#METABOLOMICS WORKBENCH TruxalCarlson_20200428_130830 DATATRACK_ID:1996 STUDY_ID:ST001372 ANALYSIS_ID:AN002292 PROJECT_ID:PR000938
VERSION             	1
CREATED_ON             	May 5, 2020, 1:21 pm
#PROJECT
PR:PROJECT_TITLE                 	Patterns in metabolite pools show that phytoplankton leave a taxon-specific
PR:PROJECT_TITLE                 	signature on particulate carbon: North Pacific Subtropical Gyre depth profile
PR:PROJECT_TYPE                  	Marine Metabolomics
PR:PROJECT_SUMMARY               	In the surface ocean, carbon is fixed by phytoplankton and respired by the
PR:PROJECT_SUMMARY               	entire marine community at an astonishingly high rate. At any point in time, the
PR:PROJECT_SUMMARY               	difference between these two processes yields a carbon pool in surface particles
PR:PROJECT_SUMMARY               	that is a combination of both freshly fixed and partially degraded material. On
PR:PROJECT_SUMMARY               	a molecular level, we have a limited knowledge of the small molecules, or
PR:PROJECT_SUMMARY               	metabolites, within this pool. Specific metabolites have been shown to be
PR:PROJECT_SUMMARY               	responsible for fueling respiration, maintaining organismal interactions, and
PR:PROJECT_SUMMARY               	transferring energy throughout the microbial community. Metabolomics, or the
PR:PROJECT_SUMMARY               	direct observation and quantification of the small molecules that are the result
PR:PROJECT_SUMMARY               	of cellular activity, provides an important lens through which we can begin to
PR:PROJECT_SUMMARY               	assess the standing stocks of small compounds that likely fuel a great deal of
PR:PROJECT_SUMMARY               	heterotrophic activity in the surface ocean. Here we describe community
PR:PROJECT_SUMMARY               	metabolomes of particulate material into the North Pacific Ocean and compare the
PR:PROJECT_SUMMARY               	metabolomes to a variety of phytoplankton grown in the lab. Using both targeted
PR:PROJECT_SUMMARY               	and untargeted metabolomics, we identify metabolites in the particulate carbon
PR:PROJECT_SUMMARY               	pool and explore their latitudinal and phylogenetic distributions. This analysis
PR:PROJECT_SUMMARY               	reveals several compounds that have not been previously recognized as abundant
PR:PROJECT_SUMMARY               	components of the marine organic carbon pool. We found that the community
PR:PROJECT_SUMMARY               	metabolome showed distinct differences between the regimes that likely reflects
PR:PROJECT_SUMMARY               	the phytoplankton community present. The community metabolome in surface waters
PR:PROJECT_SUMMARY               	of the subtropical domain was remarkably consistent even when sampled weeks
PR:PROJECT_SUMMARY               	apart, while the northern regions showed a patichier and less reproducible
PR:PROJECT_SUMMARY               	community metabolome. Some individual compounds showed distinct patterns between
PR:PROJECT_SUMMARY               	oceanographic regimes, including homarine, an abundant molecule that can
PR:PROJECT_SUMMARY               	contribute up to 4% of the total particulate carbon pool in marine surface
PR:PROJECT_SUMMARY               	waters. Glutamic acid and glutamine showed opposite patterns in the
PR:PROJECT_SUMMARY               	oceanographic regimes, suggesting differences in community-level nitrogen
PR:PROJECT_SUMMARY               	assimilation in these different regimes. Overall, this study offers a new
PR:PROJECT_SUMMARY               	perspective into particulate carbon composition in oceanographic research,
PR:PROJECT_SUMMARY               	reveals important carbon pools that may fuel the microbial loop, and suggests an
PR:PROJECT_SUMMARY               	altered community-level nitrogen assimilation capacity over the North Pacific
PR:PROJECT_SUMMARY               	transition zone.
