#METABOLOMICS WORKBENCH Simcox_Lab_20240520_120841 DATATRACK_ID:4852 STUDY_ID:ST003215 ANALYSIS_ID:AN005272 PROJECT_ID:PR002005 VERSION 1 CREATED_ON May 21, 2024, 2:05 pm #PROJECT PR:PROJECT_TITLE Protein restriction slows the development and progression of Alzheimer's disease PR:PROJECT_TITLE in mice PR:PROJECT_TYPE MS targeted lipidomics PR:PROJECT_SUMMARY Dietary protein is a critical regulator of metabolic health and aging. Low PR:PROJECT_SUMMARY protein diets are associated with healthy aging in humans, and many independent PR:PROJECT_SUMMARY groups of researchers have shown that dietary protein restriction (PR) extends PR:PROJECT_SUMMARY the lifespan and healthspan of mice. Here, we examined the effect of PR on PR:PROJECT_SUMMARY metabolic health and the development and progression of Alzheimer’s disease PR:PROJECT_SUMMARY (AD) in the 3xTg mouse model of AD. We found that PR has metabolic benefits for PR:PROJECT_SUMMARY 3xTg mice and non-transgenic controls of both sexes, promoting leanness and PR:PROJECT_SUMMARY glycemic control in 3xTg mice and rescuing the glucose intolerance of 3xTg PR:PROJECT_SUMMARY females. We found that PR induces sex-specific alterations in circulating PR:PROJECT_SUMMARY metabolites and in the brain lipidome, downregulating sphingolipid subclasses PR:PROJECT_SUMMARY including ceramides, glucosylceramides, and sphingomyelins in 3xTg females. PR:PROJECT_SUMMARY Consumption of a PR diet starting at 6 months of age reduced AD pathology in PR:PROJECT_SUMMARY conjunction with reduced mTORC1 activity, increased autophagy, and had cognitive PR:PROJECT_SUMMARY benefits for 3xTg mice. Finally, PR improved the survival of 3xTg mice. Our PR:PROJECT_SUMMARY results demonstrate that PR slows the progression of AD at molecular and PR:PROJECT_SUMMARY pathological levels, preserves cognition in this mouse model of AD, and suggests PR:PROJECT_SUMMARY that PR or pharmaceutical interventions that mimic the effects of this diet may PR:PROJECT_SUMMARY hold promise as a treatment for AD. PR:INSTITUTE University of Wisconsin-Madison PR:LAST_NAME Simcox PR:FIRST_NAME Judith PR:ADDRESS 433 Babcock Dr, Madison, WI, 53706, USA PR:EMAIL jsimcox@wisc.edu PR:PHONE - #STUDY ST:STUDY_TITLE Protein restriction slows the development and progression of Alzheimer's disease ST:STUDY_TITLE in mice ST:STUDY_SUMMARY Dietary protein is a critical regulator of metabolic health and aging. Low ST:STUDY_SUMMARY protein diets are associated with healthy aging in humans, and many independent ST:STUDY_SUMMARY groups of researchers have shown that dietary protein restriction (PR) extends ST:STUDY_SUMMARY the lifespan and healthspan of mice. Here, we examined the effect of PR on ST:STUDY_SUMMARY metabolic health and the development and progression of Alzheimer’s disease ST:STUDY_SUMMARY (AD) in the 3xTg mouse model of AD. We found that PR has metabolic benefits for ST:STUDY_SUMMARY 3xTg mice and non-transgenic controls of both sexes, promoting leanness and ST:STUDY_SUMMARY glycemic control in 3xTg mice and rescuing the glucose intolerance of 3xTg ST:STUDY_SUMMARY females. We found that PR induces sex-specific alterations in circulating ST:STUDY_SUMMARY metabolites and in the brain lipidome, downregulating sphingolipid subclasses ST:STUDY_SUMMARY including ceramides, glucosylceramides, and sphingomyelins in 3xTg females. ST:STUDY_SUMMARY Consumption of a PR diet starting at 6 months of age reduced AD pathology in ST:STUDY_SUMMARY conjunction with reduced mTORC1 activity, increased autophagy, and had cognitive ST:STUDY_SUMMARY benefits for 3xTg mice. Finally, PR improved the survival of 3xTg mice. Our ST:STUDY_SUMMARY results demonstrate that PR slows the progression of AD at molecular and ST:STUDY_SUMMARY pathological levels, preserves cognition in this mouse model of AD, and suggests ST:STUDY_SUMMARY that PR or pharmaceutical interventions that mimic the effects of this diet may ST:STUDY_SUMMARY hold promise as a treatment for AD. ST:INSTITUTE University of Wisconsin-Madison ST:LAST_NAME Simcox ST:FIRST_NAME Judith ST:ADDRESS 433 Babcock Dr, Madison, WI, 53706, USA ST:EMAIL jsimcox@wisc.edu ST:PHONE - ST:TOTAL_SUBJECTS 40 ST:NUM_MALES 20 ST:NUM_FEMALES 20 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN 