#METABOLOMICS WORKBENCH TruxalCarlson_20200527_155406 DATATRACK_ID:2022 STUDY_ID:ST001409 ANALYSIS_ID:AN002356 PROJECT_ID:PR000965
VERSION             	1
CREATED_ON             	June 26, 2020, 3:44 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 Transition Zone 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 patchier 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                         	206-616-4658
#STUDY
ST:STUDY_TITLE                   	Patterns in metabolite pools show that phytoplankton leave a taxon-specific
ST:STUDY_TITLE                   	signature on particulate carbon: North Pacific Transition Zone 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 patchier 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
ST:EMAIL                         	kheal@uw.edu
ST:PHONE                         	206-616-4658
#SUBJECT
SU:SUBJECT_TYPE                  	Other
SU:SUBJECT_SPECIES               	Natural mixed marine microbial community
SU:GENDER                        	Not applicable
#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           	-	Blk0p2_1	Depth_m:NA | Vol_L:NA	Latitude=NA; Longitude=NA; UTC=NA; Replicate=1; Type=Blk; RAW_FILE_NAME=180702_Blk_Blk0p2_1;180702_Blk_Blk0p2_1;180716_Blk_Blk0p2_1
SUBJECT_SAMPLE_FACTORS           	-	S7E41D30_1	Depth_m:30 | Vol_L:4	Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D30_1;180702_Smp_S7E41D30_1;180716_Smp_S7E41D30_1
SUBJECT_SAMPLE_FACTORS           	-	S7E41D40_1	Depth_m:40 | Vol_L:4	Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D40_1;180702_Smp_S7E41D40_1;180716_Smp_S7E41D40_1
SUBJECT_SAMPLE_FACTORS           	-	S7E41D60_1	Depth_m:60 | Vol_L:4	Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D60_1;180702_Smp_S7E41D60_1;180716_Smp_S7E41D60_1
SUBJECT_SAMPLE_FACTORS           	-	S7E41D80_1	Depth_m:80 | Vol_L:4	Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D80_1;180702_Smp_S7E41D80_1;180716_Smp_S7E41D80_1
SUBJECT_SAMPLE_FACTORS           	-	S7E41D100_1	Depth_m:100 | Vol_L:4	Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D100_1;180702_Smp_S7E41D100_1;180716_Smp_S7E41D100_1
SUBJECT_SAMPLE_FACTORS           	-	S7E41D120_1	Depth_m:120 | Vol_L:4	Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D120_1;180702_Smp_S7E41D120_1;180716_Smp_S7E41D120_1
SUBJECT_SAMPLE_FACTORS           	-	S7E41D140_1	Depth_m:140 | Vol_L:4	Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D140_1;180702_Smp_S7E41D140_1;180716_Smp_S7E41D140_1
SUBJECT_SAMPLE_FACTORS           	-	S7E41D160_1	Depth_m:160 | Vol_L:4	Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D160_1;180702_Smp_S7E41D160_1;180716_Smp_S7E41D160_1
SUBJECT_SAMPLE_FACTORS           	-	S7E41D180_1	Depth_m:180 | Vol_L:4	Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D180_1;180702_Smp_S7E41D180_1;180716_Smp_S7E41D180_1
SUBJECT_SAMPLE_FACTORS           	-	S7E41D250_1	Depth_m:250 | Vol_L:4	Latitude=41.417; Longitude=-158; UTC=2017-06-03T19;51;00; Replicate=1; Type=Smp; RAW_FILE_NAME=180702_Smp_S7E41D250_1;180702_Smp_S7E41D250_1;180716_Smp_S7E41D250_1
