#METABOLOMICS WORKBENCH yorch40_20201020_054228 DATATRACK_ID:2217 STUDY_ID:ST001518 ANALYSIS_ID:AN002521 PROJECT_ID:PR001023 VERSION 1 CREATED_ON November 4, 2020, 10:24 am #PROJECT PR:PROJECT_TITLE Metabolome analysis in the diagnosis of childhood cerebellar ataxia PR:PROJECT_SUMMARY Metabolome studies to aid in the diagnosis and molecular elucidation of a child PR:PROJECT_SUMMARY presenting chronic progressive cerebellar ataxia and an undiagnosed condition. PR:INSTITUTE CEMBIO PR:LAST_NAME Sáiz PR:FIRST_NAME Jorge PR:ADDRESS km 0, Universidad CEU-San Pablo Urbanización Montepríncipe, M-501, 28925 PR:ADDRESS Alcorcón PR:EMAIL jorge.saizgalindo@ceu.es PR:PHONE 0034913 72 47 11 #STUDY ST:STUDY_TITLE Metabolome analysis in the diagnosis of childhood cerebellar ataxia ST:STUDY_SUMMARY Metabolome studies to aid in the diagnosis and molecular elucidation of a child ST:STUDY_SUMMARY presenting chronic progressive cerebellar ataxia and an undiagnosed condition. ST:INSTITUTE CEMBIO ST:LAST_NAME Sáiz ST:FIRST_NAME Jorge ST:ADDRESS km 0, Universidad CEU-San Pablo Urbanización Montepríncipe. M-501, 28925 ST:ADDRESS Alcorcón ST:EMAIL jorge.saizgalindo@ceu.es ST:PHONE 913 72 47 11 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #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 - Control_Plasma_LC_POS Subject:Control Technique=LC; RAW_FILE_NAME=Control_Plasma_LC_POS SUBJECT_SAMPLE_FACTORS - Control_Plasma_LC_NEG Subject:Control Technique=LC; RAW_FILE_NAME=Control_Plasma_LC_NEG SUBJECT_SAMPLE_FACTORS - Control_Plasma_GC Subject:Control Technique=GC; RAW_FILE_NAME=Control_Plasma_GC SUBJECT_SAMPLE_FACTORS - Control_Plasma_CE Subject:Control Technique=CE; RAW_FILE_NAME=Control_Plasma_CE SUBJECT_SAMPLE_FACTORS - Control_Urine_LC_POS Subject:Control Technique=LC; RAW_FILE_NAME=Control_Urine_LC_POS SUBJECT_SAMPLE_FACTORS - Control_Urine_LC_NEG Subject:Control Technique=LC; RAW_FILE_NAME=Control_Urine_LC_NEG SUBJECT_SAMPLE_FACTORS - Control_Urine_CE Subject:Control Technique=CE; RAW_FILE_NAME=Control_Urine_CE SUBJECT_SAMPLE_FACTORS - Case_Plasma_LC_POS Subject:Case Technique=LC; RAW_FILE_NAME=Case_Plasma_LC_POS SUBJECT_SAMPLE_FACTORS - Case_Plasma_LC_NEG Subject:Case Technique=LC; RAW_FILE_NAME=Case_Plasma_LC_NEG SUBJECT_SAMPLE_FACTORS - Case_Plasma_GC Subject:Case Technique=GC; RAW_FILE_NAME=Case_Plasma_GC SUBJECT_SAMPLE_FACTORS - Case_Plasma_CE Subject:Case Technique=CE; RAW_FILE_NAME=Case_Plasma_CE SUBJECT_SAMPLE_FACTORS - Case_Urine_LC_POS Subject:Case Technique=LC; RAW_FILE_NAME=Case_Urine_LC_POS SUBJECT_SAMPLE_FACTORS - Case_Urine_LC_NEG Subject:Case Technique=LC; RAW_FILE_NAME=Case_Urine_LC_NEG SUBJECT_SAMPLE_FACTORS - Case_Urine_CE Subject:Case Technique=CE; RAW_FILE_NAME=Case_Urine_CE #COLLECTION CO:COLLECTION_SUMMARY Metabolite extraction was performed in plasma and urine samples from the patient CO:COLLECTION_SUMMARY and a healthy control, matched for age, sex and weight, according to standard CO:COLLECTION_SUMMARY protocols with some modifications. CO:SAMPLE_TYPE Blood (plasma) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Metabolite extraction was performed in plasma and urine samples from the patient TR:TREATMENT_SUMMARY and a healthy control, matched for age, sex and weight, according to standard TR:TREATMENT_SUMMARY protocols with some modifications. