#METABOLOMICS WORKBENCH yorch40_20201020_054228 DATATRACK_ID:2217 STUDY_ID:ST001518 ANALYSIS_ID:AN002522 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_SUMMARY Positive ionization CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Discovery® HS C18 (15 cm x 2.1 mm x 3 µm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6520 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS None MS:MS_RESULTS_FILE ST001518_AN002522_Results.txt UNITS:area Has m/z:Yes Has RT:Yes RT units:Minutes #END