was collected through a trace metal clean
CO:COLLECTION_SUMMARY            	stayfish system suspended at a depth of 8 m prefiltered through 100 µm nylon
CO:COLLECTION_SUMMARY            	mesh. Triplicate samples were collected into acid washed 2 L polycarbonate
CO:COLLECTION_SUMMARY            	bottles, spiked with 90 nM of 13C7-15N-labeled homarine, and incubated in
CO:COLLECTION_SUMMARY            	temperature and light-controlled incubators for 5 different timepoints (6, 12,
CO:COLLECTION_SUMMARY            	24, 48 and 96 hours). Triplicates of spiked and unspiked samples were filtered
CO:COLLECTION_SUMMARY            	as quickly as possible, no more than 30 minutes (T0, and unamended control
CO:COLLECTION_SUMMARY            	samples, respectively). All particulate samples (4 L) were collected using
CO:COLLECTION_SUMMARY            	peristaltic pumps onto Durapore® 0.22 μm, 47 mm, hydrophilic PVDF membrane
CO:COLLECTION_SUMMARY            	filters, flash frozen in liquid nitrogen, and stored at -80°C.
CO:SAMPLE_TYPE                   	Marine particulate matter
#TREATMENT
TR:TREATMENT_SUMMARY             	Triplicate samples were collected into acid washed 2 L polycarbonate bottles,
TR:TREATMENT_SUMMARY             	spiked with 90 nM of 13C7-15N-labeled homarine, and incubated in temperature and
TR:TREATMENT_SUMMARY             	light-controlled incubators for 5 different timepoints (6, 12, 24, 48 and 96
TR:TREATMENT_SUMMARY             	hours). Triplicates of spiked and unspiked samples were filtered as quickly as
TR:TREATMENT_SUMMARY             	possible, no more than 30 minutes (T0, and unamended control samples,
TR:TREATMENT_SUMMARY             	respectively).
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	For particulate metabolomics, cell pellets were extracted using a combination of
SP:SAMPLEPREP_SUMMARY            	mechanical and chemical disruption techniques as described in previous work
SP:SAMPLEPREP_SUMMARY            	(Boysen et al 2018). Metabolites from the supernatant were extracted using a
SP:SAMPLEPREP_SUMMARY            	cation-exchange-based solid phase extraction technique as described previously
SP:SAMPLEPREP_SUMMARY            	(Sacks et al 2022), with 1 mL of supernatant diluted into 10 mL of HPLC grade
SP:SAMPLEPREP_SUMMARY            	water. To prevent confusion using the isotope labels, we used a subset of
SP:SAMPLEPREP_SUMMARY            	isotopically-labeled internal standards, as reported in Table S17 of
SP:SAMPLEPREP_SUMMARY            	https://doi.org/10.21203/rs.3.rs-7359689/v1.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	For HILIC chromatography, a SeQuant ZIC-pHILIC column (5 um particle size, 2.1
CH:CHROMATOGRAPHY_SUMMARY        	mm x 150 mm, from Millipore) was used with 10 mM ammonium carbonate in 85:15
CH:CHROMATOGRAPHY_SUMMARY        	acetonitrile to water (Solvent A) and 10 mM ammonium carbonate in 85:15 water to
CH:CHROMATOGRAPHY_SUMMARY        	acetonitrile (Solvent B) at a flow rate of 0.15 mL/min. This column was compared
CH:CHROMATOGRAPHY_SUMMARY        	with a Waters UPLC BEH amide and a Millipore cHILIC column; the pHILIC showed
CH:CHROMATOGRAPHY_SUMMARY        	superior reproducibility and peak shapes. The column was held at 100% A for 2
CH:CHROMATOGRAPHY_SUMMARY        	minutes, ramped to 64% B over 18 minutes, ramped to 100% B over 1 minute, held
CH:CHROMATOGRAPHY_SUMMARY        	at 100% B for 5 minutes, and equilibrated at 100% A for 25 minutes (50 minutes
CH:CHROMATOGRAPHY_SUMMARY        	total). The column was maintained at 30 C. The injection volume was 2 µL for
CH:CHROMATOGRAPHY_SUMMARY        	samples and standard mixes. When starting a batch, the column was equilibrated
CH:CHROMATOGRAPHY_SUMMARY        	at the starting conditions for at least 30 minutes. To improve the performance
CH:CHROMATOGRAPHY_SUMMARY        	of the HILIC column, we maintained the same injection volume, kept the
CH:CHROMATOGRAPHY_SUMMARY        	instrument running water blanks between samples as necessary, and injected
CH:CHROMATOGRAPHY_SUMMARY        	standards in a representative matrix in addition to standards in water. After
CH:CHROMATOGRAPHY_SUMMARY        	each batch, the column was flushed with 10 mM ammonium carbonate in 85:15 water
CH:CHROMATOGRAPHY_SUMMARY        	to acetonitrile for 20 to 30 minutes.
