#METABOLOMICS WORKBENCH jroth002_20190716_224921 DATATRACK_ID:1772 STUDY_ID:ST001220 ANALYSIS_ID:AN002033 PROJECT_ID:PR000819 VERSION 1 CREATED_ON July 20, 2019, 11:28 am #PROJECT PR:PROJECT_TITLE Untargeted metabolomics of bumble bees exposed to cadmium PR:PROJECT_SUMMARY Bumble bee workers were exposed to cadmium chloride or control through diet. We PR:PROJECT_SUMMARY then ran untargeted metabolomics on the bees. PR:INSTITUTE UC Riverside PR:LAST_NAME Rothman PR:FIRST_NAME Jason PR:ADDRESS 900 University Ave., Riverside, CA, 91766, USA PR:EMAIL jroth002@ucr.edu PR:PHONE 9518275817 #STUDY ST:STUDY_TITLE Effects of cadmium exposure on the bumble bee metabolome ST:STUDY_SUMMARY We exposed worker bumble bees to cadmium chloride or control through diet. We ST:STUDY_SUMMARY then used LCMS untargeted metabolomics on pools of 3 bees each. ST:INSTITUTE UC Riverside ST:LAST_NAME Rothman ST:FIRST_NAME Jason ST:ADDRESS 900 University Ave., Riverside, CA, 91766, USA ST:EMAIL jroth002@ucr.edu ST:PHONE 9518275817 #SUBJECT SU:SUBJECT_TYPE Invertebrate SU:SUBJECT_SPECIES Bombus impatiens SU:TAXONOMY_ID 132113 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS 20181024_011 QC-1 Factor:QCPool SUBJECT_SAMPLE_FACTORS 20181024_012 BBCT-3 Factor:Control SUBJECT_SAMPLE_FACTORS 20181024_013 BBCT-2 Factor:Control SUBJECT_SAMPLE_FACTORS 20181024_014 BBCD-1 Factor:Cadmium SUBJECT_SAMPLE_FACTORS 20181024_015 QC-2 Factor:QCPool SUBJECT_SAMPLE_FACTORS 20181024_016 BBCT-5 Factor:Control SUBJECT_SAMPLE_FACTORS 20181024_017 BBCD-5 Factor:Cadmium SUBJECT_SAMPLE_FACTORS 20181024_018 BBCD-2 Factor:Cadmium SUBJECT_SAMPLE_FACTORS 20181024_019 QC-3 Factor:QCPool SUBJECT_SAMPLE_FACTORS 20181024_020 BBCD-4 Factor:Cadmium SUBJECT_SAMPLE_FACTORS 20181024_021 BBCT-1 Factor:Control SUBJECT_SAMPLE_FACTORS 20181024_022 BBCD-3 Factor:Cadmium SUBJECT_SAMPLE_FACTORS 20181024_023 BBCT-4 Factor:Control SUBJECT_SAMPLE_FACTORS 20181024_024 QC-4 Factor:QCPool #COLLECTION CO:COLLECTION_SUMMARY We immersed live bees in liquid nitrogen and stored the samples at -80 °C. CO:SAMPLE_TYPE Insect tissue #TREATMENT TR:TREATMENT_SUMMARY We obtained five commercial Bombus impatiens colonies that each contained TR:TREATMENT_SUMMARY approximately 50 workers, a gravid queen, pollen and a Biogluc® feeder from the TR:TREATMENT_SUMMARY Biobest Group (Biobest USA Inc., Romulus, MI). We immediately replaced the TR:TREATMENT_SUMMARY Biogluc with sterile 60% sucrose and allowed the bees access ad libitum. We also TR:TREATMENT_SUMMARY provided the colony with pollen patties ad libitum and kept the colonies at 29 TR:TREATMENT_SUMMARY °C under constant darkness at the University of California, Riverside. We TR:TREATMENT_SUMMARY allowed the colonies to grow undisturbed for two weeks before starting the TR:TREATMENT_SUMMARY experiment. Once the colonies had populated, we transferred 10 bees (two cohorts TR:TREATMENT_SUMMARY of five) to 475 mL polypropylene containers (WebstaurantStore, Lancaster, PA) TR:TREATMENT_SUMMARY and reared them at 29 °C under 24-hour darkness. We then exposed the bees to TR:TREATMENT_SUMMARY treatments consisting of 60% sucrose spiked with either 0.46 mg/kg cadmium TR:TREATMENT_SUMMARY chloride and used unspiked 60% sucrose as a control. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY We then pooled three bee abdomens from each cage, freeze-dried the samples, and SP:SAMPLEPREP_SUMMARY homogenized the abdomens to a fine powder at 4° C using a bead mill SP:SAMPLEPREP_SUMMARY homogenizer. Next, we extracted 10-12 mg of the powder in a 1.5 mL tube with 100 SP:SAMPLEPREP_SUMMARY µL of ice-cold extraction solvent (30:30:20:20 SP:SAMPLEPREP_SUMMARY acetonitrile:methanol:water:isopropanol) per 1 mg of tissue. We sonicated the SP:SAMPLEPREP_SUMMARY samples for 5 minutes in an ice bath, then vortexed them for 30 min at 4° C. SP:SAMPLEPREP_SUMMARY Lastly, we centrifuged the samples at 16,000 x g for 15 min at 4° C. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY We used a Synapt G2-Si quadrupole time-of-flight mass spectrometer (Waters, CH:CHROMATOGRAPHY_SUMMARY Milford, MA) coupled to an I-class UPLC system (Waters) for LC-MS analyses in CH:CHROMATOGRAPHY_SUMMARY the UC Riverside Metabolomics Core Facility. We carried out separations on a CSH CH:CHROMATOGRAPHY_SUMMARY phenyl-hexyl column (2.1 x 100 mm, 1.7 µM) (Waters, Milford, MA), with the CH:CHROMATOGRAPHY_SUMMARY following mobile phases: A. Water with 0.1% formic acid and B. Acetonitrile with CH:CHROMATOGRAPHY_SUMMARY 0.1% formic acid at a flow rate of 250 µL/min at 40° C. We injected 2 µL of CH:CHROMATOGRAPHY_SUMMARY sample extract, and the gradient was as follows: 0 min, 1% B; 1 min, 1% B; 8 CH:CHROMATOGRAPHY_SUMMARY min, 40% B; 24 min, 100% B; 26.5 min, 100% B; 27 min, 1% B. CH:CHROMATOGRAPHY_TYPE Normal phase CH:INSTRUMENT_NAME Waters Acquity I-Class CH:COLUMN_NAME Waters Acquity CSH C18 (100 x 2.1mm, 1.7um) CH:METHODS_FILENAME jroth002_LC_method.docx #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS We operated the MS in positive ion mode (50 to 1200 m/z) with a 100 ms scan time MS:MS_COMMENTS and acquired MS/MS data at 1 MS/MS scan per MS scan. We set source and MS:MS_COMMENTS desolvation temperatures to 150° C and 600° C, respectively. We set the MS:MS_COMMENTS desolvation gas flow to 1100 L/hr and cone gas flow to 150 L/h, with all gases MS:MS_COMMENTS being nitrogen except the collision gas, which was argon, and set capillary MS:MS_COMMENTS voltage to 1 kV. We generated a quality control sample by pooling equal aliquots MS:MS_COMMENTS of each sample and analyzed this pool every 3-4 injections to monitor system MS:MS_COMMENTS stability and performance. We analyzed samples in random order and used a MS:MS_COMMENTS leucine enkephalin infusion for mass correction. We processed the metabolite MS:MS_COMMENTS data (peak picking, alignment, deconvolution, integration, normalization, and MS:MS_COMMENTS spectral matching) with Progenesis Qi software (Nonlinear Dynamics, Durham, NC). MS:MS_COMMENTS We normalized the resulting data to total ion abundance and removed features MS:MS_COMMENTS with a coefficient of variation greater than 20% or an average abundance less MS:MS_COMMENTS than 200 in the quality control injections as in Barupal et al. 2018 and Dunn et MS:MS_COMMENTS al. 2011 (Barupal et al., 2018; Dunn et al., 2011). To aid in the identification MS:MS_COMMENTS of features belonging to the same metabolite, we assigned features a cluster ID MS:MS_COMMENTS using RAMClust (Broeckling, Afsar, Neumann, Ben-Hur, & Prenni, 2014). Next, we MS:MS_COMMENTS used a slightly modified version of the metabolomics standard initiative MS:MS_COMMENTS guidelines to assign annotation level confidence (Schymanski et al., 2014; MS:MS_COMMENTS Sumner et al., 2007): Annotation level 1 indicates an MS and MS/MS match or MS MS:MS_COMMENTS and retention time match to an in-house database generated with authentic MS:MS_COMMENTS standards. Level 2a indicates an MS and MS/MS match to an external database. MS:MS_COMMENTS Level 2b indicates an MS and MS/MS match to the LipidBlast database (Kind et MS:MS_COMMENTS al., 2013) or an MS match and diagnostic evidence (i.e. the dominant presence of MS:MS_COMMENTS an m/z 85 fragment ion for acylcarnitines). We searched against several mass MS:MS_COMMENTS spectral metabolite databases including Metlin, Massbank of North America MS:MS_COMMENTS (Blaženović et al., 2019; Kind et al., 2013), and an in-house database in the MS:MS_COMMENTS UC Riverside Metabolomics Core Facility. MS:MS_RESULTS_FILE ST001220_AN002033_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END