#METABOLOMICS WORKBENCH jburgess_20160303_102854 STUDY_ID:ST000357 ANALYSIS_ID:AN000584 PROJECT_ID:PR000285 VERSION 1 CREATED_ON March 11, 2016, 10:45 am #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 hypothalmus from Anxiety Prone HSV-Latently ST:STUDY_TITLE Infected Obese Mice ST:STUDY_TYPE Broad Spectrum LCMS ST:STUDY_SUMMARY Brain tissue 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 brain tissue 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 31 ST:NUM_MALES 31 #SUBJECT SU:SUBJECT_TYPE mouse SU:SUBJECT_SPECIES Mus musculus SU:GENOTYPE_STRAIN C57Bl/6 SU:AGE_OR_AGE_RANGE 5.5 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 386 Hypo386 HSV-1:no | Diet at Week 2:HF Weight (g) =34.8 SUBJECT_SAMPLE_FACTORS 415 Hypo415 HSV-1:yes | Diet at Week 2:LF Weight (g) =34.8 SUBJECT_SAMPLE_FACTORS 411 Hypo411 HSV-1:yes | Diet at Week 2:HF Weight (g) =40.2 SUBJECT_SAMPLE_FACTORS 402 Hypo402 HSV-1:no | Diet at Week 2:HF Weight (g) =38.8 SUBJECT_SAMPLE_FACTORS 390 Hypo390 HSV-1:no | Diet at Week 2:LF Weight (g) =34.5 SUBJECT_SAMPLE_FACTORS 391 Hypo391 HSV-1:no | Diet at Week 2:LF Weight (g) =31.2 SUBJECT_SAMPLE_FACTORS 398 Hypo398 HSV-1:yes | Diet at Week 2:LF Weight (g) =35.2 SUBJECT_SAMPLE_FACTORS 385 Hypo385 HSV-1:no | Diet at Week 2:HF Weight (g) =41.6 SUBJECT_SAMPLE_FACTORS 405 Hypo405 HSV-1:no | Diet at Week 2:LF Weight (g) =30.8 SUBJECT_SAMPLE_FACTORS 395 Hypo395 HSV-1:yes | Diet at Week 2:HF Weight (g) =40.9 SUBJECT_SAMPLE_FACTORS 410 Hypo410 HSV-1:yes | Diet at Week 2:HF Weight (g) =37.8 SUBJECT_SAMPLE_FACTORS 387 Hypo387 HSV-1:no | Diet at Week 2:HF Weight (g) =31.9 SUBJECT_SAMPLE_FACTORS 413 Hypo413 HSV-1:yes | Diet at Week 2:LF Weight (g) =36 SUBJECT_SAMPLE_FACTORS 396 Hypo396 HSV-1:yes | Diet at Week 2:HF Weight (g) =48.4 SUBJECT_SAMPLE_FACTORS 414 Hypo414 HSV-1:yes | Diet at Week 2:LF Weight (g) =36.6 SUBJECT_SAMPLE_FACTORS 409 Hypo409 HSV-1:yes | Diet at Week 2:HF Weight (g) =37.1 SUBJECT_SAMPLE_FACTORS 388 Hypo388 HSV-1:no | Diet at Week 2:HF Weight (g) =44.1 SUBJECT_SAMPLE_FACTORS 397 Hypo397 HSV-1:yes | Diet at Week 2:LF Weight (g) =33.1 SUBJECT_SAMPLE_FACTORS 399 Hypo399 HSV-1:yes | Diet at Week 2:LF Weight (g) =33 SUBJECT_SAMPLE_FACTORS 389 Hypo389 HSV-1:no | Diet at Week 2:LF Weight (g) =34 SUBJECT_SAMPLE_FACTORS 393 Hypo393 HSV-1:yes | Diet at Week 2:HF Weight (g) =44.1 SUBJECT_SAMPLE_FACTORS 412 Hypo412 HSV-1:yes | Diet at Week 2:HF Weight (g) =34.7 SUBJECT_SAMPLE_FACTORS 392 Hypo392 HSV-1:no | Diet at Week 2:LF Weight (g) =34 SUBJECT_SAMPLE_FACTORS 408 Hypo408 HSV-1:no | Diet at Week 2:LF Weight (g) =29.1 SUBJECT_SAMPLE_FACTORS 407 Hypo407 HSV-1:no | Diet at Week 2:LF Weight (g) =34.7 SUBJECT_SAMPLE_FACTORS 394 Hypo394 HSV-1:yes | Diet at Week 2:HF Weight (g) =48.2 SUBJECT_SAMPLE_FACTORS 400 Hypo400 HSV-1:yes | Diet at Week 2:LF Weight (g) =33.2 SUBJECT_SAMPLE_FACTORS 406 Hypo406 HSV-1:no | Diet at Week 2:LF Weight (g) =31.4 SUBJECT_SAMPLE_FACTORS 403 Hypo403 HSV-1:no | Diet at Week 2:HF Weight (g) =48.6 SUBJECT_SAMPLE_FACTORS 401 Hypo401 HSV-1:no | Diet at Week 2:HF Weight (g) =49.2 SUBJECT_SAMPLE_FACTORS 416 Hypo416 HSV-1:yes | Diet at Week 2:LF Weight (g) =35.2 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 Brain tissue samples were homogenized in 50:50 Acetonitrile:Water (10 µL to CO:COLLECTION_SUMMARY every 1 mg of tissue) with washed ceramic beads on a MagNA Lyser, using two 30 CO:COLLECTION_SUMMARY sec pulses at 2,000 rpm with a 1 min chilling step in between pulses. Samples CO:COLLECTION_SUMMARY were vortexed on a multi-tube vortexer for 2 min at 