3xTg SU:GENDER Male and female SU:ANIMAL_FEED Control, TD.180161; PR, TD.10192 #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 - F1 Sample source:Brain | Genotype:Non-transgenic | Treatment:Control RAW_FILE_NAME(Raw file name)=F1_pos_cer_dMRM_072023_063.d SUBJECT_SAMPLE_FACTORS - F2 Sample source:Brain | Genotype:Non-transgenic | Treatment:Control RAW_FILE_NAME(Raw file name)=F2_pos_cer_dMRM_072023_011.d SUBJECT_SAMPLE_FACTORS - F3 Sample source:Brain | Genotype:Non-transgenic | Treatment:Control RAW_FILE_NAME(Raw file name)=F3_pos_cer_dMRM_072023_033.d SUBJECT_SAMPLE_FACTORS - F4 Sample source:Brain | Genotype:Non-transgenic | Treatment:Control RAW_FILE_NAME(Raw file name)=F4_pos_cer_dMRM_072023_015.d SUBJECT_SAMPLE_FACTORS - F5 Sample source:Brain | Genotype:Non-transgenic | Treatment:Control RAW_FILE_NAME(Raw file name)=F5_pos_cer_dMRM_072023_025.d SUBJECT_SAMPLE_FACTORS - F6 Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=F6_pos_cer_dMRM_072023_061.d SUBJECT_SAMPLE_FACTORS - F7 Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=F7_pos_cer_dMRM_072023_065.d SUBJECT_SAMPLE_FACTORS - F8 Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=F8_pos_cer_dMRM_072023_023.d SUBJECT_SAMPLE_FACTORS - F9 Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=F9_pos_cer_dMRM_072023_079.d SUBJECT_SAMPLE_FACTORS - F10 Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=F10_pos_cer_dMRM_072023_081.d SUBJECT_SAMPLE_FACTORS - F11 Sample source:Brain | Genotype:3xTg | Treatment:Control RAW_FILE_NAME(Raw file name)=F11_pos_cer_dMRM_072023_047.d SUBJECT_SAMPLE_FACTORS - F12 Sample source:Brain | Genotype:3xTg | Treatment:Control RAW_FILE_NAME(Raw file name)=F12_pos_cer_dMRM_072023_057.d SUBJECT_SAMPLE_FACTORS - F13 Sample source:Brain | Genotype:3xTg | Treatment:Control RAW_FILE_NAME(Raw file name)=F13_pos_cer_dMRM_072023_075.d SUBJECT_SAMPLE_FACTORS - F14 Sample source:Brain | Genotype:3xTg | Treatment:Control RAW_FILE_NAME(Raw file name)=F14_pos_cer_dMRM_072023_051.d SUBJECT_SAMPLE_FACTORS - F15 Sample source:Brain | Genotype:3xTg | Treatment:Control RAW_FILE_NAME(Raw file name)=F15_pos_cer_dMRM_072023_059.d SUBJECT_SAMPLE_FACTORS - F16 Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=F16_pos_cer_dMRM_072023_031.d SUBJECT_SAMPLE_FACTORS - F17 Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=F17_pos_cer_dMRM_072023_089.d SUBJECT_SAMPLE_FACTORS - F18 Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=F18_pos_cer_dMRM_072023_009.d SUBJECT_SAMPLE_FACTORS - F19 Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=F19_pos_cer_dMRM_072023_071.d SUBJECT_SAMPLE_FACTORS - F20 Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=F20_pos_cer_dMRM_072023_019.d SUBJECT_SAMPLE_FACTORS - M1 Sample source:Brain | Genotype:Non-transgenic | Treatment:Control RAW_FILE_NAME(Raw file name)=M1_pos_cer_dMRM_072023_083.d SUBJECT_SAMPLE_FACTORS - M2 Sample source:Brain | Genotype:Non-transgenic | Treatment:Control RAW_FILE_NAME(Raw file name)=M2_pos_cer_dMRM_072023_035.d SUBJECT_SAMPLE_FACTORS - M3 Sample source:Brain | Genotype:Non-transgenic | Treatment:Control RAW_FILE_NAME(Raw file name)=M3_pos_cer_dMRM_072023_029.d SUBJECT_SAMPLE_FACTORS - M4 Sample source:Brain | Genotype:Non-transgenic | Treatment:Control RAW_FILE_NAME(Raw file name)=M4_pos_cer_dMRM_072023_041.d SUBJECT_SAMPLE_FACTORS - M5 Sample source:Brain | Genotype:Non-transgenic | Treatment:Control RAW_FILE_NAME(Raw file name)=M5_pos_cer_dMRM_072023_017.d SUBJECT_SAMPLE_FACTORS - M6 Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=M6_pos_cer_dMRM_072023_053.d SUBJECT_SAMPLE_FACTORS - M7 Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=M7_pos_cer_dMRM_072023_013.d SUBJECT_SAMPLE_FACTORS - M8 Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=M8_pos_cer_dMRM_072023_021.d SUBJECT_SAMPLE_FACTORS - M9 Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=M9_pos_cer_dMRM_072023_087.d SUBJECT_SAMPLE_FACTORS - M10 Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=M10_pos_cer_dMRM_072023_043.d SUBJECT_SAMPLE_FACTORS - M11 Sample source:Brain | Genotype:3xTg | Treatment:Control RAW_FILE_NAME(Raw file name)=M11_pos_cer_dMRM_072023_073.d SUBJECT_SAMPLE_FACTORS - M12 Sample source:Brain | Genotype:3xTg | Treatment:Control