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Plasma sample preparation for liquid chromatography–mass spectrometry SP:SAMPLEPREP_SUMMARY (LC–MS) entailed the following steps: (1) thaw sample on ice, vortex for 2 min SP:SAMPLEPREP_SUMMARY and transfer 100 µL of plasma into an Eppendorf tube; (2) for protein SP:SAMPLEPREP_SUMMARY precipitation, add 300 µL of cold (-18˚C) methanol/ethanol (1:1 v/v), vortex SP:SAMPLEPREP_SUMMARY for 1 min, incubate on ice for 5 min and vortex again briefly; (3) centrifuge at SP:SAMPLEPREP_SUMMARY 13000 rpm, 4˚C, for 20 min; and (4) transfer the supernatant into the LC–MS SP:SAMPLEPREP_SUMMARY system. Plasma sample preparation for gas chromatography–mass spectrometry SP:SAMPLEPREP_SUMMARY (GC–MS) entailed the following steps: (1) thaw sample on ice, vortex for 2 min SP:SAMPLEPREP_SUMMARY and transfer 40 µL of plasma into an Eppendorf tube; (2) for protein SP:SAMPLEPREP_SUMMARY precipitation, add 120 µL of cold acetonitrile, vortex for 2 min and incubate SP:SAMPLEPREP_SUMMARY on ice for 5 min; (3) centrifuge at 15400 rpm, 4˚C, for 10 min; and (4) SP:SAMPLEPREP_SUMMARY transfer the supernatant into a GC vial with insert; (5) evaporate in a Speedvac SP:SAMPLEPREP_SUMMARY at 30°C until dry; (6) add 10 µL of O-methoxyamine hydrochloride (15 mg/mL) in SP:SAMPLEPREP_SUMMARY pyridine and vortex for 5 min; (7) sonicate for 2 min and vortex for 2 min SP:SAMPLEPREP_SUMMARY (repeat step a total of three times); (8) incubate at room temperature for 16 h SP:SAMPLEPREP_SUMMARY in darkness for the reaction of the methoxymation; (9) add 10 µL of SP:SAMPLEPREP_SUMMARY N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1 % trimethylchlorosilane SP:SAMPLEPREP_SUMMARY (TMCS) and incubate 1 h at 70 ˚C for the silylation; (10) add 100 µL of 10 ppm SP:SAMPLEPREP_SUMMARY C18:0 methyl ester in n-heptane as internal standard, vortex for 2 min and SP:SAMPLEPREP_SUMMARY centrifuge at 2500 rpm, 20˚C, for 15 min; and (11) transfer into the GC–MS SP:SAMPLEPREP_SUMMARY system. Plasma sample preparation for capillary electrophoresis–mass SP:SAMPLEPREP_SUMMARY spectrometry (CE–MS) entailed the following steps: (1) thaw sample on ice, SP:SAMPLEPREP_SUMMARY vortex for 1 min and transfer 100 µL of plasma into an Eppendorf tube already SP:SAMPLEPREP_SUMMARY filled with 100 µL of 0.2 M formic acid with 5 % acetonitrile and 0.4 mM SP:SAMPLEPREP_SUMMARY methionine sulfone as internal standard; (2) vortex for 2 min and transfer to a SP:SAMPLEPREP_SUMMARY Millipore filter (30 kDa protein cutoff); (3) centrifuge at 2000 rpm, 4˚C, for SP:SAMPLEPREP_SUMMARY 70 min; and (4) transfer the filtrate into a CE vial where the volume is SP:SAMPLEPREP_SUMMARY directly injected into the CE–MS system. Urine samples for LC–MS were thawed SP:SAMPLEPREP_SUMMARY on ice and vortexed prior injection into the LC-MS system (without SP:SAMPLEPREP_SUMMARY pre-treatment). For CE–MS, sample procedure entailed: (1) thaw