#METABOLOMICS WORKBENCH kcontrep_20200630_101303 DATATRACK_ID:2089 STUDY_ID:ST001415 ANALYSIS_ID:AN002367 PROJECT_ID:PR000971 VERSION 1 CREATED_ON June 30, 2020, 12:18 pm #PROJECT PR:PROJECT_TITLE Untargeted metabolomics of primary mouse neutrophils PR:PROJECT_SUMMARY Untargeted metabolomics of primary neutrophils from young and old, male and PR:PROJECT_SUMMARY female mice PR:INSTITUTE Stanford University PR:DEPARTMENT Genetics PR:LAST_NAME Contrepois PR:FIRST_NAME Kevin PR:ADDRESS 300 Pasteur Dr, ALWAY bldg M302, STANFORD, California, 94305, USA PR:EMAIL kcontrep@stanford.edu PR:PHONE 6507239914 #STUDY ST:STUDY_TITLE Multi-omic profiling of primary mouse neutrophils reveals a pattern of sex and ST:STUDY_TITLE age-related functional regulation ST:STUDY_SUMMARY Neutrophils are the most abundant white blood cells in humans and constitute one ST:STUDY_SUMMARY of the first lines of defense in the innate immune response. Neutrophils are ST:STUDY_SUMMARY extremely short-lived cells, which survive less than a day after reaching ST:STUDY_SUMMARY terminal differentiation. Thus, little is known about how organismal aging, ST:STUDY_SUMMARY rather than the daily cellular aging process, may impact neutrophil biology. In ST:STUDY_SUMMARY addition, accumulating evidence suggests that both immunity and organismal aging ST:STUDY_SUMMARY are extremely sex-dimorphic. Here, we describe a multi-omic resource of mouse ST:STUDY_SUMMARY primary bone marrow neutrophils from young and old female and male animals, at ST:STUDY_SUMMARY the transcriptomic, metabolomic and lipidomic levels. Importantly, we identify ST:STUDY_SUMMARY widespread age-related and sex-dimorphic regulation of ‘omics’ in ST:STUDY_SUMMARY neutrophils, specifically regulation of chromatin metabolism. We leverage ST:STUDY_SUMMARY machine-learning and identify candidate molecular drivers of age-related and ST:STUDY_SUMMARY sex-dimorphic transcriptional regulation of neutrophils. We leverage our ST:STUDY_SUMMARY resource to predict increased levels/release of neutrophil elastase in male ST:STUDY_SUMMARY mice. To date, this dataset represents the largest multi-omic resource for the ST:STUDY_SUMMARY study of neutrophils across biological sex and ages. This resource identifies ST:STUDY_SUMMARY molecular states linked to neutrophil characteristics linked to organismal age ST:STUDY_SUMMARY or sex, which could be leveraged to improve immune responses across individuals. ST:INSTITUTE Stanford University ST:LAST_NAME Contrepois ST:FIRST_NAME Kevin ST:ADDRESS 300 Pasteur Dr ST:EMAIL kcontrep@stanford.edu ST:PHONE 6506664538 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J SU:AGE_OR_AGE_RANGE 4 months & 20 months SU:GENDER Male and female SU:ANIMAL_ANIMAL_SUPPLIER Jackson Laboratories SU:ANIMAL_HOUSING SPF animal facility at USC #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 - M_20m_4a Sex:Male | Age:Old