PR:INSTITUTE                     	University of Washington
PR:DEPARTMENT                    	School of Oceanography
PR:LABORATORY                    	Ingalls Lab
PR:LAST_NAME                     	Heal
PR:FIRST_NAME                    	Katherine
PR:ADDRESS                       	1501 NE Boat Street, Marine Science Building, Room G, Seattle, WA, 98195, USA
PR:EMAIL                         	kheal@uw.edu
PR:PHONE                         	612-616-4840
#STUDY
ST:STUDY_TITLE                   	Patterns in metabolite pools show that phytoplankton leave a taxon-specific
ST:STUDY_TITLE                   	signature on particulate carbon: North Pacific Subtropical Gyre depth profile
ST:STUDY_TYPE                    	Marine metabolomics depth profile
ST:STUDY_SUMMARY                 	In the surface ocean, carbon is fixed by phytoplankton and respired by the
ST:STUDY_SUMMARY                 	entire marine community at an astonishingly high rate. At any point in time, the
ST:STUDY_SUMMARY                 	difference between these two processes yields a carbon pool in surface particles
ST:STUDY_SUMMARY                 	that is a combination of both freshly fixed and partially degraded material. On
ST:STUDY_SUMMARY                 	a molecular level, we have a limited knowledge of the small molecules, or
ST:STUDY_SUMMARY                 	metabolites, within this pool. Specific metabolites have been shown to be
ST:STUDY_SUMMARY                 	responsible for fueling respiration, maintaining organismal interactions, and
ST:STUDY_SUMMARY                 	transferring energy throughout the microbial community. Metabolomics, or the
ST:STUDY_SUMMARY                 	direct observation and quantification of the small molecules that are the result
ST:STUDY_SUMMARY                 	of cellular activity, provides an important lens through which we can begin to
ST:STUDY_SUMMARY                 	assess the standing stocks of small compounds that likely fuel a great deal of
ST:STUDY_SUMMARY                 	heterotrophic activity in the surface ocean. Here we describe community
ST:STUDY_SUMMARY                 	metabolomes of particulate material into the North Pacific Ocean and compare the
ST:STUDY_SUMMARY                 	metabolomes to a variety of phytoplankton grown in the lab. Using both targeted
ST:STUDY_SUMMARY                 	and untargeted metabolomics, we identify metabolites in the particulate carbon
ST:STUDY_SUMMARY                 	pool and explore their latitudinal and phylogenetic distributions. This analysis
ST:STUDY_SUMMARY                 	reveals several compounds that have not been previously recognized as abundant
ST:STUDY_SUMMARY                 	components of the marine organic carbon pool. We found that the community
ST:STUDY_SUMMARY                 	metabolome showed distinct differences between the regimes that likely reflects
ST:STUDY_SUMMARY                 	the phytoplankton community present. The community metabolome in surface waters
ST:STUDY_SUMMARY                 	of the subtropical domain was remarkably consistent even when sampled weeks
ST:STUDY_SUMMARY                 	apart, while the northern regions showed a patichier and less reproducible
ST:STUDY_SUMMARY                 	community metabolome. Some individual compounds showed distinct patterns between
ST:STUDY_SUMMARY                 	oceanographic regimes, including homarine, an abundant molecule that can
ST:STUDY_SUMMARY                 	contribute up to 4% of the total particulate carbon pool in marine surface
ST:STUDY_SUMMARY                 	waters. Glutamic acid and glutamine showed opposite patterns in the
ST:STUDY_SUMMARY                 	oceanographic regimes, suggesting differences in community-level nitrogen
ST:STUDY_SUMMARY                 	assimilation in these different regimes. Overall, this study offers a new
ST:STUDY_SUMMARY                 	perspective into particulate carbon composition in oceanographic research,
ST:STUDY_SUMMARY                 	reveals important carbon pools that may fuel the microbial loop, and suggests an
ST:STUDY_SUMMARY                 	altered community-level nitrogen assimilation capacity over the North Pacific
ST:STUDY_SUMMARY                 	transition zone.
ST:INSTITUTE                     	University of Washington
ST:DEPARTMENT                    	School of Oceanography
ST:LABORATORY                    	Ingalls Lab
ST:LAST_NAME                     	Heal
ST:FIRST_NAME                    	Katherine
ST:ADDRESS                       	1501 NE Boat Street, Marine Science Building, Room G
ST:EMAIL                         	kheal@uw.edu
ST:PHONE                         	612-616-4840
#SUBJECT
SU:SUBJECT_TYPE                  	Other
SU:SUBJECT_SPECIES               	Natural mixed marine microbial community
#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           	-	FilterA	Depth_m:NA | Vol_L:NA	Latitude=NA; Longitude=NA; UTC=NA; Replicate=A; Type=Blk; RAW_FILE_NAME=170410_Blk_FilterBlk_A;170410_Blk_FilterBlk_A;170413_Blk_FilterBlk_A
