Serum Sample Preparation for UPLC-TOF-MS Based Broad Spectrum Analysis. Table of Contents

Transcription

1 NIH Eastern Regional Comprehensive Metabolomics Resource Core at RTI International Page 1 of 5 Table of Contents Page 1.0 BACKGROUND SCOPE MATERIALS Equipment Reagents PROCEDURE Sample Preparation Pooled QC Samples Reversed Phase Method HILIC Method REFERENCES... 4

2 NIH Eastern Regional Comprehensive Metabolomics Resource Core at RTI International Page 2 of BACKGROUND Serum samples for UPLC-TOF-MS broad spectrum metabolomics analysis can be prepared using a methanolic protein precipitation. The protein precipitation approach is a simple yet effective way of extracting all classes of metabolites present in the serum samples. This procedure describes serum sample preparation steps for both reversed-phase and HILIC UPLC-TOF-MS broad spectrum metabolomics analysis. This procedure should be used as a guide for sample preparation. Any required adjustments should be worked out during method development stage. 2.0 SCOPE This center specific protocol (CSP) describes the serum sample preparation steps for UPLC-TOF-MS broad spectrum metabolomics analysis. 3.0 MATERIALS 3.1 Equipment 200µL, 1000µL Pipettes Centrifuge Centrifugal vacuum evaporator Ice 2.0 ml Protein Low-bind Eppendorf tubes Multi-tube vortexer 3.2 Reagents HPLC Grade Water 95:5 Water:Methanol HPLC Grade Methanol L-tryptophan-d5 (ISWS)- 0.05mg/ml 95:5 Acetonitrile:Water 4.0 PROCEDURE This procedure assumes there is adequate volume of serum available to perform all the described analysis. The minimum volume of all the study samples should be checked ahead of time. If there are samples with low volume, then the volumes described in this protocol can be adjusted as long as the ratios of the reagents are kept constant. 4.1 Sample Preparation Daily Checks 1) Ensure daily pipette checks have been performed on all the pipettes that are going be used during the sample preparation Pooled QC Samples 1) Prepare a whole-study pooled QC sample by combining aliquots of equal volume from each study sample. If the sample volume is not enough to

3 NIH Eastern Regional Comprehensive Metabolomics Resource Core at RTI International Page 3 of 5 generate pooled QC sample, then use an equivalent matrix (eg. species/gender matched serum) as external QC sample. 2) Prepare phenotypic pooled QC samples for each study phenotype by combining aliquots of equal volume of sample from each study groups. Preparation of phenotypic pool is optional and should be prepared when there is ample sample available for analysis. 3) Thoroughly mix the pooled QC samples by vortexing for 30 sec Reversed Phase Method 1) Allow serum samples to thaw on ice for min. 2) Transfer 100µL of serum to a new pre-labeled Low-bind Eppendorf tube for each sample, including pooled QC samples. Use cooled sample storage vials and perform pipetting steps on ice. 3) Add 50µL of ice cold internal standard working solution (ISWS) to each tube containing the sample. 4) Add 400 µl of methanol to each tube containing the samples. 5) Thoroughly mix on multiple tube vortexer for 2 mins at 5000 rpm. 6) Pellet the protein precipitate using a centrifuge operating at room temperature and at 16,000 rcf for 4 min. 7) Transfer 400 µl of the supernatant into pre-labeled 2.0 ml Low-bind eppendorf tube and dry down sample in a centrifugal vacuum evaporator operating at 30 C for 3 hrs. 8) If samples are not analyzed immediately, freeze the vials at -80 C after they are dry. 9) For immediate analysis, add the 100 µl of 95:5 Water:Methanol to each sample. 10) Thoroughly mix on multiple tube vortexer for 2 mins at 5000 rpm. 11) Centrifuge samples at room temperature and at 16,000 rcf for 4 min. 12) Transfer the supernatant to pre-labeled autosampler vials. 13) Inject 10 µl into the UPLC-MS system for broad spectrum metabolomics analysis. 14) After sample analysis, place new caps on the autosampler vials and store them at -80 C HILIC Method 1) Allow serum samples to thaw on ice for min. 2) Transfer 50 µl of serum to a new pre-labeled Low-bind eppendorf tube (including the QC samples). Use cooled sample storage vials and perform pipetting steps on ice.

4 NIH Eastern Regional Comprehensive Metabolomics Resource Core at RTI International Page 4 of 5 3) Add 50 µl of ice cold internal standard working solution (ISWS) to each tube containing the sample. 4) Add 400 µl of methanol to each tube containing the samples. 5) Thoroughly mix on multiple tube vortexer for 2 mins at 5000 rpm. 6) Pellet the protein precipitate using a centrifuge operating at room temperature and at 16,000 rcf for 4 min. 7) Transfer 400 µl of the supernatant into pre-labeled 2.0 ml Low-bind eppendorf tube and dry down sample in a centrifugal vacuum evaporator operating at 30 C for 3 hrs. 8) If sample are not analyzed immediately, freeze the vials at -80 C after they are dry. 9) For immediate analysis, add the 200 µl of 95:5 Acetonitrile:Water to each sample. 10) Thoroughly mix on multiple tube vortexer for 2 mins at 5000 rpm. 11) Centrifuge samples at room temperature and at 16,000 rcf for 4 min. 12) Transfer the supernatant to pre-labeled autosampler vials. 13) Inject 2-5 µl into the UPLC-MS system for HILIC broad spectrum metabolomics analysis. 14) After sample analysis, place new caps on the autosampler vials and store them at -80 C. 5.0 REFERENCES Dunn, et al. Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry. Nat Protoc 2011, 6, Spagou, et al. HILIC-UPLC-MS for exploratory urinary metabolic profiling in toxicological studies. Anal Chem 2011, 83, (1),

5 NIH Eastern Regional Comprehensive Metabolomics Resource Core at RTI International Page 5 of 5 REVIEW & REVISION HISTORY Version Describe Major Changes or Indicate Reviewed with No Revisions Effective Date/ Review Date 0 New CSP