RAW_FILE_NAME=pHILIC_M_20m_4a; RAW_FILE_NAME=nHILIC_M_20m_4a; RAW_FILE_NAME=pRPLC_M_20m_4a; RAW_FILE_NAME=nRPLC_M_20m_4a SUBJECT_SAMPLE_FACTORS - F_20m_3a Sex:Female | Age:Old RAW_FILE_NAME=pHILIC_F_20m_3a; RAW_FILE_NAME=nHILIC_F_20m_3a; RAW_FILE_NAME=pRPLC_F_20m_3a; RAW_FILE_NAME=nRPLC_F_20m_3a SUBJECT_SAMPLE_FACTORS - F_4m_1b Sex:Female | Age:Young RAW_FILE_NAME=pHILIC_F_4m_1b; RAW_FILE_NAME=nHILIC_F_4m_1b; RAW_FILE_NAME=pRPLC_F_4m_1b; RAW_FILE_NAME=nRPLC_F_4m_1b SUBJECT_SAMPLE_FACTORS - M_4m_1a Sex:Male | Age:Young RAW_FILE_NAME=pHILIC_M_4m_1a; RAW_FILE_NAME=nHILIC_M_4m_1a; RAW_FILE_NAME=pRPLC_M_4m_1a; RAW_FILE_NAME=nRPLC_M_4m_1a SUBJECT_SAMPLE_FACTORS - F_4m_1a Sex:Female | Age:Young RAW_FILE_NAME=pHILIC_F_4m_1a; RAW_FILE_NAME=nHILIC_F_4m_1a; RAW_FILE_NAME=pRPLC_F_4m_1a; RAW_FILE_NAME=nRPLC_F_4m_1a SUBJECT_SAMPLE_FACTORS - M_20m_3a Sex:Male | Age:Old RAW_FILE_NAME=pHILIC_M_20m_3a; RAW_FILE_NAME=nHILIC_M_20m_3a; RAW_FILE_NAME=pRPLC_M_20m_3a; RAW_FILE_NAME=nRPLC_M_20m_3a SUBJECT_SAMPLE_FACTORS - M_4m_2b Sex:Male | Age:Young RAW_FILE_NAME=pHILIC_M_4m_2b; RAW_FILE_NAME=nHILIC_M_4m_2b; RAW_FILE_NAME=pRPLC_M_4m_2b; RAW_FILE_NAME=nRPLC_M_4m_2b SUBJECT_SAMPLE_FACTORS - F_20m_4b Sex:Female | Age:Old RAW_FILE_NAME=pHILIC_F_20m_4b; RAW_FILE_NAME=nHILIC_F_20m_4b; RAW_FILE_NAME=pRPLC_F_20m_4b; RAW_FILE_NAME=nRPLC_F_20m_4b SUBJECT_SAMPLE_FACTORS - M_20m_3b Sex:Male | Age:Old RAW_FILE_NAME=pHILIC_M_20m_3b; RAW_FILE_NAME=nHILIC_M_20m_3b; RAW_FILE_NAME=pRPLC_M_20m_3b; RAW_FILE_NAME=nRPLC_M_20m_3b SUBJECT_SAMPLE_FACTORS - M_4m_2a Sex:Male | Age:Young RAW_FILE_NAME=pHILIC_M_4m_2a; RAW_FILE_NAME=nHILIC_M_4m_2a; RAW_FILE_NAME=pRPLC_M_4m_2a; RAW_FILE_NAME=nRPLC_M_4m_2a SUBJECT_SAMPLE_FACTORS - M_4m_1b Sex:Male | Age:Young RAW_FILE_NAME=pHILIC_M_4m_1b; RAW_FILE_NAME=nHILIC_M_4m_1b; RAW_FILE_NAME=pRPLC_M_4m_1b; RAW_FILE_NAME=nRPLC_M_4m_1b SUBJECT_SAMPLE_FACTORS - F_20m_4a Sex:Female | Age:Old RAW_FILE_NAME=pHILIC_F_20m_4a; RAW_FILE_NAME=nHILIC_F_20m_4a; RAW_FILE_NAME=pRPLC_F_20m_4a; RAW_FILE_NAME=nRPLC_F_20m_4a SUBJECT_SAMPLE_FACTORS - F_4m_2b Sex:Female | Age:Young RAW_FILE_NAME=pHILIC_F_4m_2b; RAW_FILE_NAME=nHILIC_F_4m_2b; RAW_FILE_NAME=pRPLC_F_4m_2b; RAW_FILE_NAME=nRPLC_F_4m_2b SUBJECT_SAMPLE_FACTORS - F_20m_3b Sex:Female | Age:Old RAW_FILE_NAME=pHILIC_F_20m_3b; RAW_FILE_NAME=nHILIC_F_20m_3b; RAW_FILE_NAME=pRPLC_F_20m_3b; RAW_FILE_NAME=nRPLC_F_20m_3b SUBJECT_SAMPLE_FACTORS - M_20m_4b Sex:Male | Age:Old RAW_FILE_NAME=pHILIC_M_20m_4b; RAW_FILE_NAME=nHILIC_M_20m_4b; RAW_FILE_NAME=pRPLC_M_20m_4b; RAW_FILE_NAME=nRPLC_M_20m_4b SUBJECT_SAMPLE_FACTORS - F_4m_2a Sex:Female | Age:Young RAW_FILE_NAME=pHILIC_F_4m_2a; RAW_FILE_NAME=nHILIC_F_4m_2a; RAW_FILE_NAME=pRPLC_F_4m_2a; RAW_FILE_NAME=nRPLC_F_4m_2a #COLLECTION CO:COLLECTION_SUMMARY The hindlimb bones of each mouse were harvested and kept on ice in D-PBS CO:COLLECTION_SUMMARY (Corning) supplemented with 1% Penicillin/Streptomycin (Corning) until further CO:COLLECTION_SUMMARY