{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002238","ANALYSIS_ID":"AN003654","VERSION":"1","CREATED_ON":"02-08-2024"},

"PROJECT":{"PROJECT_TITLE":"LC-HRMS based plasma metabolomics analysis for biomarker discovery of neuroblastoma: 3-O-methyldopa is a new biomarker of poor prognosis of metastatic disease","PROJECT_TYPE":"Biomarker Discovery","PROJECT_SUMMARY":"Neuroblastoma (NB) is the most common extra-cranial malignant tumor in children. Although the survival rate of NB has improved over the years, the outcome of NB still remains unfavorable in a high percentage of cases. Prognosis is currently based on a combination of clinical, histo-pathological and biological features, on which patients are classified in different risk groups and addressed to different treatment protocols. A more accurate risk stratification remains a key point in the study of NB: in particular, the availability of novel prognostic biomarkers of metastatic “high risk” NB at diagnosis could help in improving patient stratification, accurately predicting outcome, relapse or response to treatments and also reducing unnecessary therapies and related toxicities. In this study an HRMS-based approach was applied for the first time to study NB with a goal of developing prognostic biomarkers that could help in improving patient stratification and providing novel therapeutic targets. Starting from an untargeted approach the differences in the metabolomic profiles of localized and metastatic patients were investigated. Key metabolites of metastatic NB were identified through differential expression analysis. Among the metabolites of L-DOPA degradation pathway 3-o-methyldopa (3-O-MD) was selected and analysed in a second cohort of patients using a targeted approach based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).","INSTITUTE":"IRCCS Gaslini","LAST_NAME":"Lavarello","FIRST_NAME":"Chiara","ADDRESS":"Via Gaslini 5, Genoa, GE, 16147, Italy","EMAIL":"chiaralavarello@gaslini.org","PHONE":"+3901056362911","DOI":"http://dx.doi.org/10.21228/M84Q4W"},

"STUDY":{"STUDY_TITLE":"LC-HRMS based plasma metabolomics analysis for biomarker discovery of neuroblastoma: 3-O-methyldopa is a new biomarker of poor prognosis of metastatic disease","STUDY_TYPE":"Biomarker Discovery","STUDY_SUMMARY":"In this paper we show for the first time a metabolomic-based biomarker discovery using HRMS applied to plasma of NB patients and its validation on a second independent cohort of patients using a different analytical method.","INSTITUTE":"Istituto Giannina Gaslini","LAST_NAME":"Lavarello","FIRST_NAME":"Chiara","ADDRESS":"Via Gaslini 5, Genoa, GE, 16147, Italy","EMAIL":"chiaralavarello@gaslini.org","PHONE":"+3901056362911","SUBMIT_DATE":"2021-10-15"},