RAW_FILE_NAME(Raw file name)=M12_pos_cer_dMRM_072023_067.d SUBJECT_SAMPLE_FACTORS - M13 Sample source:Brain | Genotype:3xTg | Treatment:Control RAW_FILE_NAME(Raw file name)=M13_pos_cer_dMRM_072023_037.d SUBJECT_SAMPLE_FACTORS - M14 Sample source:Brain | Genotype:3xTg | Treatment:Control RAW_FILE_NAME(Raw file name)=M14_pos_cer_dMRM_072023_077.d SUBJECT_SAMPLE_FACTORS - M15 Sample source:Brain | Genotype:3xTg | Treatment:Control RAW_FILE_NAME(Raw file name)=M15_pos_cer_dMRM_072023_091.d SUBJECT_SAMPLE_FACTORS - M16 Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=M16_pos_cer_dMRM_072023_049.d SUBJECT_SAMPLE_FACTORS - M17 Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=M17_pos_cer_dMRM_072023_069.d SUBJECT_SAMPLE_FACTORS - M18 Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=M18_pos_cer_dMRM_072023_045.d SUBJECT_SAMPLE_FACTORS - M19 Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=M19_pos_cer_dMRM_072023_039.d SUBJECT_SAMPLE_FACTORS - M20 Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction RAW_FILE_NAME(Raw file name)=M20_pos_cer_dMRM_072023_027.d #COLLECTION CO:COLLECTION_SUMMARY Mice were euthanized by cervical dislocation after a 3 hour fast, and the right CO:COLLECTION_SUMMARY hemisphere was fixed in formalin for histology whereas the left hemisphere was CO:COLLECTION_SUMMARY snap-frozen for mass spectrometry analysis. CO:SAMPLE_TYPE Brain #TREATMENT TR:TREATMENT_SUMMARY At the start of the experiment, mice were randomized to receive either a 21% TR:TREATMENT_SUMMARY protein diet (Control, TD.180161) or a 7% protein diet (PR, TD.10192) obtained TR:TREATMENT_SUMMARY from Envigo. Our diets were formulated to be isocaloric, meaning that any TR:TREATMENT_SUMMARY reduction in protein content was compensated for by adjusting the carbohydrate TR:TREATMENT_SUMMARY content and fat percent stayed constant. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Whole brain samples were pulverized and 20 mg were homogenized in 215 µL MeOH SP:SAMPLEPREP_SUMMARY plus 10 µL of a 30 µM solution of each of the following internal standards: SP:SAMPLEPREP_SUMMARY Cer d18:1(d7)_15:0 (Avanti #67492-15-3), Cer d18:1(d7)_16:0 (Avanti # SP:SAMPLEPREP_SUMMARY 1840942-13-3), Cer d18:1(d7)_18:0 (Avanti #1840942-14-4), Cer d18:1(d7)_24:0 SP:SAMPLEPREP_SUMMARY (Avanti #1840942-15-5), Cer d18:1(d7)_24:1 (Avanti # 1840942-16-6), and SM SP:SAMPLEPREP_SUMMARY d18:1(d7)_18:1 (Avanti # 2342574-42-7). The samples were homogenized in bead SP:SAMPLEPREP_SUMMARY tubes (1.4mm, Qiagen, #13113-50) in a Qiagen TissueLyzer II (catalog no.: SP:SAMPLEPREP_SUMMARY 9244420) for 2 cycles in blocks chilled to 4°C. 250 µL H2O and 750 µL MTBE SP:SAMPLEPREP_SUMMARY were then added, and the samples were inverted to mix and placed on ice. After SP:SAMPLEPREP_SUMMARY 15 minutes, the samples were centrifuged at 4 °C at 16000 x g for 5 minutes, SP:SAMPLEPREP_SUMMARY and 500 µL of the top organic phase was removed into a new tube and dried using SP:SAMPLEPREP_SUMMARY a speedvac. Lipids were then resuspended in 150 µL IPA and stored at -20°C SP:SAMPLEPREP_SUMMARY until analysis. An insoluble precipitate was also observed when the extracts SP:SAMPLEPREP_SUMMARY were resuspended in IPA. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY For targeted sphingolipid separation CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) CH:SOLVENT_A 60:40 acetonitrile:water; 10 mM ammonium formate; 0.1% formic acid CH:SOLVENT_B 9:1:90 acetonitrile:water:isopropyl alcolhol; 10 mM ammonium formate; 0.1% CH:SOLVENT_B formic acid CH:FLOW_GRADIENT The gradient began with 30% B and increased to 60% over 1.8 min, then increased CH:FLOW_GRADIENT to 80% until 7 min and 99% until 7.14 min, which was maintained until 10 min. CH:FLOW_RATE 0.5 mL/min CH:COLUMN_TEMPERATURE 60 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6495 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Data was processed in the Agilent MassHunter Wokstation, and sphingolipids were MS:MS_COMMENTS quantified by peak height based on the relative concentration of the appropriate MS:MS_COMMENTS internal standard within the samples. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ng lipid/mg brain MS_METABOLITE_DATA_START Samples M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20 Factors Sample source:Brain | Genotype:Non-transgenic | Treatment:Control Sample source:Brain | Genotype:Non-transgenic | Treatment:Control Sample source:Brain | Genotype:Non-transgenic | Treatment:Control Sample source:Brain | Genotype:Non-transgenic | Treatment:Control Sample source:Brain | Genotype:Non-transgenic | Treatment:Control Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction Sample source:Brain | Genotype:3xTg | Treatment:Control Sample source:Brain | Genotype:3xTg | Treatment:Control Sample source:Brain | Genotype:3xTg | Treatment:Control Sample source:Brain | Genotype:3xTg | Treatment:Control Sample source:Brain | Genotype:3xTg | Treatment:Control Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction Sample source:Brain | Genotype:Non-transgenic | Treatment:Control Sample source:Brain | Genotype:Non-transgenic | Treatment:Control Sample source:Brain | Genotype:Non-transgenic | Treatment:Control Sample source:Brain | Genotype:Non-transgenic | Treatment:Control Sample source:Brain | Genotype:Non-transgenic | Treatment:Control Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction Sample source:Brain | Genotype:Non-transgenic | Treatment:Protein restriction Sample source:Brain | Genotype:3xTg | Treatment:Control Sample source:Brain | Genotype:3xTg | Treatment:Control Sample source:Brain | Genotype:3xTg | Treatment:Control Sample source:Brain | Genotype:3xTg | Treatment:Control Sample source:Brain | Genotype:3xTg | Treatment:Control Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction Sample source:Brain | Genotype:3xTg | Treatment:Protein restriction GlcCer (d18:1/16:0) 0.493310748 0.111154286 0.182621565 0.283442689 0.100922297 0.349289151 0.299077602 0.292251389 0.11110967 0.450875785 0.232678906 0.438960427 0.364104906 0.447560846 0.356358471 0.198676128 0.069583065 0.296666883 0.240376402 0.479533582 0.646643125 0.229603125 0.22069949 0.341980282 0.237967284 0.461080839 0.399525 0.124375949 0.236158163 0.236926339 0.292086782 0.2060369 0.35053192 0.565696111 0.10333575 0.180239231 0.168794764 0.004992647 0.132707813 0.106744133 Cer(m18:1/20:0) 0.034235748 0.009159286 0.013149618 0.023293868 0.004675 0.028721462 0.016947285 0.027097222 0.009798113 0.034368161 0.0153375 0.041955213 0.008015094 0.033777574 0.018570248 0.013629699 0.007033065 0.018165584 0.019290421 0.022435821 0.02456 0.027344196 0.035497959 0.020128873 0.011447531 0.02612573 0.034551446 0.0082 0.02319898 0.018589286 0.033583333 0.011531181 0.037409598 0.01742 0.01116975 0.012142308 0.013728534 0.011672794 0.008494531 0.0071625 GlcCer (d18:1/18:0) 3.204663084 0.927654286 1.208562595 1.307155189 0.626527703 1.991479245 1.583640271 2.27081875 0.640395991 3.404666368 1.604405469 2.327909716 1.507575283 2.509574449 2.175344008 0.937006015 0.485845968 1.917056494 1.710046262 2.450165672 2.532105625 1.383780134 1.684941837 2.148062676 1.297035185 2.624319526 1.86633905 0.564332278 1.671532653 1.217817188 1.624350575 0.960691328 1.888603125 2.23855 0.6233055 1.174626154 1.009130105 0 0.737214844 0.68380102 SM(d18:0/20:0) 769.6173722 157.1894721 330.937855 52.38265542 87.70778851 214.562683 110.8947387 362.0200493 67.04126816 181.034517 441.5364898 69.36666682 164.9988826 173.2971822 364.9225915 36.05131523 0 169.7878468 406.7311107 344.4805828 110.2055538 231.9263658 133.0018255 576.9531085 83.58740864 449.9469881 161.8275607 26.69744304 343.4852148 144.5247757 212.4233971 50.26437011 316.5675703 173.0900544 0 160.5264754 4.758688743 4988.891314 32.89289453 40.92917143 SM(d18:1/18:0) 81.83938178 69.21338571 52.58277252 64.58442594 57.76279459 76.7031934 44.76641267 96.50479236 54.54555142 71.10490628 86.85003047 82.68110118 53.62887962 66.26244154 67.01215289 48.87834474 85.88208065 55.0861526 57.48635257 77.63338433 81.23560875 81.91672969 60.97116939 69.34629366 44.59798457 47.8325688 49.81685888 59.41609114 58.26477143 92.24904174 60.66875115 49.50418284 75.85782321 57.49927556 65.90381025 113.6054777 69.7334678 676.0960132 88.4791375 108.4422995 Cer(d18:1/16:0) 0.036659579 0.031958571 0.020330725 0.060353774 0.028597973 0.039512972 0.065224208 0.031859028 0.050305896 0.032686547 0.062646875 0.04586872 0.021628302 0.020648162 0.046217975 0.067034774 0.037998387 