#METABOLOMICS WORKBENCH blfitz_20170626_153526 DATATRACK_ID:1104 STUDY_ID:ST000786 ANALYSIS_ID:AN001245 PROJECT_ID:PR000572
VERSION             	1
CREATED_ON             	June 26, 2017, 4:12 pm
#PROJECT
PR:PROJECT_TITLE                 	Urine Metabolite Identification
PR:PROJECT_TYPE                  	MS and MS/MS analyses
PR:PROJECT_SUMMARY               	Structural identification of unknown metabolites in human urine
PR:INSTITUTE                     	Colorado State University
PR:DEPARTMENT                    	MIP
PR:LABORATORY                    	Belisle
PR:LAST_NAME                     	Fitzgerald
PR:FIRST_NAME                    	Bryna
PR:ADDRESS                       	3185 Rampart Rd, Fort Collins, CO, 80521, USA
PR:EMAIL                         	blfitz@colostate.edu
PR:PHONE                         	9704918905
#STUDY
ST:STUDY_TITLE                   	N-acetylisoputreanine-g-lactam Identification
ST:STUDY_SUMMARY                 	An untargeted metabolomics approach was utilized to determine urinary
ST:STUDY_SUMMARY                 	metabolites that could serve as small molecule biomarkers for treatment response
ST:STUDY_SUMMARY                 	to standard tuberculosis treatment. However, the majority of metabolites that
ST:STUDY_SUMMARY                 	most accurately distinguished patient samples at time of diagnosis from those at
ST:STUDY_SUMMARY                 	one month after the start of therapy lacked structural identification. The
ST:STUDY_SUMMARY                 	detection of unknown metabolite structures is a well-known limitation of
ST:STUDY_SUMMARY                 	untargeted metabolomics, and underscores a need for continued elucidation of
ST:STUDY_SUMMARY                 	novel metabolite structures. In this study, we sought to define the structure of
ST:STUDY_SUMMARY                 	a urine metabolite with an experimentally determined mass of 202.1326 Da,
ST:STUDY_SUMMARY                 	classified as molecular feature (MF) 202.1326. A hypothesized structure of
ST:STUDY_SUMMARY                 	N1-acetylisoputreanine was developed for MF 202.1326 using in silico tools and
ST:STUDY_SUMMARY                 	liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the absence of a
ST:STUDY_SUMMARY                 	commercial standard, synthetic N1-acetylisoputreanine was generated using
ST:STUDY_SUMMARY                 	enzymatic and chemical synthesis and LC-MS/MS was used to confirm the structure
ST:STUDY_SUMMARY                 	of MF 202.1326 as N1-acetylisoputreanine, a proposed terminal polyamine
ST:STUDY_SUMMARY                 	catabolite that had not been previously detected in biological samples. Further
ST:STUDY_SUMMARY                 	analysis demonstrated that N1-acetylisoputreanine and an alternative form of
ST:STUDY_SUMMARY                 	this metabolite, N1-acetylisoputreanine-γ-lactam, are both present in human
ST:STUDY_SUMMARY                 	urine and are likely end-products of polyamine metabolism.
ST:INSTITUTE                     	Colorado State University
ST:LAST_NAME                     	Fitzgerald
ST:FIRST_NAME                    	Bryna
ST:ADDRESS                       	3185 Rampart Rd
ST:EMAIL                         	blfitz@colostate.edu
ST:PHONE                         	9704918905
#SUBJECT
SU:SUBJECT_TYPE                  	Human
SU:SUBJECT_SPECIES               	Homo sapiens
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	Synthetic Standard	ACISOGA-r002	Treatment :MS	
SUBJECT_SAMPLE_FACTORS           	Synthetic Standard	MSMS_20_Acisoga	Treatment :MS/MS	
SUBJECT_SAMPLE_FACTORS           	Patient Urine	PooledUrine_MSMS_20	Treatment :MS/MS	
SUBJECT_SAMPLE_FACTORS           	Urine + Synthetic Standard	Urine-ACISOGA_012417	Treatment :MS	
SUBJECT_SAMPLE_FACTORS           	Urine	Urine-Water_012417	Treatment :MS	
#COLLECTION
CO:COLLECTION_SUMMARY            	Urine was collected and frozen at -20 for storage. Prior to analysis, urine was
CO:COLLECTION_SUMMARY            	thawed and centrifuged to remove particulates. Synthetic NACIP-gl material was
CO:COLLECTION_SUMMARY            	stored in water at 4 and diluted in water prior to analysis.
#TREATMENT
TR:TREATMENT_SUMMARY             	Urine, urine spiked with synthetic standard, and synthetic standard alone were
TR:TREATMENT_SUMMARY             	analyzed by LC-MS and LC-MS/MS
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Urine was thawed on ice and centrifuged to remove particulates prior to
SP:SAMPLEPREP_SUMMARY            	analysis. N1-acetylisoputreanine-γ-lactam was synthesized as previously
SP:SAMPLEPREP_SUMMARY            	described. Acetylation was performed with N-(3-Aminopropyl) pyrrolidin-2-one
SP:SAMPLEPREP_SUMMARY            	(0.75 M) and acetyl chloride (0.75 M) in methanol and 0.5 M PBS (1:1) adjusted
SP:SAMPLEPREP_SUMMARY            	to pH 7.0 with sodium hydroxide. The reaction products were extracted with
SP:SAMPLEPREP_SUMMARY            	dichloromethane, dried, suspended in 2 ml LC-MS grade water and stored at 4˚C.
SP:SAMPLEPREP_SUMMARY            	Prior to LC-MS analyses, the synthetic compound was diluted 1:100 in water or
SP:SAMPLEPREP_SUMMARY            	human urine.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Agilent 1200
CH:COLUMN_NAME                   	Atlantis T3 reverse-phase C18 3.5µm column (2.1 by 150mm
CH:FLOW_GRADIENT                 	100% Solvent A to 90% Solvent B
CH:FLOW_RATE                     	0.25 ml/min
CH:COLUMN_TEMPERATURE            	30
CH:SOLVENT_A                     	0.1% Formic Acid in Water
CH:SOLVENT_B                     	0.1% Formic Acid in Methanol
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:MS_COMMENTS                   	-
MS:INSTRUMENT_NAME               	Agilent
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_RESULTS_FILE               	ST000786_AN001245_Results.txt	UNITS:counts*min
#END