Preparing Urine Samples for NMR-Based Metabolomics Analysis Using Chenomx ISTD Solution. Table of Contents

Transcription

1 NIH Eastern Regional Comprehensive Metabolomics Resource Core at RTI International Page 1 of 6 Table of Contents Page 1.0 BACKGROUND SCOPE MATERIALS Equipments Reagents PROCEDURE General Guidelines Daily pipette and balance check Volume of the samples for the analysis Pooled QC Samples Preparing the Pooled QC samples NMR Samples NMR sample preparation REFERENCES... 5

2 NIH Eastern Regional Comprehensive Metabolomics Resource Core at RTI International Page 2 of BACKGROUND Urine samples are aqueous solutions and they can readily be mixed with the Chenomx Internal Standard (ISTD) Solution, Deuterium Oxide (D 2 O), and Phosphate Buffer in D 2 O. Aqueous NMR solutions should contain at least 10% of D 2 O to maintain a good lock signal on the NMR Spectrometer. Chenomx ISTD contain 5mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS-d6, chemical shift indicator), 100 mm imidazole (ph indicator), and 0.2% NaN 3 (to inhibit bacterial growth) in D 2 O.It is also necessary to prepare pooled samples (to represent the whole study as well as the sub-study phenotypic groups) for quality control measures. This procedure should be used as a guide based on previously described methods 1 and adjusted to individual studies as needed. 2.0 SCOPE This CSP describes the preparation of urine samples using phosphate buffer for broad spectrum metabolomics analysis. 3.0 MATERIALS 3.1 Equipments 100 L Pipette 200 L Pipette 1000 L Pipette 2 ml Eppendorf tubes 15 ml disposable tubes Pipette tips Centrifuge apparatus 5mm/ 3mm NMR tubes 10 ml Volumetric flasks Weighing balance ph meter Labels 3.2 Reagents Chenomx ISTD (IS-2, 5mM DSS-d6, 0.2% w/v NaN3, > 98% v/v D2O) Dry Ice 0.2M Phosphate Buffer (ph=7.4) in 99.9% D 2 O Ice 99.9% Deuterium Oxide (D 2 O) Sigma-Aldrich

3 NIH Eastern Regional Comprehensive Metabolomics Resource Core at RTI International Page 3 of PROCEDURE 4.1 General Guidelines Daily pipette and balance check 1) Ensure daily pipette checks have been performed on all the pipettes that are going be used during the sample preparation Volume of the samples for the analysis 1) Check for the minimum volume of all the samples received to determine the maximum sample volume to be used. 2) Determine the number of pooled QC samples needed for each study phenotype as well as for the combined phenotypes. Typically the total number of QC samples is 10% of the study samples. 3) To be consistent with the analysis, use a fixed volume from each of the samples for sample preparation (including pooled QC samples). Typically, 400 L of urine is needed for the sample preparation. 4) Determine the volume of each sample needed for preparing QC samples. Use a volume that will be sufficient to prepare the total number of aliquots from pooled samples. Typical minimum number of replicate samples per QC group is 3. 5) If sample volume is an issue, use alternative source of QC samples for the analysis. 6) For limited sample volumes, use 3mm NMR tubes and adjust the volumes accordingly. The filling volume for a 5mm NMR tube is 600 L, whereas the volume needed for 3mm NMR tube is 200 L. 7) Complete the QC Checklist - Urine Sample Preparation for NMR (using the template provided), sign, and date. Keep it in the study binder 4.2 Preparing 0.2M Phosphate Buffer (ph=7.4) containing 0.2% NaN 3 1) Weigh 200 mg of NaN 3 into a 15 ml tube. 2) Prepare a 2% solution of NaN 3 in D 2 O by adding 10 ml of D 2 O.

4 NIH Eastern Regional Comprehensive Metabolomics Resource Core at RTI International Page 4 of 6 3) Weigh mg of Na 2 HPO 4 and transfer into a 10 ml volumetric flask. 4) Weigh 52.5 mg of NaH 2 PO 4 and transfer into the same 10 ml volumetric flask. 5) Add 1 ml of 2% NaN 3 prepared in step 2 into the same 10 ml volumetric flask. 4.3 Pooled QC Samples 6) Fill the 10 ml volumetric flask to the line by adding D 2 O. 7) Check the ph of the solution and adjust it to ph=7.4, if needed, by adding acid or base Preparing the Pooled QC samples 1) If frozen, thaw the samples on ice. Vortex for 30s before use. 2) Transfer an aliquot of samples belonging to each phenotype into a separate 15 ml tube. 3) Vortex for 30s.These will be Pooled QC (Individual Phenotype) samples. 4) Transfer an aliquot (fixed volume) from each of the Pooled QC Sample in step 2 into a 15 ml tube. 5) Vortex for 30s. This will be the Pooled QC (Combined Phenotypes) sample. 4.4 NMR Samples NMR sample preparation 1) If frozen, thaw the samples on ice. Vortex for 30s before use. 2) Transfer aliquots of 400 L of urine samples into Eppendorf tubes. If the minimum volume is not 400 L, make up the volume to 400 L using D 2 O. 3) Add 230 l of 0.2M Phosphate Buffer of D 2 O into each of the Eppendorf tube. The urine may have a variable ph so the use of a buffer permits for better spectral alignment. If ph is desired as an independent variable, D 2 O should be used to determine the native ph through the imidazole chemical shift. 2 4) Add 70 L of Chenomx ISTD solution into each Eppendorf tube.

5 NIH Eastern Regional Comprehensive Metabolomics Resource Core at RTI International Page 5 of 6 5) Vortex for 30s and centrifuge at rcf for 5 minutes. 6) Transfer 600 L aliquot of the supernatant into 5mm NMR tube (or 200 L aliquot into 3mm NMR tube). Store at 4 C. If the samples are not analyzed immediately, the samples need to be stored in cryo vials at -80 C until data acquisition. Ensure that the tubes are freezer-friendly labeled, flash-freeze the samples in N 2 (l), and store in -80 C freezer in an appropriately labeled box until analysis. 7) If previously frozen samples are to be analyzed, thaw the samples on ice and vortex for 30s in preparation for sample analysis. 9) After data acquisition is completed, transfer the samples back into labeled Eppendorf tubes and store them in the -80 C freezer. 5.0 REFERENCES 1. Beckonert, O., et al. Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts. Nature protocols 2, (2007). 2. Baryshnikova, O.K., Williams, T.C. & Sykes, B.D. Internal ph indicators for biomolecular NMR. Journal of biomolecular NMR 41, 5-7 (2008).

6 NIH Eastern Regional Comprehensive Metabolomics Resource Core at RTI International Page 6 of 6 REVIEW & REVISION HISTORY Version Describe Major Changes or Indicate Reviewed with No Revisions Effective Date/ Review Date New Review Date 0 New CSP