#METABOLOMICS WORKBENCH jburgess_20160306_210457 STUDY_ID:ST000363 ANALYSIS_ID:AN000596 PROJECT_ID:PR000285 VERSION 1 CREATED_ON March 10, 2016, 9:48 pm #PROJECT PR:PROJECT_TITLE Metabolic Profiling of Anxiety Prone HSV-Latently Infected Obese Mice PR:PROJECT_TYPE Broad Spectrum LCMS PR:PROJECT_SUMMARY The biological factors that lead children from low socioeconomic backgrounds to PR:PROJECT_SUMMARY be at greater risk for the development of anxiety and learning problems are not PR:PROJECT_SUMMARY well understood. While it is clear that there are genetic components to the risk PR:PROJECT_SUMMARY of developing mental health disorders, a role for environmental factors in PR:PROJECT_SUMMARY inducing these problems has been suggested. Many of these factors that affect PR:PROJECT_SUMMARY the brain likely involve exposures that occur early in life. Two such factors PR:PROJECT_SUMMARY are the higher rates of herpes simplex virus (HSV)-1 seropositivity and PR:PROJECT_SUMMARY prevalence of obesity among these children. HSV-1 infection has been associated PR:PROJECT_SUMMARY with impaired cognition during childhood and mental health problems in PR:PROJECT_SUMMARY adulthood. Additionally, obese adults are shown to have higher HSV-1 titers. PR:PROJECT_SUMMARY Similarly, other studies have correlated a diet high in saturated fat and PR:PROJECT_SUMMARY obesity with increased risk of mood disorders and anxiety. A mouse model of PR:PROJECT_SUMMARY obese HSV-1 latent infection was developed. Broad spectrum metabolomics analysis PR:PROJECT_SUMMARY was performed to better understand the metabolomic profile of hippocampus and to PR:PROJECT_SUMMARY compare this metabolomics profile with that of the hypothalamus, microglia, and PR:PROJECT_SUMMARY peripheral blood mononuclear cells. Brain tissue samples for metabolomics PR:PROJECT_SUMMARY experiments were generated in the following manner: 3-week old mice were placed PR:PROJECT_SUMMARY on a 10% low fat (LF) diet and acclimated for one week prior to intranasal HSV-1 PR:PROJECT_SUMMARY infection or mock infection with PBS. Fourteen days post-infection mice were PR:PROJECT_SUMMARY randomized to either a 45% high fat diet (HF) or remained on the LF diet. Eight PR:PROJECT_SUMMARY weeks post-diet transition, mice were euthanized and brain tissue samples were PR:PROJECT_SUMMARY collected and processed for metabolomics. PR:INSTITUTE University of North Carolina , Chapel Hill PR:DEPARTMENT Gillings School of Public Health PR:LABORATORY Department of Nutrition PR:LAST_NAME Sheridan PR:FIRST_NAME Patricia PR:ADDRESS 2002 Hooker Research Center, CB#7461, Chapel Hill NC, 27599 PR:EMAIL patricia_sheridan@med.unc.edu PR:PHONE 919-843-6434 PR:FUNDING_SOURCE NIH, Common Fund, Pilot and Feasibility #STUDY ST:STUDY_TITLE Broad Spectrum MS analysis of mouse peripheral blood mononuclear cells (PBMC) ST:STUDY_TITLE from Anxiety Prone HSV-Latently Infected Obese Mice ST:STUDY_TYPE Broad Spectrum LCMS ST:STUDY_SUMMARY Mononuclear cell samples for metabolomics experiments were generated in the ST:STUDY_SUMMARY following manner: 3-week old mice were placed on a 10% low fat (LF) diet and ST:STUDY_SUMMARY acclimated for one week prior to intranasal HSV-1 infection or mock