SUBJECT_SAMPLE_FACTORS           	-	TruePoo_Half1	Depth_m:NA | Vol_L:NA	Latitude=NA; Longitude=NA; UTC=NA; Replicate=1; Type=Pool; RAW_FILE_NAME=180702_Poo_TruePoo_Half1;180702_Poo_TruePoo_Half1;180716_Poo_TruePoo10_Half1
SUBJECT_SAMPLE_FACTORS           	-	TruePoo_Half2	Depth_m:NA | Vol_L:NA	Latitude=NA; Longitude=NA; UTC=NA; Replicate=2; Type=Pool; RAW_FILE_NAME=180702_Poo_TruePoo_Half2;180702_Poo_TruePoo_Half2;180726_Poo_TruePoo10_Half2
SUBJECT_SAMPLE_FACTORS           	-	TruePoo_Half3	Depth_m:NA | Vol_L:NA	Latitude=NA; Longitude=NA; UTC=NA; Replicate=3; Type=Pool; RAW_FILE_NAME=180702_Poo_TruePoo_Half3;180702_Poo_TruePoo_Half3;180716_Poo_TruePoo10_Half3
SUBJECT_SAMPLE_FACTORS           	-	TruePoo_Full1	Depth_m:NA | Vol_L:NA	Latitude=NA; Longitude=NA; UTC=NA; Replicate=1; Type=Pool; RAW_FILE_NAME=180702_Poo_TruePoo_Full1;180702_Poo_TruePoo_Full1;180716_Poo_TruePoo10_Full1
SUBJECT_SAMPLE_FACTORS           	-	TruePoo_Full2	Depth_m:NA | Vol_L:NA	Latitude=NA; Longitude=NA; UTC=NA; Replicate=2; Type=Pool; RAW_FILE_NAME=180702_Poo_TruePoo_Full2;180702_Poo_TruePoo_Full2;180716_Poo_TruePoo10_Full2
SUBJECT_SAMPLE_FACTORS           	-	TruePoo_Full3	Depth_m:NA | Vol_L:NA	Latitude=NA; Longitude=NA; UTC=NA; Replicate=3; Type=Pool; RAW_FILE_NAME=180702_Poo_TruePoo_Full3;180702_Poo_TruePoo_Full3;180716_Poo_TruePoo10_Full3
#COLLECTION
CO:COLLECTION_SUMMARY            	Samples for particulate metabolites were collected from 10 depths between 30 and
CO:COLLECTION_SUMMARY            	250 m on June 3, 2017 aboard the R/V Marcus Langseth. At each sampling location
CO:COLLECTION_SUMMARY            	and depth, single, duplicate, or triplicate filters were collected using either
CO:COLLECTION_SUMMARY            	niskins attached to a conductivity, temperature, depth array (CTD) or the
CO:COLLECTION_SUMMARY            	underway intake. Samples (4 L) were collected into polycarbonate carboys,
CO:COLLECTION_SUMMARY            	filtered onto 147 mm 0.2 μm PTFE filters using peristaltic pumps, polycarbonate
CO:COLLECTION_SUMMARY            	filter holders, and Masterflex PharMed BPT tubing (Cole-Parmer). Filters were
CO:COLLECTION_SUMMARY            	flash frozen in liquid nitrogen and stored at -80°C until extraction. In
CO:COLLECTION_SUMMARY            	addition to our samples, we filtered seawater through two 0.2 μm PTFE filters
CO:COLLECTION_SUMMARY            	in series, using the second filter as a methodological blank. This blank is
CO:COLLECTION_SUMMARY            	especially important to parse signals from the salt matrix that would be present
CO:COLLECTION_SUMMARY            	after sorption to the filter.
CO:SAMPLE_TYPE                   	Suspended Marine Particulate Matter
CO:STORAGE_CONDITIONS            	Described in summary
#TREATMENT
TR:TREATMENT_SUMMARY             	No treatment, this is a study of the natural marine microbial population in the
TR:TREATMENT_SUMMARY             	North Pacific Transition Zone.
#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                      	NEGATIVE
MS:MS_COMMENTS                   	See attached protocol.
MS:MS_RESULTS_FILE               	ST001409_AN002356_Results.txt	UNITS:Adjusted and normalized peak areas	Has m/z:Yes	Has RT:Yes	RT units:Seconds
#END