0.054994156 0.032005374 0.042064925 0.049416875 0.054391741 0.063482143 0.060307746 0.049948148 0.040138139 0.03933781 0.042635443 0.035568367 0.039876563 0.034686207 0.040794465 0.036165402 0.056526111 0.0588795 0.026392308 0.075358115 0.040988971 0.043251563 0.038835459 Cer(d18:1/18:1) 1.152312617 0.3812 0.542324427 0.515145991 0.347589189 0.700835377 0.731902941 0.817404861 0.373799292 1.336665471 0.731360938 1.225273934 0.788510377 1.041776471 0.956691322 0.402703759 0.220970161 0.994200649 0.911653037 1.078000746 1.00206625 0.494281473 0.587441327 0.78245 0.37176358 0.82009927 0.791785537 0.35856519 0.527108163 0.684251786 0.775806897 0.463200554 1.038808259 1.266835556 0.45695925 0.725044615 0.502826702 0.000578676 0.412326563 0.577414286 SM(d18:0/22:0) 607.4100091 180.9410379 270.7176475 185.4976097 131.9638804 239.6865467 176.8151457 346.9133035 116.7346733 229.7632735 352.5264508 215.7293275 230.5886853 260.1118858 352.3693376 115.9874679 87.15049435 231.0245584 358.9883902 348.1584642 216.6518125 267.71189 177.4140888 419.3641528 110.6563586 397.0378538 201.413736 116.7699639 262.846 215.0361984 263.8004052 125.3350251 339.4349444 253.7514756 91.556235 216.9547538 131.598973 1518.146974 127.8063336 147.9955814 Cer(d18:1/17:0) 0.005264019 0.000839286 0.000570802 0.008231604 0.001915541 0.001432783 0.002686425 0.001678472 0.007517689 0.000838117 0.002451563 0.002441943 0.002499057 0.005126471 0.002704339 0.000775376 0.000209677 0.0137 0.002080374 0.007580597 0.00785375 0.009328125 0.010696429 0.001180282 0.003004938 0.000579745 0.005788636 0.000757595 0.002166837 0.004230804 0.00440977 0.00158524 0.00348817 0.007651111 0.000219 0.004637692 0.007321728 0.000620588 0.001826563 0.006339031 GlcCer (d18:0/24:0) 0.084800467 0.034983571 0.067636832 0.045111085 0.011386486 0.105991981 0.04299638 0.115002778 0.023390094 0.244706502 0.109892969 0.071018957 0.036444906 0.178861765 0.051694215 0.022244549 0.003709677 0.084587662 0.083972664 0.067490299 0.103478125 0.074515848 0.078508163 0.077859859 0.04787037 0.091200547 0.089713636 0.009852532 0.076995408 0.025774554 0.088709195 0.018236347 0.083613616 0.081659444 0.02245575 0.017966154 0.02870733 0 0.013797656 0.021561735 SM(d18:0/24:0) 54.71621846 12.258425 18.66742672 10.62323561 4.743110135 22.97929741 7.878099774 22.21751319 3.71663066 22.05200179 29.98741563 15.1219891 11.04620717 21.26270294 23.52744731 5.059130639 1.671771774 11.67420065 20.42500444 25.42144104 13.1740475 23.26142411 18.77232092 34.62194225 6.47099321 40.24781606 13.7076031 3.78491519 25.31231939 9.315780804 20.34594253 5.410195572 23.00731071 15.02001111 2.29512375 8.400829231 3.670143455 22.55805588 3.516828906 4.338123214 SM(d18:0/24:1) 41.0970715 6.394956429 12.1617521 2.373338208 0.339116216 9.168827123 2.211232805 14.44577083 0 10.84449484 19.31830938 5.038056398 4.475724906 10.68669926 13.41901798 0.711448872 0 5.738327273 13.50392033 13.25958881 5.139493125 10.74179063 6.409875 22.49444577 1.569759877 22.46295164 6.175183884 0 14.95787347 3.091478571 10.88385172 0.437867712 11.25436205 7.185275 0 2.207073077 0 51.55812794 0 0.346391327 SM(d18:1/20:0) 10.89197103 4.085607143 5.306100573 4.038788208 3.410304054 6.399352358 3.695074208 7.690263889 3.573471226 8.587922422 4.722774219 8.291033175 2.633437358 8.957262684 7.160360331 2.812442481 3.202206452 5.308386364 4.527717757 6.407312687 8.05952125 7.422155357 6.726332143 6.479226761 2.534647531 6.365900912 6.73863657 1.910944304 5.848832143 4.707210938 4.438923563 3.231178229 6.165496875 4.19987 2.29831275 4.676177692 2.798089005 827.2419801 2.79075625 5.052332908 GlcCer (d18:0/24:1) 0.424157944 0.122067143 0.184650573 0.013575 0.006788514 0.018928302 0.16786086 0.0292 0.080997877 0.036876457 0.010482031 0.03245545 0.15216 0.027464338 0.244306612 0.006702632 0.003971774 0.016435065 0.228760514 0.018097015 0.273529375 0.222831696 0.15355 0.280462676 0.092419753 0.379841058 0.290928719 0.071206962 0.254077041 0.142291741 0.241316092 0.109662177 0.220962054 0.215607778 0.06421725 0.112533846 0.080500524 0.000168382 0.063599219 0.074589031 Cer(d18:1/18:0) 0.787914252 0.955373571 0.733378053 2.097128774 1.211647973 1.094979481 2.296368326 1.110295833 1.582582783 1.016428924 0.75875625 2.502087441 1.269389434 