Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA355256

Local Sample ID:	gluc_pyr_3_24hr
Subject ID:	SU003402
Subject Type:	Cultured cells
Subject Species:	Bos taurus
Taxonomy ID:	9913

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Sample Preparation:

Sampleprep ID:	SP003409
Sampleprep Summary:	DMTMM mediated coupling of amine tags to biological acids is well studied and previous reports were used as a basis for acid tagging. Briefly, the coupling of biological acids to the amine tag was initiated with the addition of 25µL of a DMTMM/NMM solution (75mM DMTMM, 100mM NMM in 95/5 ACN/H2O)). The 96-well plate was allowed to react for 30 minutes at room temperature before another addition (12.5µL) of DMTMM/NMM to ensure reaction completion and account for water-mediated DMTMM degradation. After 4 hours total reaction time, 50µL of 50mM myristic acid was added and allowed to react for an additional 30 minutes to quench the reaction. All 96 well lysates are mixed into a 15mL falcon tube after the reaction is quenched. This reaction solution is mixed 1:1 with chloroform, vortexed for 30s, then centrifuged at 2,000 rpm for 5 minutes to ensure phase separation. The top aqueous layer is then taken for WCX enrichment. Supelclean 500 mg WCX columns were used to selectively enrich quaternary amine-tagged metabolites. Columns were conditioned with 4 mL of 90/10 (MeOH/H2O) and then equilibrated with 4 mL of loading buffer (10% MeOH in 20mM ammonium formate pH 8.5). The extracted reaction solution was mixed 1:1 with loading buffer before injection. The column was then rinsed with 4 mL of loading buffer, followed by elution with 8 mL of 90/10 (MeOH/H2O) + 1% formic acid. While singly tagged analytes readily elute at mL 2-3 under these conditions, doubly tagged analytes require more fractions to be collected. The first 1mL of this elution was discarded and the rest were pooled, dried, and reconstituted into 50µL of 25/25/50 (MeOH/H2O/ACN) to ensure solubility of all tagged analytes.

Sampleprep ID:

SP003409

Sampleprep Summary:

DMTMM mediated coupling of amine tags to biological acids is well studied and previous reports were used as a basis for acid tagging. Briefly, the coupling of biological acids to the amine tag was initiated with the addition of 25µL of a DMTMM/NMM solution (75mM DMTMM, 100mM NMM in 95/5 ACN/H2O)). The 96-well plate was allowed to react for 30 minutes at room temperature before another addition (12.5µL) of DMTMM/NMM to ensure reaction completion and account for water-mediated DMTMM degradation. After 4 hours total reaction time, 50µL of 50mM myristic acid was added and allowed to react for an additional 30 minutes to quench the reaction. All 96 well lysates are mixed into a 15mL falcon tube after the reaction is quenched. This reaction solution is mixed 1:1 with chloroform, vortexed for 30s, then centrifuged at 2,000 rpm for 5 minutes to ensure phase separation. The top aqueous layer is then taken for WCX enrichment. Supelclean 500 mg WCX columns were used to selectively enrich quaternary amine-tagged metabolites. Columns were conditioned with 4 mL of 90/10 (MeOH/H2O) and then equilibrated with 4 mL of loading buffer (10% MeOH in 20mM ammonium formate pH 8.5). The extracted reaction solution was mixed 1:1 with loading buffer before injection. The column was then rinsed with 4 mL of loading buffer, followed by elution with 8 mL of 90/10 (MeOH/H2O) + 1% formic acid. While singly tagged analytes readily elute at mL 2-3 under these conditions, doubly tagged analytes require more fractions to be collected. The first 1mL of this elution was discarded and the rest were pooled, dried, and reconstituted into 50µL of 25/25/50 (MeOH/H2O/ACN) to ensure solubility of all tagged analytes.