infection ST:STUDY_SUMMARY with PBS. Fourteen days post-infection mice were randomized to either a 45% high ST:STUDY_SUMMARY fat diet (HF) or remained on the LF diet. Eight weeks post-diet transition, mice ST:STUDY_SUMMARY were euthanized and mononuclear cell samples were collected and processed for ST:STUDY_SUMMARY metabolomics. ST:INSTITUTE RTI ST:DEPARTMENT Discovery Sciences ST:LABORATORY System and Translational Sciences ST:LAST_NAME Sumner ST:FIRST_NAME Susan ST:ADDRESS 3040 Cornwallis Rd, RTP,NC ST:EMAIL ssumner@rti.org ST:PHONE 919-541-7479 ST:NUM_GROUPS 4 ST:TOTAL_SUBJECTS 30 ST:NUM_MALES 30 #SUBJECT SU:SUBJECT_TYPE mouse SU:SUBJECT_SPECIES Mus musculus SU:GENOTYPE_STRAIN C57Bl/6 SU:AGE_OR_AGE_RANGE 4.5 mo SU:WEIGHT_OR_WEIGHT_RANGE 29.1 g-49.2 g SU:GENDER male SU:ANIMAL_ANIMAL_SUPPLIER JAX SU:ANIMAL_HOUSING 4/cage SU:ANIMAL_FEED Purina chow #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS 444 Blood444 HSV-1:No | Diet at Week 2:HF Weight (g) =38.1 SUBJECT_SAMPLE_FACTORS 430 Blood430 HSV-1:yes | Diet at Week 2:LF Weight (g) =25.2 SUBJECT_SAMPLE_FACTORS 431 Blood431 HSV-1:no | Diet at Week 2:LF Weight (g) =32.7 SUBJECT_SAMPLE_FACTORS 443 Blood443 HSV-1:No | Diet at Week 2:HF Weight (g) =37.8 SUBJECT_SAMPLE_FACTORS 441 Blood441 HSV-1:yes | Diet at Week 2:HF Weight (g) =38.6 SUBJECT_SAMPLE_FACTORS 428 Blood428 HSV-1:yes | Diet at Week 2:LF Weight (g) =28.9 SUBJECT_SAMPLE_FACTORS 427 Blood427 HSV-1:yes | Diet at Week 2:HF Weight (g) =31.4 SUBJECT_SAMPLE_FACTORS 445 Blood445 HSV-1:No | Diet at Week 2:HF Weight (g) =37.7 SUBJECT_SAMPLE_FACTORS 422 Blood422 HSV-1:no | Diet at Week 2:LF Weight (g) =30.3 SUBJECT_SAMPLE_FACTORS 436 Blood436 HSV-1:yes | Diet at Week 2:LF Weight (g) =29.8 SUBJECT_SAMPLE_FACTORS 442 Blood442 HSV-1:yes | Diet at Week 2:HF Weight (g) =33.4 SUBJECT_SAMPLE_FACTORS 418 Blood418 HSV-1:no | Diet at Week 2:HF Weight (g) =30.8 SUBJECT_SAMPLE_FACTORS 433 Blood433 HSV-1:no | Diet at Week 2:LF Weight (g) =26.4 SUBJECT_SAMPLE_FACTORS 439 Blood439 HSV-1:yes | Diet at Week 2:HF Weight (g) =33.8 SUBJECT_SAMPLE_FACTORS 435 Blood435 HSV-1:yes | Diet at Week 2:LF Weight (g) =24.9 SUBJECT_SAMPLE_FACTORS 425 Blood425 HSV-1:yes | Diet at Week 2:HF Weight (g) =33.2 SUBJECT_SAMPLE_FACTORS 434 Blood434 HSV-1:no | Diet at Week 2:LF Weight (g) =26.2 SUBJECT_SAMPLE_FACTORS 421 Blood421 HSV-1:no | Diet at Week 2:LF Weight (g) =30.8 SUBJECT_SAMPLE_FACTORS 440 Blood440 HSV-1:yes | Diet at Week 2:HF Weight (g) =38.3 SUBJECT_SAMPLE_FACTORS 437 Blood437 HSV-1:yes | Diet at Week 2:LF Weight (g) =27.6 SUBJECT_SAMPLE_FACTORS 420 Blood420 HSV-1:no | Diet at Week 2:HF Weight (g) =33.8 SUBJECT_SAMPLE_FACTORS 423 Blood423 HSV-1:no | Diet at Week 2:LF Weight (g) =30.7 SUBJECT_SAMPLE_FACTORS 424 Blood424 HSV-1:no | Diet at Week 2:LF Weight (g) =29.8 SUBJECT_SAMPLE_FACTORS 429 Blood429 HSV-1:yes | Diet at Week 2:LF Weight (g) =28.6 SUBJECT_SAMPLE_FACTORS 419 Blood419 HSV-1:no | Diet at Week 2:HF Weight (g) =33.2 SUBJECT_SAMPLE_FACTORS 432 Blood432 HSV-1:no | Diet at Week 2:LF Weight (g) =29.6 SUBJECT_SAMPLE_FACTORS 438 Blood438 HSV-1:yes | Diet at Week 2:LF Weight (g) =29.9 SUBJECT_SAMPLE_FACTORS 417 Blood417 HSV-1:no | Diet at Week 2:HF Weight (g) =32.6 SUBJECT_SAMPLE_FACTORS 426 Blood426 