{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000786","ANALYSIS_ID":"AN001245","VERSION":"1","CREATED_ON":"June 26, 2017, 4:12 pm"},

"PROJECT":{"PROJECT_TITLE":"Urine Metabolite Identification","PROJECT_TYPE":"MS and MS/MS analyses","PROJECT_SUMMARY":"Structural identification of unknown metabolites in human urine","INSTITUTE":"Colorado State University","DEPARTMENT":"MIP","LABORATORY":"Belisle","LAST_NAME":"Fitzgerald","FIRST_NAME":"Bryna","ADDRESS":"3185 Rampart Rd, Fort Collins, CO, 80521, USA","EMAIL":"blfitz@colostate.edu","PHONE":"9704918905"},

"STUDY":{"STUDY_TITLE":"N-acetylisoputreanine-g-lactam Identification","STUDY_SUMMARY":"An untargeted metabolomics approach was utilized to determine urinary metabolites that could serve as small molecule biomarkers for treatment response to standard tuberculosis treatment. However, the majority of metabolites that most accurately distinguished patient samples at time of diagnosis from those at one month after the start of therapy lacked structural identification. The detection of unknown metabolite structures is a well-known limitation of untargeted metabolomics, and underscores a need for continued elucidation of novel metabolite structures. In this study, we sought to define the structure of a urine metabolite with an experimentally determined mass of 202.1326 Da, classified as molecular feature (MF) 202.1326. A hypothesized structure of N1-acetylisoputreanine was developed for MF 202.1326 using in silico tools and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the absence of a commercial standard, synthetic N1-acetylisoputreanine was generated using enzymatic and chemical synthesis and LC-MS/MS was used to confirm the structure of MF 202.1326 as N1-acetylisoputreanine, a proposed terminal polyamine catabolite that had not been previously detected in biological samples. Further analysis demonstrated that N1-acetylisoputreanine and an alternative form of this metabolite, N1-acetylisoputreanine-γ-lactam, are both present in human urine and are likely end-products of polyamine metabolism.","INSTITUTE":"Colorado State University","LAST_NAME":"Fitzgerald","FIRST_NAME":"Bryna","ADDRESS":"3185 Rampart Rd","EMAIL":"blfitz@colostate.edu","PHONE":"9704918905"},

"SUBJECT":{"SUBJECT_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"Synthetic Standard",
"Sample ID":"ACISOGA-r002",
"Factors":{"Treatment":"MS"}
},
{
"Subject ID":"Synthetic Standard",
"Sample ID":"MSMS_20_Acisoga",
"Factors":{"Treatment":"MS/MS"}
},
{
"Subject ID":"Patient Urine",
"Sample ID":"PooledUrine_MSMS_20",
"Factors":{"Treatment":"MS/MS"}
},
{
"Subject ID":"Urine + Synthetic Standard",
"Sample ID":"Urine-ACISOGA_012417",
"Factors":{"Treatment":"MS"}
},
{
"Subject ID":"Urine",
"Sample ID":"Urine-Water_012417",
"Factors":{"Treatment":"MS"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Urine was collected and frozen at -20 for storage. Prior to analysis, urine was thawed and centrifuged to remove particulates. Synthetic NACIP-gl material was stored in water at 4 and diluted in water prior to analysis."},

"TREATMENT":{"TREATMENT_SUMMARY":"Urine, urine spiked with synthetic standard, and synthetic standard alone were analyzed by LC-MS and LC-MS/MS"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Urine was thawed on ice and centrifuged to remove particulates prior to analysis. N1-acetylisoputreanine-γ-lactam was synthesized as previously described. Acetylation was performed with N-(3-Aminopropyl) pyrrolidin-2-one (0.75 M) and acetyl chloride (0.75 M) in methanol and 0.5 M PBS (1:1) adjusted to pH 7.0 with sodium hydroxide. The reaction products were extracted with dichloromethane, dried, suspended in 2 ml LC-MS grade water and stored at 4˚C. Prior to LC-MS analyses, the synthetic compound was diluted 1:100 in water or human urine."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Agilent 1200","COLUMN_NAME":"Atlantis T3 reverse-phase C18 3.5µm column (2.1 by 150mm","FLOW_GRADIENT":"100% Solvent A to 90% Solvent B","FLOW_RATE":"0.25 ml/min","COLUMN_TEMPERATURE":"30","SOLVENT_A":"0.1% Formic Acid in Water","SOLVENT_B":"0.1% Formic Acid in Methanol"},

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

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Agilent","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_RESULTS_FILE":"ST000786_AN001245_Results.txt UNITS:counts*min"}

}