1.217794301 1.423731818 1.895443985 1.852112903 1.640073377 1.284499065 1.56648806 1.65427875 0.991810714 1.231009694 0.742096479 1.412795062 0.826573358 1.710686777 1.762371519 0.841226531 2.802683036 1.27838908 1.456130258 1.113296652 1.439027222 2.01988275 1.269779231 2.132612042 0.027477941 2.005960156 1.618275765 GlcCer (d18:1/20:0) 1.273874299 0.227052857 0.274872137 0.494125472 0.120253378 0.409393868 0.451525113 0.628186806 0.160908962 0.754287444 0.261196875 0.8017109 0.445266226 1.013017279 0.550376033 0.234846429 0.12618871 0.469785065 0.310404673 0.631226866 1.069931875 0.362309598 0.461735204 0.606219718 0.512542593 0.948857847 0.694556405 0.110087342 0.517581122 0.258883929 0.591844253 0.299562731 0.606229018 0.897315 0.143016 0.300232308 0.232562042 0.001470588 0.170642969 0.161846173 Cer(m18:1/24:1) 0.022586916 0.009207857 0.012372137 0.020321462 0.00345473 0.021263915 0.008807919 0.02886875 0.005269811 0.044938117 0.008805469 0.030651896 0.012072453 0.037339522 0.01916219 0.009202444 0.00601371 0.020955844 0.016093458 0.025354478 0.03120625 0.009861161 0.010881633 0.014338028 0.008529012 0.022689416 0.03656095 0.003913291 0.016402041 0.010560268 0.024195402 0.004888007 0.020762277 0.013397222 0.00717 0.007876154 0.013348429 0 0.006370313 0.005032653 Cer(d18:0/18:0) 0.037099065 0.02216 0.033905153 0.066793868 0.023835811 0.040943632 0.055924887 0.034179167 0.038171462 0.041724215 0.027586719 0.07805545 0.03594566 0.076356618 0.037427479 0.040155451 0.045879032 0.0646 0.034428505 0.049428358 0.039745 0.054105134 0.056785204 0.018607746 0.024187654 0.023354015 0.048891322 0.033920253 0.032795408 0.083497098 0.041077586 0.038850554 0.037906473 0.048733889 0.03751575 0.040596923 0.049243194 0.000572794 0.047395313 0.036687245 GlcCer (d18:0/20:0) 0.033784346 0.004477143 0.004266412 0.011463679 0.002136486 0.009353774 0.006561991 0.010420833 0.00348184 0.015226009 0.010409375 0.011303318 0.006074717 0.016100735 0.015251653 0.004998496 0.001090323 0.008216234 0.008573131 0.013186567 0.020863125 0.009257813 0.011375 0.010485915 0.006988272 0.01302427 0.014732851 0.002416456 0.010944898 0.00624442 0.011745402 0.008937454 0.013530134 0.013733889 0.0028275 0.006982308 0.005934817 0.000620588 0.006390625 0.0057 Cer(d18:0/20:0) 0.033970093 0.008073571 0.01207729 0.011521698 0.001809459 0.012581604 0.007616742 0.012761806 0 0.021907399 0.013128906 0.01618436 0.00834283 0.025287684 0.011166322 0.004733459 1.69355E-05 0.015193506 0.009190654 0.016608955 0.02449875 0.007852902 0.014902551 0.015807746 0.01153642 0.015052555 0.012358884 0.001424684 0.015952041 0.004184598 0.01545977 0.006782657 0.009009375 0.015468889 0.002541 0.002623077 0.002736911 0 0.00225625 0.002165816 SM(d18:1/24:1) 174.6004486 21.21610714 51.48270115 26.35357642 3.988313514 51.61915047 17.62541674 66.90917292 1.219086085 88.72605807 60.87833672 47.71610829 18.51956774 82.10518015 53.27127459 9.969406579 0 27.59700195 58.72668505 84.63489403 63.0209175 33.8900183 38.80706173 87.70519789 21.78926543 110.7279942 45.65512624 3.819763291 87.67103622 26.30391362 62.4809431 9.741241328 59.8215683 46.31070889 0 17.11468077 0 338.5674015 1.139948438 6.01318852 GlcCer (d18:1/24:0) 33.38416822 6.205402857 14.24587844 12.4209559 2.976037162 20.38238491 9.353566968 24.73801528 4.344427358 26.24162623 13.26954141 16.94245024 11.24083585 21.8648079 18.35561653 5.709060338 0.937229839 13.02304026 18.82736916 19.01549925 15.23757688 14.58853728 11.99204388 24.00541338 6.258541975 28.94892482 21.39391674 2.753826582 19.41521122 7.905158705 19.26557011 5.463564576 17.0232817 16.56120611 2.26722525 6.357248462 4.095820681 0 2.804126563 3.501271684 Cer(d18:1/22:1) 168.7924605 54.42525929 90.32267462 105.6073592 36.43820068 91.76404599 73.81627941 136.4037625 42.18419929 173.4976507 82.04379609 153.4947732 93.17587642 159.0066767 127.9514659 49.29324868 17.6264129 103.2558519 101.8573528 125.6249888 145.8799125 72.74828371 81.76053929 125.5177563 48.86734815 122.1437797 134.1855143 33.42256772 84.48550204 62.13855402 122.5321408 64.48556347 126.1990051 152.7509939 41.4653025 71.76382538 53.18709738 0.050602941 40.80052109 50.99434056 Cer(m18:1/24:0) 0.416723832 0.11178 0.096662977 0.097041509 0.042004054 0.216763443 0.092657919 0.3213375 