5,000 rpm and centrifuged at CO:COLLECTION_SUMMARY room temperature for 4 min at 16,000 rcf. A 50 µL aliquot of each experimental CO:COLLECTION_SUMMARY brain tissue homogenized supernatant was transferred to a labeled 2.0 mL Lo-Bind CO:COLLECTION_SUMMARY eppendorf tube. Analytical pooled samples were created by combining 15 µL CO:COLLECTION_SUMMARY aliquot from each experimental sample in a 2.0 mL Lo-Bind eppendorf tube. The QC CO:COLLECTION_SUMMARY pooled sample was vortexed for 30 sec and 50 µL aliquots were transferred to 5 CO:COLLECTION_SUMMARY labeled 2.0 mL Lo-Bind eppendorf tubes. Next, 350 µL of acetonitrile was added CO:COLLECTION_SUMMARY to study sample and QC pooled sample tubes. The samples were vortexed on a CO:COLLECTION_SUMMARY multi-tube vortexer for 2 min at 5,000 rpm, and centrifuged at room temperature CO:COLLECTION_SUMMARY for 4 min at 16,000 rcf. The supernatants were transferred to new labeled tubes CO:COLLECTION_SUMMARY and dried on a lyophilizer overnight. Dried samples were reconstituted in 50 µL CO:COLLECTION_SUMMARY of 95:5 Water:Methanol, mixed on an analog vortex mixer for 1 min with speed set CO:COLLECTION_SUMMARY at 10. Then, the samples were centrifuged at room temperature for 4 min at CO:COLLECTION_SUMMARY 16,000 rcf, and the supernatants were transferred to autosampler vials for data CO:COLLECTION_SUMMARY acquisition. UPLC-MS Methods: UPLC-MS spectra were collected for all samples. CO:COLLECTION_SUMMARY UPLC was performed on a Waters Acquity UPLC with an Acquity HSS T3 column (2.1x CO:COLLECTION_SUMMARY 100mm x 1.8 µm) at 50 °C using the reversed-phase separation. Water with 0.1% CO:COLLECTION_SUMMARY formic acid (mobile phase A) and methanol with 0.1% formic acid (mobile phase B) CO:COLLECTION_SUMMARY were used as mobile phases and the metabolites were chromatographically CO:COLLECTION_SUMMARY separated using a gradient separation: (see the 3. CO:COLLECTION_SUMMARY Sheridan-Mice-Hypothalamus_RP-Metadata and Analytical Metadata.xlsx file for the CO:COLLECTION_SUMMARY flow gradient). Mass spectroscopy analysis was performed using a Synapt G2 Si CO:COLLECTION_SUMMARY ESI-Q-TOF using a 10 µL injection volume. UPLC-MS data were collected over CO:COLLECTION_SUMMARY 70-1000 m/z in both positive and negative modes. CO:SAMPLE_TYPE brain homogenate #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 Brain tissue samples were homogenized in 50:50 Acetonitrile:Water (10 µL to SP:SAMPLEPREP_SUMMARY every 1 mg of tissue) with washed ceramic beads on a MagNA Lyser, using two 30 SP:SAMPLEPREP_SUMMARY sec pulses at 2,000 rpm with a 1 min chilling step in between pulses. Samples SP:SAMPLEPREP_SUMMARY were vortexed on a multi-tube vortexer for 2 min at 5,000 rpm and centrifuged at SP:SAMPLEPREP_SUMMARY room temperature for 4 min at 16,000 rcf. A 50 µL aliquot of each experimental SP:SAMPLEPREP_SUMMARY brain tissue homogenized supernatant was transferred to a labeled 2.0 mL Lo-Bind SP:SAMPLEPREP_SUMMARY eppendorf tube. Analytical pooled samples were created by combining 15 µL SP:SAMPLEPREP_SUMMARY aliquot from each experimental sample in a 2.0 mL Lo-Bind eppendorf tube. The QC SP:SAMPLEPREP_SUMMARY pooled sample was vortexed for 30 sec and 50 µL aliquots were transferred to 5 SP:SAMPLEPREP_SUMMARY labeled 2.0 mL Lo-Bind eppendorf tubes. Next, 350 µL of acetonitrile was added SP:SAMPLEPREP_SUMMARY to study sample and QC pooled sample tubes. The samples were vortexed on a SP:SAMPLEPREP_SUMMARY multi-tube vortexer for 2 min at 5,000 rpm, and centrifuged at room temperature