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Waters Acquity I-Class
CH:COLUMN_NAME                   	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)
CH:SOLVENT_A                     	85% acetonitrile/15% water; 10 mM ammonium carbonate
CH:SOLVENT_B                     	15% acetonitrile/85% water; 10 mM ammonium carbonate
CH:FLOW_GRADIENT                 	The column was held at 100% A for 2 minutes, ramped to 64% B over 18 minutes,
CH:FLOW_GRADIENT                 	ramped to 100% B over 1 minute, held at 100% B for 5 minutes, and equilibrated
CH:FLOW_GRADIENT                 	at 100% A for 25 minutes
CH:FLOW_RATE                     	0.15 mL/min
CH:COLUMN_TEMPERATURE            	30
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive HF hybrid Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	MS acquisition Comments: Polarity switching was used with a scan range of 60 to
MS:MS_COMMENTS                   	900 m/z and a resolution of 60,000. MS parameters were as follows: capillary
MS:MS_COMMENTS                   	temperature was 320 ∞C, the H-ESI spray voltage was 3.3 kV, and the auxiliary
MS:MS_COMMENTS                   	gas heater temperature was 100 ∞C. The S-lens RF level was 65. Sheath gas,
MS:MS_COMMENTS                   	auxiliary gas, and sweep gas flow rates were maintained at 16, 3, and 1,
MS:MS_COMMENTS                   	respectively.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	normalized peak area
MS_METABOLITE_DATA_START
Samples	240926_Smp_RC104_HomFate_Con_A_posDDA	240926_Smp_RC104_HomFate_Con_B_posDDA	240926_Smp_RC104_HomFate_Con_C_posDDA	240926_Smp_RC104_HomFate_T0_A_posDDA	240926_Smp_RC104_HomFate_T0_B_posDDA	240926_Smp_RC104_HomFate_T0_C_posDDA	240926_Smp_RC104_HomFate_T6_A_posDDA	240926_Smp_RC104_HomFate_T6_B_posDDA	240926_Smp_RC104_HomFate_T6_C_posDDA	240926_Smp_RC104_HomFate_T12_A_posDDA	240926_Smp_RC104_HomFate_T12_B_posDDA	240926_Smp_RC104_HomFate_T12_C_posDDA	240926_Smp_RC104_HomFate_T24_A_posDDA	240926_Smp_RC104_HomFate_T24_B_posDDA	240926_Smp_RC104_HomFate_T24_C_posDDA	240926_Smp_RC104_HomFate_T48_A_posDDA	240926_Smp_RC104_HomFate_T48_B_posDDA	240926_Smp_RC104_HomFate_T48_C_posDDA	240926_Smp_RC104_HomFate_T96_A_posDDA	240926_Smp_RC104_HomFate_T96_B_posDDA	240926_Smp_RC104_HomFate_T96_C_posDDA
Factors	Sample source:Filtered Cells | treatment:Control | timepoint:0 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Control | timepoint:0 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Control | timepoint:0 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:0 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:0 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:0 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:6 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:6 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:6 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:12 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:12 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:12 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:24 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:24 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:24 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:48 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:48 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:48 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:96 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:96 | lat:48.15104 | lon:-123.00699	Sample source:Filtered Cells | treatment:Homarine (13C7,15N-labelled) | timepoint:96 | lat:48.15104 | lon:-123.00699