0.04279033 0.304307623 0.197286719 0.200220853 0.099343019 0.307090809 0.267159917 0.058360714 0.013133871 0.164605195 0.215151869 0.260376119 0.246849375 0.185142857 0.152988776 0.20818169 0.089188889 0.305345803 0.27637624 0.055032278 0.203136224 0.097860938 0.247523563 0.101800185 0.188117411 0.180721667 0.04070775 0.084879231 0.07886623 0 0.023225781 0.057945918 Cer(d18:2/24:1) 0.258004206 0.05584 0.192036641 0.330062972 0.125723649 0.095688679 0.274789819 0.224802083 0.134511792 0.147327578 0.096530469 0.347522986 0.193391887 0.28061636 0.325713223 0.209277068 0.075016129 0.273161688 0.228274065 0.262884328 0.283146875 0.097007813 0.10192449 0.178388732 0.135362346 0.188063869 0.309455579 0.123041139 0.097144898 0.170533929 0.227128736 0.160299631 0.225622098 0.300282222 0.151539 0.228460769 0.201940052 0.009246324 0.14909375 0.150151531 Cer(d18:1/20:0) 0.085886215 0.067147857 0.054405916 0.10615684 0.079694595 0.068288208 0.113542534 0.086640278 0.094342217 0.070670179 0.04385625 0.154456635 0.074132264 0.113031618 0.112213636 0.097573308 0.094435484 0.117404545 0.062453271 0.09729403 0.0928775 0.097605134 0.08519898 0.088309859 0.08602716 0.073861314 0.115881818 0.078009494 0.065796429 0.181749107 0.096617241 0.076349446 0.095861384 0.106652222 0.11333925 0.113696923 0.151617801 0.043044853 0.10335 0.108718622 GlcCer (d18:1/22:0) 37.57023294 7.892732143 13.8041376 15.94790448 3.609883108 17.942025 9.311664027 25.24957361 4.286499764 26.93670874 15.08212813 21.09830403 13.95781528 27.73962518 19.33377149 6.657841917 1.756982258 14.13592273 12.9628514 19.99929925 22.71192438 12.51212746 14.36739337 21.79770141 10.20821235 25.21783467 22.89662417 2.854868987 17.89253878 6.468548438 22.04368448 7.199173616 17.06484107 20.395375 3.80242425 6.968604615 5.253118586 0.006491912 3.529615625 4.805223214 Cer(d18:2/22:0) 0.117879673 0.062932143 0.09008645 0.110892453 0.053952027 0.120844104 0.130123982 0.178565278 0.046413679 0.073839013 0.040772656 0.141791943 0.124242453 0.091501103 0.144055165 0.100378195 0.070958065 0.110329221 0.133013551 0.186328358 0.139995 0.126046875 0.046355102 0.136059155 0.081790123 0.056532482 0.171648347 0.073181646 0.075130612 0.104911607 0.146474138 0.120155535 0.141829688 0.174483333 0.131226 0.151468462 0.130906021 0.134125735 0.085526563 0.126018367 GlcCer (d18:1/24:1) 24.81741939 6.491834286 11.13865305 12.06150212 4.084866216 16.33387854 10.11443348 18.56755417 4.307047642 18.57670561 10.13978594 14.13056943 8.585253396 18.38029963 13.8196593 6.248144549 1.597498387 11.19235065 12.94326449 17.46681866 16.41416438 12.72579509 8.989752041 16.41965704 5.725869753 23.35561478 18.00301488 3.996499367 14.73755408 8.94984442 14.12253851 5.819246679 13.55669129 13.48627389 3.567171 7.360303077 5.113812565 0.005102206 3.567767969 3.910464031 Cer(d18:0/24:1) 0.226155841 0.048645 0.08084084 0.082771698 0.01517973 0.11609717 0.039082805 0.113091667 0.01425566 0.16451704 0.065423438 0.137539336 0.051997358 0.202624081 0.073713223 0.033799624 0.008395161 0.068764286 0.094115888 0.113969403 0.112898125 0.072 0.079135714 0.100375352 0.034459259 0.179115876 0.089589669 0.019518354 0.120483673 0.043840848 0.121327011 0.024137269 0.078786161 0.067411111 0.01582125 0.032586923 0.020665445 0.000988235 0.013883594 0.017647194 Cer(d18:0/22:0) 1.640756776 0.395603571 0.541690076 0.592219811 0.098018919 0.755779245 0.263779412 0.865548611 0.096122406 1.424666368 0.61155625 1.024726777 0.590245472 1.661724265 0.686158264 0.211281767 0.067041935 0.453562338 0.563817757 0.88808806 0.96131375 0.425480357 0.656971429 0.778140141 0.342141358 1.069801095 0.637875 0.126724684 0.835438776 0.190435714 1.067918966 0.223941144 0.698982589 0.603516111 0.06700725 0.235664615 0.17183089 0 0.131485938 0.124050765 Cer(d18:1/26:0) 0.344801636 0.085055714 0.220110687 0.247831132 0.075989865 0.301658491 0.246023756 0.416399306 0.097519104 0.627793049 0.248747656 0.375762085 0.229233396 0.545023897 0.420328512 0.112763346 0.043809677 0.292564286 0.34984486 0.363363433 0.380746875 0.188221205 0.168067347 0.318698592 0.093248148 0.53795365 0.30855 0.081213924 0.276668367 0.201908705 0.27403908 0.087058118 0.364769866 0.418657778 0.09857325 0.165568462 0.168731152 0 0.103299219 0.144345918 