#METABOLOMICS WORKBENCH katherine_heal_20251107_135300 DATATRACK_ID:6638 STUDY_ID:ST004354 ANALYSIS_ID:AN007270 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-X Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
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_negDDA	240926_Smp_RC104_HomFate_Con_B_negDDA	240926_Smp_RC104_HomFate_Con_C_negDDA	240926_Smp_RC104_HomFate_T0_A_negDDA	240926_Smp_RC104_HomFate_T0_B_negDDA	240926_Smp_RC104_HomFate_T0_C_negDDA	240926_Smp_RC104_HomFate_T6_A_negDDA	240926_Smp_RC104_HomFate_T6_B_negDDA	240926_Smp_RC104_HomFate_T6_C_negDDA	240926_Smp_RC104_HomFate_T12_A_negDDA	240926_Smp_RC104_HomFate_T12_B_negDDA	240926_Smp_RC104_HomFate_T12_C_negDDA	240926_Smp_RC104_HomFate_T24_A_negDDA	240926_Smp_RC104_HomFate_T24_B_negDDA	240926_Smp_RC104_HomFate_T24_C_negDDA	240926_Smp_RC104_HomFate_T48_A_negDDA	240926_Smp_RC104_HomFate_T48_B_negDDA	240926_Smp_RC104_HomFate_T48_C_negDDA	240926_Smp_RC104_HomFate_T96_A_negDDA	240926_Smp_RC104_HomFate_T96_B_negDDA	240926_Smp_RC104_HomFate_T96_C_negDDA
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
L-Cysteic acid	5581100	3173321	2126887	3693699	4368997	2594727	2428381	1571229	1911049	3140132	1719387	4267654	4492363	4114836	11466377	4269419	8530205	8741777	14691083	17350050	18812582
UDP-N-acetylglucosamine	120952	183604	61747	248814	324969	298272	401820	155732	209903	509821	146054	747754	273549	558037	794027	78650	831804	567163	2466944	2749468	3227518
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	molecule_id	retention_time	mz	mz
L-Cysteic acid	Cysteic acid	12.15	167.9967	167.9967
UDP-N-acetylglucosamine	UDP-N-acetylglucosamine	12.75	606.0738	606.0738
METABOLITES_END
#END