SP:SAMPLEPREP_SUMMARY for 4 min at 16,000 rcf. The supernatants were transferred to new labeled tubes SP:SAMPLEPREP_SUMMARY and dried on a lyophilizer overnight. Dried samples were reconstituted in 50 µL SP:SAMPLEPREP_SUMMARY of 95:5 Water:Methanol, mixed on an analog vortex mixer for 1 min with speed set SP:SAMPLEPREP_SUMMARY at 10. Then, the samples were centrifuged at room temperature for 4 min at SP:SAMPLEPREP_SUMMARY 16,000 rcf, and the supernatants were transferred to autosampler vials for data SP:SAMPLEPREP_SUMMARY 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 #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE #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 Hypo385 Hypo386 Hypo387 Hypo388 Hypo401 Hypo402 Hypo403 Hypo393 Hypo394 Hypo395 Hypo396 Hypo409 Hypo410 Hypo411 Hypo412 Hypo389 Hypo390 Hypo391 Hypo392 Hypo405 Hypo406 Hypo407 Hypo408 Hypo397 Hypo398 Hypo399 Hypo400 Hypo413 Hypo414 Hypo415 Hypo416 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: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: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 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 2.50_175.0461m/z 74.16137632 76.13577011 93.43945562 81.17861884 85.26437858 82.13403451 86.16148689 56.89102537 129.0140038 249.751723 109.7622163 155.9852699 106.2193899 142.4067095 119.0454741 129.6006931 57.73589832 127.8362545 82.66920109 28.76655 33.95410273 26.85225508 43.32282621 36.92539498 50.63837966 51.80986142 65.76334788 18.86524187 53.21465554 104.500108 22.77622671 33.94731516 55.45684129 55.11764451 95.13981206 27.38221112 0.87_160.1321m/z 245.2359824 205.1115045 260.5100864 214.103176 211.7471308 86.09285526 128.0704134 73.23823857 64.59421475 71.52487282 142.9409762 74.64658595 208.0304028 271.0043344 459.3028967 259.7569665 72.04363924 100.4424275 90.68423008 78.64376069 292.7385637 316.8555943 238.1780366 243.0955291 516.7278471 182.683081 97.44824644 433.679724 298.7879971 1055.546925 270.2305334 309.9591447 277.4756359 279.4503969 386.7867852 89.55688747 9.35_382.7348m/z 14.26180475 12.5038147 14.09099071 19.62894748 24.54626781 39.91910752 15.67555516 16.62626509 30.84792833 15.42275632 7.953024145 15.38348274 5.849619575 54.64478911 26.28670454 20.7011902 40.15117622 5.851711672 16.43500875 4.63747368 5.493192391 0 0 25.45881739 12.83026882 0 0 0 18.79481007 10.51440108 18.37676747 6.943996169 0 0 0 0 11.77_301.2706m/z 24.56018702 18.68229853 2.792470663 2.360096732 14.12848443 67.35492452 5.691763098 0.830145434 0.571061692 101.6027611 106.4279543 952.8386692 1.172421077 0.185595003 1.459599702 0.539059213 0.131108309 73.43252253 0.525960932 0.286533566 0.465346054 0.097564586 0.039517095 3.610184874 0.094419518 0.539814582 2.225193742 0.529056526 0.498609213 0.475400378 1.52033804 1.740020782 0.017651046 1.428754857 3.069053908 0.833139015 3.85_239.0901m/z 69.8482839 55.2768681 64.53577906 63.44397893 60.50278448 62.55214006 70.34890354 52.40661659 70.94164989 81.9635256 98.76425505 64.74933631 73.07929738 83.56280564 51.48697147 84.748444 65.94512116 70.03573106 62.32553984 58.74968711 43.64887927 38.82861517 68.66255274 62.76419574 49.69151426 52.09935627 52.34550647 26.00095592 55.51351692 41.75041871 18.3124652 39.34954089 57.4804616 43.03768156 61.3648132 36.01291081 0.74_160.1322m/z 338.7793549 289.5123156 284.7196344 270.3732461 249.5563884 134.0545101 179.3278097 121.341614 77.61917084 79.65949744 182.6589497 76.41154182 292.1805622 338.7999091 597.3588539 429.2544271 99.17670528 111.7767888 126.5846863 122.9171895 410.5413251 497.5732615 