Cer(d18:1/24:1) 6.694316355 2.142588571 2.951779008 3.474619811 1.442933784 2.943896462 2.251769457 4.168379861 1.655344811 7.36758565 2.905870313 6.381909242 2.457393396 7.613729779 4.828690909 1.812575188 0.914630645 3.645280519 2.95735514 3.75501791 5.389480625 1.965268527 2.831808673 3.886961972 1.860280247 4.938238139 5.164188843 1.12571519 2.792344898 1.672019866 4.152478161 1.640402768 3.267604688 3.638486111 1.6033965 2.651368462 1.892308115 0.024366912 1.1990375 1.545382653 Cer(d18:1/22:0) 12.5192979 2.633535714 4.977786641 5.217847642 1.145960135 5.555450943 3.020985068 8.081075694 1.383385613 9.015321525 4.395715625 6.75096327 5.057343962 9.565720588 6.48292314 2.215030827 0.597041129 4.048188961 4.290880374 6.301270896 7.798521875 4.00633192 4.200341327 7.015780986 3.123785185 8.47155219 7.173560331 1.052781013 5.793669898 2.098570313 7.000663218 2.5094131 5.469315402 6.369411667 1.3837125 2.476698462 1.757372513 0.302444118 1.249932813 1.669737245 Cer(d18:2/24:0) 0.807016121 0.108346429 0.419103435 0.654579481 0.118082432 0.303351651 0.328476244 0.569913194 0.200695755 0.3132713 0.283696094 0.71376327 0.419607736 0.715004228 0.787511157 0.238272744 0.068722581 0.490585714 0.439045093 0.594324627 1.05856125 0.23538683 0.254089286 0.516624648 0.547846914 0.578435036 0.707237603 0.12128481 0.325922959 0.234548438 0.52802931 0.252809779 0.482256027 0.486312778 0.05122725 0.280473846 0.145721466 0.056327941 0.129846094 0.168440051 Cer(d18:0/24:0) 0.741335748 0.270967857 0.367567557 0.241473113 0.057647297 0.381832075 0.12931629 0.457770833 0.097372642 0.60917287 0.416055469 0.367633649 0.19557283 0.604337316 0.321784711 0.11607688 0.042201613 0.274988961 0.323198832 0.342170896 0.4034 0.226441741 0.318866837 0.422533803 0.31849321 0.469604562 0.418930165 0.048067722 0.397233163 0.099575223 0.516044828 0.107986716 0.347620313 0.196298333 0.06032325 0.127661538 0.079591099 0.000385294 0.057353906 0.073644643 Cer(d18:1/23:0) 6.398620794 1.300777143 2.477630153 2.523638208 0.604458784 2.851587028 1.282227828 3.745777778 0.855883726 5.724193722 2.595572656 3.372703791 2.006495094 5.522870956 3.510972521 0.962292293 0.322677419 1.849012987 2.19115514 2.892381343 3.2924725 1.962616741 2.151335204 3.618439437 1.591722222 5.307002737 3.92985062 0.472637975 3.166752551 0.78556942 3.544585057 0.819599446 2.374811384 2.178893889 0.60265125 1.056253077 0.750121728 0.001027206 0.64179375 0.689814796 Cer(d18:1/24:0) 15.08005864 3.007962857 5.097407634 4.927114387 1.001344595 5.640330425 1.770483258 7.787967361 1.600033019 10.07137265 5.308738281 6.755279147 3.419643962 11.96002996 6.034960537 2.018731015 0.701405645 3.810154545 4.022967056 5.725614179 7.60739 3.405254464 5.015921429 7.408628873 4.804655556 9.482735584 8.250653306 0.658472152 5.335737245 1.309804688 6.899938506 1.482296679 4.577235938 3.398101667 1.040586 1.823913846 1.57466466 0.155975735 1.104374219 1.057777806 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Precursor Ion Product Ion Ret Time (min) Cer(d18:0/18:0) 568.6 284.3 4.7 Cer(d18:0/20:0) 596.6 284.4 5 Cer(d18:0/22:0) 624.6 284.4 5.5 Cer(d18:0/24:0) 652.7 284.4 6 Cer(d18:0/24:1) 650.7 284.3 5.5 Cer(d18:1/16:0) 538.5 282.3 4.6 Cer(d18:1/17:0) 552.5 264.2 4.4 Cer(d18:1/18:0) 566.6 264.2 4.4 Cer(d18:1/18:1) 564.5 282.3 4.3 Cer(d18:1/20:0) 594.6 282.3 5 Cer(d18:1/22:0) 622.6 282.3 5.3 Cer(d18:1/22:1) 620.6 282.3 5.1 Cer(d18:1/23:0) 636.6 282.3 5.8 Cer(d18:1/24:0) 650.6 282.3 6 Cer(d18:1/24:1) 648.6 264.2 5.8 Cer(d18:1/26:0) 678.7 282.3 5.6 Cer(d18:2/22:0) 620.6 280.3 5.2 Cer(d18:2/24:0) 648.6 280.3 5.7 Cer(d18:2/24:1) 646.6 280.3 5 Cer(m18:1/20:0) 578.6 266.3 3.6 Cer(m18:1/24:0) 634.6 266.3 5 Cer(m18:1/24:1) 632.6 266.3 4.5 GlcCer (d18:0/20:0) 758.6 284.3 4.5 GlcCer (d18:0/24:0) 814.7 284.3 5 GlcCer (d18:0/24:1) 812.7 284.3 4.9 GlcCer (d18:1/16:0) 700.6 282.3 3.8 GlcCer (d18:1/18:0) 728.6 282.3 4.2 GlcCer (d18:1/20:0) 756.6 282.3 4.6 GlcCer (d18:1/22:0) 784.7 282.3 5.1 GlcCer (d18:1/24:0) 812.7 282.3 5.2 GlcCer (d18:1/24:1) 810.7 282.3 5.3 SM(d18:0/20:0) 761.6 266.3 3.9 SM(d18:0/22:0) 789.7 266.3 4.4 SM(d18:0/24:0) 817.7 266.3 4.8 SM(d18:0/24:1) 815.7 266.3 4.4 SM(d18:1/18:0) 731.6 264.3 4 SM(d18:1/20:0) 759.6 184.4 4.9 SM(d18:1/24:1) 813.7 184.4 5.1 METABOLITES_END #END