processing. Muscle tissue was removed from the bones, and the bone marrow from CO:COLLECTION_SUMMARY cleaned bones was collected into clean tubes (Amend et al., 2016). Red blood CO:COLLECTION_SUMMARY cells from the marrow were removed using Red Blood Cell Lysis (Miltenyi Biotech CO:COLLECTION_SUMMARY #130-094-183), according to the manufacturer’s instructions, albeit with no CO:COLLECTION_SUMMARY vortexing step to avoid unscheduled neutrophil activation. Neutrophils were CO:COLLECTION_SUMMARY isolated from other bone marrow cells using magnetic-assisted cell sorting CO:COLLECTION_SUMMARY (Miltenyi Biotech kit #130-097-658). Viability and yield were assessed using CO:COLLECTION_SUMMARY trypan blue exclusion and an automated COUNTESS cell counter (Thermo-Fisher CO:COLLECTION_SUMMARY Scientific). Purified cells were pelleted at 300g and snap-frozen in liquid CO:COLLECTION_SUMMARY nitrogen until processing for RNA, lipid or metabolite isolation. CO:SAMPLE_TYPE Bone marrow #TREATMENT TR:TREATMENT_SUMMARY There was no treatment. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites and lipids were extracted from neutrophil cell pellets and analyzed SP:SAMPLEPREP_SUMMARY in a randomized order. Extraction was performed using a biphasic separation SP:SAMPLEPREP_SUMMARY protocol with ice-cold methanol, methyl tert-butyl ether (MTBE) and water SP:SAMPLEPREP_SUMMARY (Contrepois et al., 2018). Briefly, 300μL of methanol spiked-in with 54 SP:SAMPLEPREP_SUMMARY deuterated internal standards provided with the Lipidyzer platform (SCIEX, cat SP:SAMPLEPREP_SUMMARY #5040156, LPISTDKIT-101) was added to the cell pellet, samples were vigorously SP:SAMPLEPREP_SUMMARY vortexed for 20 seconds and sonicated in a water bath 3 times for 30 seconds on SP:SAMPLEPREP_SUMMARY ice. Lipids were solubilized by adding 1000μL of MTBE and incubated under SP:SAMPLEPREP_SUMMARY agitation for 1h at 4°C. After addition of 250μL of ice-cold water, the SP:SAMPLEPREP_SUMMARY samples were vortexed for 1 min and centrifuged at 14,000g for 5 min at 20°C. SP:SAMPLEPREP_SUMMARY The upper phase containing the lipids was then collected and dried down under SP:SAMPLEPREP_SUMMARY nitrogen. The dry lipid extracts were reconstituted with 300μL of 10 mM SP:SAMPLEPREP_SUMMARY ammonium acetate in 9:1 methanol:toluene for analaysis. The lower phase SP:SAMPLEPREP_SUMMARY containing metabolites was subjected to further protein precipitation by adding SP:SAMPLEPREP_SUMMARY 4 times of ice-cold 1:1:1 isopropanol:acetonitrile:water spiked in with 17 SP:SAMPLEPREP_SUMMARY labeled internal standards and incubating for 2 hours at -20°C. The supernatant SP:SAMPLEPREP_SUMMARY was dried down to completion under nitrogen and re-suspended in 100μL of 1:1 SP:SAMPLEPREP_SUMMARY MeOH:Water for analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY RPLC experiments were performed using a Zorbax SBaq column 2.1 x 50 mm, 1.7 