SUBJECT_SAMPLE_FACTORS           	-	FilterB	Depth_m:NA | Vol_L:NA	Latitude=NA; Longitude=NA; UTC=NA; Replicate=B; Type=Blk; RAW_FILE_NAME=170410_Blk_FilterBlk_B;170410_Blk_FilterBlk_B;170413_Blk_FilterBlk_B
SUBJECT_SAMPLE_FACTORS           	-	KM1513-15m_A	Depth_m:15 | Vol_L:10.7	Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=A; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-15m_A;170410_Smp_KM1513-15m_A;170413_Smp_KM1513-15m_A
SUBJECT_SAMPLE_FACTORS           	-	KM1513-15m_B	Depth_m:15 | Vol_L:10.7	Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=B; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-15m_B;170410_Smp_KM1513-15m_B;170413_Smp_KM1513-15m_B
SUBJECT_SAMPLE_FACTORS           	-	KM1513-15m_C	Depth_m:15 | Vol_L:10.7	Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=C; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-15m_C;170410_Smp_KM1513-15m_C;170413_Smp_KM1513-15m_C
SUBJECT_SAMPLE_FACTORS           	-	KM1513-45m_A	Depth_m:45 | Vol_L:9	Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=A; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-45m_A;170410_Smp_KM1513-45m_A;170413_Smp_KM1513-45m_A
SUBJECT_SAMPLE_FACTORS           	-	KM1513-45m_B	Depth_m:45 | Vol_L:9	Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=B; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-45m_B;170410_Smp_KM1513-45m_B;170413_Smp_KM1513-45m_B
SUBJECT_SAMPLE_FACTORS           	-	KM1513-45m_C	Depth_m:45 | Vol_L:9	Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=C; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-45m_C;170410_Smp_KM1513-45m_C;170413_Smp_KM1513-45m_C
SUBJECT_SAMPLE_FACTORS           	-	KM1513-75m_A	Depth_m:75 | Vol_L:12.7	Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=A; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-75m_A;170410_Smp_KM1513-75m_A;170413_Smp_KM1513-75m_A
SUBJECT_SAMPLE_FACTORS           	-	KM1513-75m_B	Depth_m:75 | Vol_L:12.7	Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=B; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-75m_B;170410_Smp_KM1513-75m_B;170413_Smp_KM1513-75m_B
SUBJECT_SAMPLE_FACTORS           	-	KM1513-75m_C	Depth_m:75 | Vol_L:12.7	Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=C; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-75m_C;170410_Smp_KM1513-75m_C;170413_Smp_KM1513-75m_C
SUBJECT_SAMPLE_FACTORS           	-	KM1513-125m_A	Depth_m:125 | Vol_L:12.17	Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=A; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-125m_A;170410_Smp_KM1513-125m_A;170413_Smp_KM1513-125m_A
SUBJECT_SAMPLE_FACTORS           	-	KM1513-125m_B	Depth_m:125 | Vol_L:12.17	Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=B; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-125m_B;170410_Smp_KM1513-125m_B;170413_Smp_KM1513-125m_B
SUBJECT_SAMPLE_FACTORS           	-	KM1513-125m_C	Depth_m:125 | Vol_L:12.17	Latitude=24.5548; Longitude=-156.3298; UTC=2015-07-31T19;54;20; Replicate=C; Type=Smp; RAW_FILE_NAME=170410_Smp_KM1513-125m_C;170410_Smp_KM1513-125m_C;170413_Smp_KM1513-125m_C
SUBJECT_SAMPLE_FACTORS           	-	April11AqExtractsHalf_1	Depth_m:NA | Vol_L:NA	Latitude=NA; Longitude=NA; UTC=NA; Replicate=1; Type=Pool; RAW_FILE_NAME=170410_Poo_April11AqExtractsHalf_1;170410_Poo_April11AqExtractsHalf_1;170413_Poo_April11AqExtractsHalf_1
SUBJECT_SAMPLE_FACTORS           	-	April11AqExtractsHalf_2	Depth_m:NA | Vol_L:NA	Latitude=NA; Longitude=NA; UTC=NA; Replicate=2; Type=Pool; RAW_FILE_NAME=170410_Poo_April11AqExtractsHalf_2;170410_Poo_April11AqExtractsHalf_2;170413_Poo_April11AqExtractsHalf_2
SUBJECT_SAMPLE_FACTORS           	-	April11AqExtractsHalf_3	Depth_m:NA | Vol_L:NA	Latitude=NA; Longitude=NA; UTC=NA; Replicate=3; Type=Pool; RAW_FILE_NAME=170410_Poo_April11AqExtractsHalf_3;170410_Poo_April11AqExtractsHalf_3;170413_Poo_April11AqExtractsHalf_3
SUBJECT_SAMPLE_FACTORS           	-	April11AqExtractsFull_1	Depth_m:NA | Vol_L:NA	Latitude=NA; Longitude=NA; UTC=NA; Replicate=1; Type=Pool; RAW_FILE_NAME=170410_Poo_April11AqExtractsFull_1;170410_Poo_April11AqExtractsFull_1;170413_Poo_April11AqExtractsFull_1
SUBJECT_SAMPLE_FACTORS           	-	April11AqExtractsFull_2	Depth_m:NA | Vol_L:NA	Latitude=NA; Longitude=NA; UTC=NA; Replicate=2; Type=Pool; RAW_FILE_NAME=170410_Poo_April11AqExtractsFull_2;170410_Poo_April11AqExtractsFull_2;170413_Poo_April11AqExtractsFull_2
SUBJECT_SAMPLE_FACTORS           	-	April11AqExtractsFull_3	Depth_m:NA | Vol_L:NA	Latitude=NA; Longitude=NA; UTC=NA; Replicate=3; Type=Pool; RAW_FILE_NAME=170410_Poo_April11AqExtractsFull_3;170410_Poo_April11AqExtractsFull_3;170413_Poo_April11AqExtractsFull_3
#COLLECTION
CO:COLLECTION_SUMMARY            	Samples for particulate metabolites were collected from different water depths
CO:COLLECTION_SUMMARY            	by niskin bottles attached to a conductivity, temperature, depth array (CTD).