Adenine	828414464	295846144	406546592	411181888	968990144	243125216	269796992	48041616	99898000	342686688	152346928	565503040	804003968	270455936	1884706048	1062670464	1443562496	2628069120	2697674752	2232317952	5164254208
Adenosine	166473088	142824688	81013544	74691080	99442192	71067720	68458344	8199271	15688428	101402856	82044360	145093728	193966288	42568336	280313888	202571216	470253344	260249616	156656752	154836336	253869440
L-Arginine	31264798	22471122	14056305	21531446	37040824	18408434	49665096	15598582	28618906	36408972	17130984	105804864	44072032	46631720	275810368	44804652	97982080	172885952	97966048	142818224	125349104
L-Asparagine	3241724	2920008	2497653	3706032	4967825	3412938	6638667	4216379	5751145	4888445	2569660	7262101	4664782	5724035	15593819	4966474	10156087	12669571	13280271	20498868	17753260
Citrulline	3562698	2786515	2128497	5609863	4523260	3818637	13640088	15725640	8919207	6212647	3107490	6998211	3994547	5209240	10352401	6753040	9243076	10622665	8590296	5925123	10862579
Cytosine	94144488	39746612	57992500	38399340	52351268	33845668	57734380	26608962	41510296	32997486	51795176	82841712	118419792	50175928	251967664	225712880	126251832	267473248	92899440	136378864	148913600
Deoxyadenosine	56932832	17484288	16346839	9762271	26827448	5293435	9670068	1250989	1258495	9840207	7299677	21880432	60727400	15190359	269789632	63392392	107223280	286240288	52479824	151710304	127509800
L-Glutamic acid	231116400	171324608	127739240	185533328	266533488	153850416	229827280	147431552	90356488	245893168	164541744	327687040	281486112	339122560	742921088	225579328	569914112	597241088	710672448	956347776	1000910848
Glutathione disulfide																					
Guanosine	2801434.7412675135	2254462.737257274	1254295.086082665	738738.7089111267	1859563.2631587344	1310256.7971445282	2606546.805902405	737988.7319066369	1083700.268	1460835.258125292	1499847.6570836152	5002509.266	4423604.762622863	3347356.3324783314	29246784.25245206	4333355.556	12447904.8	9172528.345631478	3531177.2605298804	10649278.539247513	7962932.657
L-Homoserine	6336433	4197646	4090230	5187977	7496378	4773560	19077056	5016232	16969282	12660430	6776393	21575730	4776402	4949253	13668226		7338758	10076362		5819197	4371571
L-Lysine	4924292	4428010	3079624	4666290	5799325	3851332	8787672	5135056	6042681	5129846	3041199	12782263	7615735	5588177	29647472	8964242	15512556	24685404	13048796	22599632	20585572
L-Phenylalanine	48287149.34636332	35222584.14	26417942.34156816	37042449.53866895	69904447.07684037	38826791.14619943	106553289.58630218	56203779.549263254	94731002.24805328	70777948.66747315	35936007.110181384	114557912.99340454	77842484.37619577	38583657.77991144	320139323.54412407	92179739.84801202	225884007.47298676	395411757.37576973	125332121.3809266	340222163.8	253885854.97243777