μm, CH:CHROMATOGRAPHY_SUMMARY 100Å (Agilent Technologies) and mobile phase solvents consisting of 0.06% CH:CHROMATOGRAPHY_SUMMARY acetic acid in water (A) and 0.06% acetic acid in methanol (B). (Contrepois et CH:CHROMATOGRAPHY_SUMMARY al., 2015) CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME Agilent Zorbax SBaq (50 x 2.1 mm, 1.7 µm) CH:FLOW_RATE 0.6 ml/min CH:COLUMN_TEMPERATURE 60 CH:SOLVENT_A 0.06% acetic acid in water CH:SOLVENT_B 0.06% acetic acid in methanol #ANALYSIS AN:ANALYSIS_TYPE MS AN:OPERATOR_NAME Kevin Contrepois AN:DETECTOR_TYPE Orbitrap AN:DATA_FORMAT .RAW #MS MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Data processing. Data from each mode were independently analyzed using MS:MS_COMMENTS Progenesis QI software v2.3 (Nonlinear Dynamics). Metabolic features from blanks MS:MS_COMMENTS and that didn’t show sufficient linearity upon dilution were discarded. Only MS:MS_COMMENTS metabolic features present in >33% of the samples in each group were kept for MS:MS_COMMENTS further analysis and missing values were imputed by drawing from a random MS:MS_COMMENTS distribution of small values in the corresponding sample (Tyanova et al., 2016). MS:MS_COMMENTS Metabolic feature annotation. Annotation confidence levels for each metabolite MS:MS_COMMENTS were provided following the Metabolomics Standards Initiative (MSI) confidence MS:MS_COMMENTS scheme. Peak annotation was first performed by matching experimental m/z, MS:MS_COMMENTS retention time and MS/MS spectra to an in-house library of analytical-grade MS:MS_COMMENTS standards (level 1). Remaining peaks were identified by matching experimental MS:MS_COMMENTS m/z and fragmentation spectra to publicly available databases including HMDB MS:MS_COMMENTS (http://www.hmdb.ca/), MoNA (http://mona.fiehnlab.ucdavis.edu/) and MassBank MS:MS_COMMENTS (http://www.massbank.jp/) using the R package ‘MetID’ (v0.2.0) (PMID: MS:MS_COMMENTS 30944337) (level 2). Briefly, metabolic feature tables from Progenesis QI were MS:MS_COMMENTS matched to fragmentation spectra with a m/z and a retention time window of ±15 MS:MS_COMMENTS ppm and ±30 s (HILIC) and ± 20 s (RPLC), respectively. When multiple MS/MS MS:MS_COMMENTS spectra match a single metabolic feature, all matched MS/MS spectra were used MS:MS_COMMENTS for the identification. Next, MS1 and MS2 pairs were searched against public MS:MS_COMMENTS databases and a similarity score was calculated using the forward dot–product MS:MS_COMMENTS algorithm which takes into account both fragments and intensities. Metabolites MS:MS_COMMENTS were reported if the similarity score was above 0.4. Level 3 corresponds to MS:MS_COMMENTS unknown metabolites. MS:CAPILLARY_TEMPERATURE 375C MS:CAPILLARY_VOLTAGE 3.4kV MS:COLLISION_ENERGY 25 & 50 NCE MS:COLLISION_GAS N2 MS:DRY_GAS_TEMP 310C MS:MS_RESULTS_FILE ST001415_AN002367_Results.txt UNITS:MS count Has m/z:Yes Has RT:Yes RT units:Minutes #END