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{
"Subject ID":"-",
"Sample ID":"NB_Plasma_POST_IROA_DDA_PriorityList_70150_C18_Neg_090",
"Factors":{"Type":"Sample"},
"Additional sample data":{"Class ID":"ID","RAW_FILE_NAME":"NB_Plasma_POST_IROA_DDA_PriorityList_70150_C18_Neg_090.mzML","Batch":"1","Analytical order":"290","Inject. volume (µL)":"1","Included":"TRUE"}
},
{
"Subject ID":"-",
"Sample ID":"NB_Plasma_POST_IROA_DDA_PriorityList_70150_C18_Pos_027",
"Factors":{"Type":"Sample"},
"Additional sample data":{"Class ID":"POST_IROA_DDA_PriorityList","RAW_FILE_NAME":"NB_Plasma_POST_IROA_DDA_PriorityList_70150_C18_Pos_027.mzML","Batch":"1","Analytical order":"251","Inject. volume (µL)":"1","Included":"TRUE"}
},
{
"Subject ID":"-",
"Sample ID":"NB_Plasma_POST_IROA_DDA_PriorityList_70150_HILIC_Neg_208",
"Factors":{"Type":"Sample"},
"Additional sample data":{"Class ID":"ID","RAW_FILE_NAME":"NB_Plasma_POST_IROA_DDA_PriorityList_70150_HILIC_Neg_208.mzML","Batch":"1","Analytical order":"208","Inject. volume (µL)":"1","Included":"TRUE"}
},
{
"Subject ID":"-",
"Sample ID":"NB_Plasma_POST_IROA_DDA_PriorityList_70150_HILIC_Pos_151",
"Factors":{"Type":"Sample"},
"Additional sample data":{"Class ID":"ID","RAW_FILE_NAME":"NB_Plasma_POST_IROA_DDA_PriorityList_70150_HILIC_Pos_151.mzML","Batch":"1","Analytical order":"351","Inject. volume (µL)":"1","Included":"TRUE"}
},
{
"Subject ID":"-",
"Sample ID":"NB_Plasma_QC_C18_Neg_027",
"Factors":{"Type":"Sample"},
"Additional sample data":{"Class ID":"QC","RAW_FILE_NAME":"NB_Plasma_QC_C18_Neg_027.mzML","Batch":"1","Analytical order":"27","Inject. volume (µL)":"1","Included":"TRUE"}
},
{
"Subject ID":"-",
"Sample ID":"NB_Plasma_QC_C18_Pos_066",
"Factors":{"Type":"Sample"},
"Additional sample data":{"Class ID":"QC","RAW_FILE_NAME":"NB_Plasma_QC_C18_Pos_066.mzML","Batch":"1","Analytical order":"242","Inject. volume (µL)":"1","Included":"TRUE"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Plasma samples were obtained from patients with NB at the diagnosis from peripheral venous blood collected in 3 mL EDTA K3-containing tubes, centrifuged at 4000 g for 5 min at 4 °C and stored at -80°C until analysed.","SAMPLE_TYPE":"Blood (plasma)","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"-"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"A 50 µL aliquot of plasma was extracted adding 150 µL cold (-20 C) methanol containing as internal standard mixture of creatine (methyl-D3, 98%), vitamin B3 (D4, 98%), uracil (1,3-15N2, 98%), chenodeoxycholic acid (2,2,4,4-D4, 98%). After over-night protein precipitation, proteins were removed by centrifugation for 10 minutes at 14,000 x g and 4 °C. The supernatant was collected and stored at -80 °C until analysis when was adding to the samples a second internal standard mixture of L-alanine (13C3, 99%), chenodeoxycholic acid (2,2,4,4-D4, 98%), L-leucine (13C6, 99%), L-phenylalanine (13C6, 99%), L-tryptophan (13C11, 99%), L-tyrosine (13C6, 99%), caffeine (13C3, 99%). stearic acid, sodium salt (13C18, 98%), sodium benzoate (13C6, 99%). Quality control (QC) samples were prepared by mixing equal volumes of all the NB plasma samples. In addition, a procedural blank, used to monitor contamination acquired during all stages of sample preparation, and external quality control (EQC) samples were included in the study and were prepared in the same way as the study samples. To avoid bias due to instrument drift, the analytical study design involves analyzing the 99 samples in a randomized way, with 16 QC and 9 EQC inserted every 6 and 12 runs respectively to assess analytical precision. Subsequently, 18 identification runs and a procedural blank were analyzed.","EXTRACT_STORAGE":"-20℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"HILIC column was eluted at a flow rate of 200 µl/min with 90% of mobile phase B (acetonitrile) for 0.1 min followed by a linear gradient to 70% of mobile phase A ( H2O 5 mM ammonium formate, pH 3) over 15 min, then kept constant for 5 min, brought back to the initial conditions in 0.5 min, and maintained for 9 min.","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Waters Acquity BEH Amide (150 x 2.1mm,1.7um)","FLOW_GRADIENT":"90% of mobile phase B for 0.1 min followed by a linear gradient to 70% of mobile phase A over 15 min, then kept constant for 5 min, brought back to the initial conditions in 0.5 min, and maintained for 9 min.","FLOW_RATE":"200 µl/min","INJECTION_TEMPERATURE":"25","SOLVENT_A":"100% water; 5 mM ammonium formate, pH 3","SOLVENT_B":"100% acetonitrile","RANDOMIZATION_ORDER":"yes","CHROMATOGRAPHY_TYPE":"HILIC"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Plus Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","MS_COMMENTS":"Data processing was performed using MS-DIAL for peak picking, alignment, and identification. For metabolite analysis, in house m/z and retention time libraries were used in addition to MS/MS spectra databases in msp format. MS-DIAL parameters were set as follows: MS1 tolerance, 0.05Da; MS2 tolerance, 0.025 Da; retention time begin, 0 min; retention time end, 100 min; minimum peak height, 10000; mass slice width, 0.1 Da; smoothing level, 3 scans; minimum peak width, 5 scans; sigma window value, 0.5. We considered M−H, M–H2O−H, M+Na-2H, M+Cl, M+FA-H, 2M−H, 2M+FA-H, M−2H, 3M-H adduct in negative ionization mode and M+H, M+Na, M+ACN+H, M+H–H2O, M+H–2H2O, M+2Na-H, M+ACN+Na, M+2ACN+H, 2M+H, M+2H, 2M+ACN+Na in positive ionization mode. Execute retention time correction on IS and IS kit with a RT tolerance of 0.1 min and a mass tolerance of 0.015 Da were performed. In supplementary table X all parameter settings for MS-Dial were reported. For in silico compound annotation of ion features with an acquired tandem mass spectrum MS-FINDER The MS1 and MS2 tolerances were set to 5 and 15 ppm, respectively. Formula finder were exclusively processed with C, H, O, N, P and S atoms.","ION_MODE":"NEGATIVE","MS_RESULTS_FILE":"ST002238_AN003654_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes"}

}