309.9593304 430.7732259 522.0952634 243.8813861 118.389801 609.8656814 349.3183748 1138.671073 420.3778486 362.1756197 372.3595875 309.0872015 496.782363 110.7404361 3.85_89.0476n 348.7095889 294.2544381 356.9784927 313.8038322 322.0018628 333.8202625 348.2679994 258.7195131 379.8785178 431.6113774 504.5090503 332.9206963 419.6742451 429.3447314 266.4612224 440.4975086 328.7266776 362.0491001 363.6920666 317.2120453 246.7844512 236.5536709 385.3561572 331.9951979 270.2648801 253.8881724 248.8381337 147.5288155 272.8672293 252.1381203 103.4373265 202.3621074 310.3758057 238.3766173 310.1265018 190.8902178 6.47_322.6988m/z 54.65560344 48.17584248 51.60078218 46.5933666 57.08325809 23.2319439 20.48952811 23.48279402 38.84105213 48.34446284 32.01806076 47.3095068 42.82983742 40.93237 30.34574551 40.9983729 38.68770997 33.02781968 27.35164237 47.39057581 36.63763643 46.83069843 52.8399769 55.45602226 43.07588509 76.29050112 55.4390166 54.23304664 49.4198146 48.62589621 29.38242592 48.67670246 47.03726793 41.71214689 31.01212719 43.34695633 15.54_293.6496m/z 22.99132736 35.41749573 28.09345906 33.99608856 32.24938106 40.24162132 43.03731585 43.41115695 23.77684175 32.87668214 44.52967899 32.01975727 33.17513855 42.77365222 28.72920464 39.74860566 34.30297285 37.50966239 43.94991226 42.94892661 23.16835236 20.58041318 19.96122628 35.2542707 9.89547427 21.76026436 28.44889322 41.09814579 22.23456797 21.94007652 16.65893279 19.60567572 27.46060401 25.33716956 26.48435316 32.80344978 3.85_460.2017n 344.7959948 256.1287346 348.59483 275.0132626 289.4272031 292.4576522 254.0116073 191.8006848 381.9126841 397.0821937 426.9146404 288.7254513 403.4265469 398.819066 193.7883335 379.7314219 303.283561 291.4600396 244.8581227 313.718484 187.2289421 141.8356171 342.4854276 264.7290484 221.4229417 208.1437287 176.3368929 71.12961616 267.5397229 147.4941736 29.72373642 123.1869426 232.0104183 192.5852748 196.0448958 78.13875074 7.69_349.1600m/z 47.30240813 46.48149109 49.22023948 53.73366483 50.44046903 30.3750448 35.30562318 39.48378216 37.31899463 44.71623919 42.8908201 53.11159251 43.9436028 37.44381364 31.04126427 43.23514979 43.0465799 38.67815153 30.855703 43.51173735 54.27799523 45.88727824 61.81030705 45.51115305 44.52263733 72.42249033 53.32756103 45.05662313 46.92954057 37.74558405 33.65977568 49.28316065 42.02710567 49.48970459 47.25459349 45.5773436 3.85_274.0363m/z 73.37680123 53.11347032 58.29668461 52.68861025 56.57694239 73.05183017 80.36944378 69.49507254 67.41499696 68.24551681 104.0512366 66.36086582 76.78606108 73.60654102 56.40251516 85.12762469 57.84638712 72.09465478 77.98810272 64.88250267 56.48640839 51.8211869 70.36288427 63.22716545 50.83312718 49.12227547 54.36063726 37.08526669 59.16307222 57.74133383 27.72070209 43.1729599 64.58261337 45.54144667 75.23976708 29.48047403 13.00_315.3004n 127392.4468 133930.3052 107583.9429 138184.0591 141206.1547 106708.2264 106843.6126 93468.51395 112693.6645 118666.503 132237.7469 108099.2304 150659.6094 142229.8837 125470.8606 158273.8946 127102.2883 130315.1383 126244.0653 163079.1842 118405.3363 130342.058 118992.331 142455.8086 99565.95842 125434.8457 126343.8569 137227.1209 120725.9397 133868.8188 160887.9391 146557.5745 118666.8871 127919.356 140489.0015 138880.5607 14.89_279.6353m/z 317.2024501 317.6867409 297.0677177 329.2815223 337.039465 349.6128523 399.5471508 288.6294016 341.7701321 420.3580858 660.1594993 267.3709218 432.60504 635.2763692 265.0642897 352.6635502 446.1392771 487.239467 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