CO:COLLECTION_SUMMARY            	Metabolite samples were filtered onto 142 mm 0.2 µm Durapore filters using
CO:COLLECTION_SUMMARY            	peristaltic, polycarbonate filter holders, and Masterflex PharMed BPT tubing
CO:COLLECTION_SUMMARY            	(Cole-Parmer). Filters were quenched in liquid nitrogen immediately after
CO:COLLECTION_SUMMARY            	filtration and stored at -80°C until extraction. Each sample was 30-40 L
CO:COLLECTION_SUMMARY            	filtered seawater; each filter was split into three equal parts for triplicate
CO:COLLECTION_SUMMARY            	extractions. A blank PTFE filter was extracted alongside samples as a
CO:COLLECTION_SUMMARY            	methodological blank.
CO:SAMPLE_TYPE                   	Suspended Marine Particulate Matter
CO:STORAGE_CONDITIONS            	Described in summary
#TREATMENT
TR:TREATMENT_SUMMARY             	No treatment - this was a study of the natural marine microbial population at
TR:TREATMENT_SUMMARY             	different depths in the North Pacific Subtropical Gyre.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Each sample was extracted using a modified Bligh-Dyer extraction. Briefly,
SP:SAMPLEPREP_SUMMARY            	filters were cut up and put into 15 mL teflon centrifuge tubes containing a
SP:SAMPLEPREP_SUMMARY            	mixture of 100 µm and 400 µm silica beads. Heavy isotope-labeled internal
SP:SAMPLEPREP_SUMMARY            	standards were added along with ~2 mL of cold aqueous solvent (50:50
SP:SAMPLEPREP_SUMMARY            	methanol:water) and ~3 mL of cold organic solvent (dichloromethane). The samples
SP:SAMPLEPREP_SUMMARY            	were shaken on a FastPrep-24 Homogenizer for 30 seconds and chilled in a -20 °C
SP:SAMPLEPREP_SUMMARY            	freezer repeatedly for three cycles of bead-beating and a total of 30 minutes of
SP:SAMPLEPREP_SUMMARY            	chilling. The organic and aqueous layers were separated by spinning samples in a
SP:SAMPLEPREP_SUMMARY            	centrifuge at 4,300 rpm for 2 minutes at 4 °C. The aqueous layer was removed to
SP:SAMPLEPREP_SUMMARY            	a new glass centrifuge tube. The remaining organic fraction was rinsed three
SP:SAMPLEPREP_SUMMARY            	more times with additions of 1 to 2 mL of 50:50 methanol:water. All aqueous
SP:SAMPLEPREP_SUMMARY            	rinses were combined for each sample and dried down under N2 gas. The remaining
SP:SAMPLEPREP_SUMMARY            	organic layer was transferred into a clean glass centrifuge tube and the
SP:SAMPLEPREP_SUMMARY            	remaining bead beating tube was rinsed two more times with cold organic solvent.
SP:SAMPLEPREP_SUMMARY            	The combined organic rinses were centrifuged, transferred to a new tube, and
SP:SAMPLEPREP_SUMMARY            	dried under N2 gas. Dried aqueous fractions were re-dissolved in 380 µL of
SP:SAMPLEPREP_SUMMARY            	water. Dried organic fractions were re-dissolved in 380 µL of 1:1
SP:SAMPLEPREP_SUMMARY            	water:acetonitrile. 20 µL of isotope-labeled injection standards in water were
SP:SAMPLEPREP_SUMMARY            	added to both fractions. Blank filters were extracted alongside samples as
SP:SAMPLEPREP_SUMMARY            	methodological blanks.
SP:PROCESSING_STORAGE_CONDITIONS 	On ice
SP:EXTRACTION_METHOD             	Bligh-Dyer
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	See attached summary
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Waters Acquity I-Class
CH:COLUMN_NAME                   	SeQuant ZIC- pHILIC (150 x 2.1mm, 5um)
#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                   	See attached protocol
MS:MS_RESULTS_FILE               	ST001372_AN002292_Results.txt	UNITS:Adjusted and normalized peak areas	Has m/z:Yes	Has RT:Yes	RT units:Seconds
#END