L-Proline	329975168	301233632	216131696	401994240	455411264	357139200	525398848	316644128	474747456	397994496	234771024	647378752	424999648	458523456	970851648	460387072	778554112	1034918208	1437732224	1742752640	1610709888
L-Serine	3360378	3272496	3200228	10361907	5572320	5938972	19043234	29328824	18260200	7086851	3502884	7512285	5589081	4754384	13992740	11156934	15402212	22258358	17292324	18214878	24793656
L-Tryptophan	2269853	2481216	1421996	1940047	3629708	1225397	4537787	994926	811011	3095877	1692857	5816447	4112184	1758581	16297657	3535653	11049527	15407719	3509991	9277409	5834805
L-Tyrosine	17004986	16171970	8928605	18132440	21175726	11825397	16610814	12085627	13608955	10511611	6508503	20597748	17874864	11825271	50900464	21429972	52630184	65196152	23295852	46389096	34919560
glutamic_acid_13C_15N				2036748	4358174	2548540	42649072	21933872	20375994	34031348	39159580	56446748	78077920	102402312	73534056	19118380	21508956	18295256		88714	53003
homarine_13C_15N	468111	1656955	138020	193787232	385618400	378575008	5037727232	2450482688	5752175616	5171395072	4343264256	7596877824	11860980736	11783908352	12899290112	12286804992	16738979840	11915720704	5323034624	4960471552	4711018496
Homarine	3334265600	3186465280	1925811200	2935372544	4581720064	4649757184	4438765568	1672967424	4159005440	3710355456	2226615552	5852463616	6030387200	4711577088	8244518912	5865862656	10939744256	9317761024	16247639040	21826738176	22711207936
n-methyl glutamic acid_13C_15N				7936199	14628324	10014775	68711344	40002576	32658574	79071208	64007064	95112640	129747792	185618160	247868880	61898968	136128832	81336024	2036118	5461728	3181033
N-Methyl-L-glutamic acid	22891796	23445922	14175991	24828420	36718128	22778976	20235522	11306632	14444931	27127190	7205047	25348784	15847087	22162646	59112880	11727931	51062928	39438344	13627127	20577740	11403732
n-methyl glutamine_13C_15N							261396	285002	391441	788233	568550	1019863	3942288	4947222	4424420	1563048	1209343	626060			
n-methyl glutamine	10923832	11560118	4103614	6726185		12285374			4760998		633951								24945624		
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	molecule_id	stable_isotope_version	retention_time	mz
Adenine	Adenine		5.25	136.0623
Adenosine	Adenosine		5.04	268.1046
L-Arginine	Arginine		19.78	175.1195
L-Asparagine	Asparagine		11.66	133.0613
Citrulline	Citrulline		12.43	176.1035
Cytosine	Cytosine		7.57	112.0511
Deoxyadenosine	Deoxyadenosine		3.85	252.1097
L-Glutamic acid	Glutamic acid		12.18	148.061
Glutathione disulfide	Glutathione disulfide		14.71	613.1598
Guanosine	Guanosine		9.65	284.0995
L-Homoserine	Homoserine		11.58	120.0661
L-Lysine	Lysine		18.9	147.1134
L-Phenylalanine	Phenylalanine		5.94	166.0868
L-Proline	Proline		9.58	116.0712
L-Serine	Serine		12	106.0504
L-Tryptophan	Tryptophan		8.19	205.0977
L-Tyrosine	Tyrosine		9.83	182.0817
glutamic_acid_13C_15N	Glutamic acid	13C6, 15N1	12.18	154.0748
homarine_13C_15N	Homarine	13C6, 15N1	6.25	146.0754
Homarine	Homarine		6.25	138.0555
n-methyl glutamic acid_13C_15N	n-Methyl Glutamic Acid	13C6, 15N1	11.06	169.0931
N-Methyl-L-glutamic acid	n-Methyl Glutamic Acid		10.65	162.0766
n-methyl glutamine_13C_15N	n-Methyl Glutamine	13C6, 15N1	10.3	168.1093
n-methyl glutamine	n-Methyl Glutamine		9.85	161.0921
METABOLITES_END
#END