HSV-1:yes | Diet at Week 2:HF Weight (g) =30.9 SUBJECT_SAMPLE_FACTORS 446 Blood446 HSV-1:No | Diet at Week 2:HF Weight (g) =39 SUBJECT_SAMPLE_FACTORS - Total_Pool_01 HSV-1:- | Diet at Week 2:- Weight (g) =- SUBJECT_SAMPLE_FACTORS - Total_Pool_02 HSV-1:- | Diet at Week 2:- Weight (g) =- SUBJECT_SAMPLE_FACTORS - Total_Pool_03 HSV-1:- | Diet at Week 2:- Weight (g) =- SUBJECT_SAMPLE_FACTORS - Total_Pool_04 HSV-1:- | Diet at Week 2:- Weight (g) =- SUBJECT_SAMPLE_FACTORS - Total_Pool_05 HSV-1:- | Diet at Week 2:- Weight (g) =- #COLLECTION CO:COLLECTION_SUMMARY For extraction, 500 µL of an ice-cold solution of 90:10 Methanol:Chloroform CO:COLLECTION_SUMMARY (v/v) ¬was added to the tubes containing the thawed cell pellets. MagNA lyser CO:COLLECTION_SUMMARY ceramic beads were added to the tubes, and a MagNA lyser was used to beat the CO:COLLECTION_SUMMARY samples for two 30 seconds pulses at 2,000 rpm placing samples on cold block for CO:COLLECTION_SUMMARY 5 min in between pulses. Sonication for 5 min was performed to the beaten cell CO:COLLECTION_SUMMARY samples followed by centrifugation at room temperature and at 16,000rcf for 4 CO:COLLECTION_SUMMARY min. A 350 µL aliquot of each experimental cell sample homogenized supernatant CO:COLLECTION_SUMMARY was transferred to a labeled 2.0 mL Lo-Bind Eppendorf tube. #TREATMENT TR:TREATMENT_SUMMARY 3-week old mice were placed on a 10% low fat (LF) diet and acclimated for one TR:TREATMENT_SUMMARY week prior to intranasal HSV-1 infection or mock infection with PBS. Fourteen TR:TREATMENT_SUMMARY days post-infection mice were randomized to either a 45% high fat diet (HF) or TR:TREATMENT_SUMMARY remained on the LF diet. Eight weeks post-diet transition, mice were euthanized TR:TREATMENT_SUMMARY and brain tissue samples were collected and processed for metabolomics. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For extraction, 500 µL of an ice-cold solution of 90:10 Methanol:Chloroform SP:SAMPLEPREP_SUMMARY (v/v) ¬was added to the tubes containing the thawed cell pellets. MagNA lyser SP:SAMPLEPREP_SUMMARY ceramic beads were added to the tubes, and a MagNA lyser was used to beat the SP:SAMPLEPREP_SUMMARY samples for two 30 seconds pulses at 2,000 rpm placing samples on cold block for SP:SAMPLEPREP_SUMMARY 5 min in between pulses. Sonication for 5 min was performed to the beaten cell SP:SAMPLEPREP_SUMMARY samples followed by centrifugation at room temperature and at 16,000rcf for 4 SP:SAMPLEPREP_SUMMARY min. A 350 µL aliquot of each experimental cell sample homogenized supernatant SP:SAMPLEPREP_SUMMARY was transferred to a labeled 2.0 mL Lo-Bind Eppendorf tube. Analytical pooled SP:SAMPLEPREP_SUMMARY samples were created by combining a 125 µL aliquot from each experimental SP:SAMPLEPREP_SUMMARY sample into a labeled 20 mL glass vial. The QC pooled sample was vortexed for 30 SP:SAMPLEPREP_SUMMARY sec and 350 µL aliquots were transferred to 5 labeled 2.0 mL Lo-Bind eppendorf SP:SAMPLEPREP_SUMMARY tubes. Next, the supernatants of the study and QC pooled samples were dried SP:SAMPLEPREP_SUMMARY overnight using a lyophilizer. The residue was reconstituted in 50 µL of SP:SAMPLEPREP_SUMMARY reconstitution buffer (95:5 Water:Methanol, v/v) and mixed on an analog vortex SP:SAMPLEPREP_SUMMARY mixer for 1 min with speed set at 10. Then, the samples were centrifuged at room SP:SAMPLEPREP_SUMMARY temperature for 4 min at 16,000 rcf, and the supernatants were transferred to SP:SAMPLEPREP_SUMMARY autosampler vials for data acquisition. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY UPLC was performed on a Waters Acquity UPLC with an Acquity HSS T3 column (2.1x CH:CHROMATOGRAPHY_SUMMARY 100mm x 1.8 µm) at 50 °C using the reversed-phase separation. Water with 0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid (mobile phase A) and methanol with 0.1% formic acid (mobile phase B) CH:CHROMATOGRAPHY_SUMMARY were used as mobile phases and the metabolites were chromatographically CH:CHROMATOGRAPHY_SUMMARY separated using a gradient separation: CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Synapt G2 Si ESI-Q-TOF CH:COLUMN_NAME Acquity HSS T3 CH:METHODS_FILENAME RTI-RCMRC-RP #ANALYSIS AN:ANALYSIS_TYPE MS AN:ACQUISITION_DATE 7/21/15 #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS intensity MS_METABOLITE_DATA_START Samples Total_Pool_01 Total_Pool_02 Total_Pool_03 Total_Pool_04 Total_Pool_05 Blood417 Blood418 Blood419 Blood420 Blood443 Blood444 Blood445 Blood446 Blood425 Blood426 Blood427 Blood439 Blood440 Blood441 Blood442 Blood421 Blood422 Blood423 Blood424 Blood431 Blood432 Blood433 Blood434 Blood428 Blood429 Blood430 Blood435 Blood436 Blood437 Blood438 Factors HSV-1:- | Diet at Week 2:- HSV-1:- | Diet at Week 2:- HSV-1:- | Diet at Week 2:- HSV-1:- | Diet at Week 2:- HSV-1:- | Diet at Week 2:- HSV-1:no | Diet at Week 2:HF HSV-1:no | Diet at Week 2:HF HSV-1:no | Diet at Week 2:HF HSV-1:no | Diet at Week 2:HF HSV-1:No | Diet at Week 2:HF HSV-1:No | Diet at Week 2:HF HSV-1:No | Diet at Week 2:HF HSV-1:No | Diet at Week 2:HF HSV-1:yes | Diet at Week 2:HF HSV-1:yes | Diet at Week 2:HF HSV-1:yes | Diet at Week 2:HF HSV-1:yes | Diet at Week 2:HF HSV-1:yes | Diet at Week 2:HF HSV-1:yes | Diet at Week 2:HF HSV-1:yes | Diet at Week 2:HF HSV-1:no | Diet at Week 2:LF HSV-1:no | Diet at Week 2:LF HSV-1:no | Diet at Week 2:LF HSV-1:no | Diet at Week 2:LF HSV-1:no | Diet at Week 2:LF HSV-1:no | Diet at Week 2:LF HSV-1:no | Diet at Week 2:LF HSV-1:no | Diet at Week 2:LF HSV-1:yes | Diet at Week 2:LF HSV-1:yes | Diet at Week 2:LF HSV-1:yes | Diet at Week 2:LF HSV-1:yes | Diet at Week 2:LF HSV-1:yes | Diet 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18969.57667 4693.542091 31772.53614 54860.53681 47003.86352 22282.36777 52780.05916 46751.74372 22605.26177 60858.41876 20294.09921 31130.05211 35133.54914 18490.42579 30657.99753 44923.01631 38286.11369 12421.66154 34636.48404 15.72_976.2852m/z 91.82083133 87.20746449 95.52811315 97.80294313 78.36444407 104.6456849 128.5566813 124.8173779 99.76182125 54.0733898 38.47123084 70.50034957 101.4852608 30.80193695 66.37494728 96.03894328 121.9710488 106.0807139 115.7515845 109.3416531 124.7923984 70.296151 95.58968119 124.8885948 78.58092042 102.8953624 26.38829184 56.69372456 69.8819402 99.30844468 104.8755296 19.00561704 116.1614758 150.6878588 74.80828357 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z Retention time (min) 0.74_793.0138m/z 793.0138088 0.7368 