on ice and SP:SAMPLEPREP_SUMMARY vortex for 1 min and transfer 200 µL into an Eppendorf tube; (2) centrifuge at SP:SAMPLEPREP_SUMMARY 13200 rpm, 4˚C, for 20 min; (3) transfer 100 µL of the supernatant into an SP:SAMPLEPREP_SUMMARY Eppendorf tube already filled with 400 µL of 0.125 M formic acid with 0.25 mM SP:SAMPLEPREP_SUMMARY methionine sulfone as internal standard; (4) vortex for 1 min and centrifuge at SP:SAMPLEPREP_SUMMARY 13200 rpm, 4˚C, for 10 min; and (5) transfer 200 µL of the supernatant into a SP:SAMPLEPREP_SUMMARY CE vial where the volume is directly injected into the CE–MS system. Quality SP:SAMPLEPREP_SUMMARY control (QC) samples were independently prepared for each technique and followed SP:SAMPLEPREP_SUMMARY the same procedure as applied for experimental samples. QC samples were always SP:SAMPLEPREP_SUMMARY injected at the beginning of each analytical run, followed by samples randomized SP:SAMPLEPREP_SUMMARY independently. A QC sample was rerun after each block of 5 samples. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890A CH:COLUMN_NAME Agilent DB5-MS (30m x 0.25mm, 0.25um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 5975C MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS None #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS area MS_METABOLITE_DATA_START Samples Case_Plasma_GC Control_Plasma_GC Factors Subject:Case Subject:Control oleic acid 192004 34107 L-(+) lactic acid 11826347 2117776 3-Hydroxybutyric acid 14884 79152 p-cresol 53004 10836 L-tryptophan 121711 30344 2-Hydroxy-3-methylbutyric acid 5209 19596 Propanoic acid 267915 76515 Asparagine 6539 1883 linoleic acid 42314 12647 glycerol 479290 152527 Pyranose 9632558 30186272 Furanose 9320 28048 Glyceric acid 7638 2570 urea 12002771 4061942 Alanine 995970 345286 L-glutamine 33676 11894 L-Lysine 12857 4776 Aminomalonic acid 31934 12183 tyrosine 155552 61473 citric acid 14529 6334 L-glutamic acid 33580 15077 palmitic acid 209222 107254 Myo-Inositol 53517 28179 phosphoric acid 840792 456766 Glycine 286499 159994 oxalic acid 2616 1514 Valine 710113 423326 L-Serine 174466 105774 L-threonine 99828 61661 2-ketoisocaproic acid 34128 21903 meso-Erythritol 6744 4398 Creatinine 37271 24804 DL-isoleucine 192207 280429 L-methionine 28408 21049 Phenylalanine 120124 89048 stearic acid 55374 45908 Methyl stearate 273257 236308 2-hydroxybutyric acid 98807 106100 L-proline 391737 365035 glycolic acid 3149 3346 L-pyroglutamic acid 203628 191868 L-leucine 511836 499602 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name oleic acid L-(+) lactic acid 3-Hydroxybutyric acid p-cresol L-tryptophan 2-Hydroxy-3-methylbutyric acid Propanoic acid Asparagine linoleic acid glycerol Pyranose Furanose Glyceric acid urea Alanine L-glutamine L-Lysine Aminomalonic acid tyrosine citric acid L-glutamic acid palmitic acid Myo-Inositol phosphoric acid Glycine oxalic acid Valine L-Serine L-threonine 2-ketoisocaproic acid meso-Erythritol Creatinine DL-isoleucine L-methionine Phenylalanine stearic acid Methyl stearate 2-hydroxybutyric acid L-proline glycolic acid L-pyroglutamic acid L-leucine METABOLITES_END #END