15.11_973.6249m/z 973.6248681 15.10966667 1.06_638.1461m/z 638.1461105 1.05705 0.70_203.1018m/z 203.1018133 0.695833333 14.89_812.2825m/z 812.2825266 14.89178333 12.70_499.1869m/z 499.1868607 12.70206667 0.74_778.0504m/z 778.0504174 0.7368 0.70_643.1102m/z 643.1102445 0.695833333 0.74_527.9673m/z 527.9672871 0.7368 0.74_822.0174m/z 822.0174017 0.7368 0.74_800.0312m/z 800.0311506 0.7368 0.70_159.1121m/z 159.1120907 0.695833333 0.74_784.0592m/z 784.0592413 0.7368 0.72_740.0952m/z 740.0951646 0.716316667 14.83_816.4788m/z 816.4788193 14.83035 15.09_947.6043m/z 947.6042519 15.08918333 0.70_726.0393m/z 726.0392905 0.695833333 1.95_663.5914m/z 663.5914112 1.950683333 12.70_458.1808m/z 458.1807798 12.70206667 12.70_498.1919m/z 498.1919426 12.70206667 15.82_303.2300m/z 303.2300437 15.81908333 1.95_660.6274n 641.6095827 1.950683333 0.70_621.1292m/z 621.1292256 0.695833333 0.70_605.1571m/z 605.1570577 0.695833333 0.70_731.0388m/z 731.038769 0.695833333 0.70_291.2241m/z 291.2240723 0.695833333 12.70_421.1531m/z 421.1531229 12.70206667 0.70_292.0895n 291.0821696 0.695833333 0.70_646.1567n 627.1388134 0.695833333 0.72_718.1099m/z 718.1098515 0.716316667 0.72_667.0678m/z 667.0678083 0.716316667 0.70_248.0989n 247.0920965 0.695833333 14.65_836.2780m/z 836.2779553 14.64596667 12.70_431.1822m/z 431.1822192 12.70206667 0.70_507.0076m/z 507.007573 0.695833333 14.54_269.2092m/z 269.2092159 14.54358333 6.45_205.0600m/z 205.059984 6.453033333 14.56_166.9917m/z 166.9917085 14.56405 0.74_762.0777m/z 762.0777276 0.7368 15.25_566.3429m/z 566.3428541 15.253 14.91_367.2434m/z 367.2433864 14.91226667 3.59_145.0751m/z 145.0750674 3.594733333 14.87_792.4816m/z 792.4815764 14.87131667 0.74_806.0375m/z 806.0374685 0.7368 15.25_506.3211m/z 506.3210694 15.253 4.16_495.1008m/z 495.1007863 4.160716667 12.70_385.1767m/z 385.1767091 12.70206667 0.74_192.0250n 191.0178052 0.7368 0.70_753.0255m/z 753.0255247 0.695833333 15.07_792.4754m/z 792.4754197 15.06871667 14.61_393.1687m/z 393.1687076 14.605 0.70_201.0861m/z 201.0861067 0.695833333 12.70_501.1865m/z 501.1864792 12.70206667 14.71_977.5624m/z 977.5623662 14.7074 15.80_327.2296m/z 327.2296257 15.7986 13.67_435.2034m/z 435.2033996 13.67033333 0.72_669.0627m/z 669.062706 0.716316667 13.92_281.2456m/z 281.2456452 13.91606667 14.69_476.2756m/z 476.2755761 14.68691667 1.95_652.6012m/z 652.601193 1.950683333 12.05_403.1865m/z 403.186475 12.0464 8.85_729.3137m/z 729.3136631 8.847616667 0.70_215.0639m/z 215.0638625 0.695833333 0.74_771.0242m/z 771.0241957 0.7368 14.71_881.5399m/z 881.5398706 14.7074 0.70_313.0643m/z 313.0643337 0.695833333 10.75_357.0609m/z 357.0609011 10.75061667 12.70_423.1508m/z 423.1507695 12.70206667 14.42_720.4553m/z 720.4552813 14.42071667 15.82_259.2396m/z 259.2396148 15.81908333 13.08_243.1933m/z 243.1933151 13.08376667 14.77_175.9612m/z 175.9611774 14.76883333 14.67_588.3270m/z 588.3270231 14.66643333 14.95_590.3359m/z 590.3358797 14.95321667 15.70_404.2678m/z 404.2677785 15.70358333 0.70_972.1460m/z 972.1460266 0.695833333 13.04_327.1713m/z 327.1713239 13.0428 7.87_213.0125m/z 213.0125181 7.8719 14.87_880.2643m/z 880.264305 14.87131667 12.13_301.1999m/z 301.1998805 12.12831667 13.47_365.1549m/z 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