This protocol is designed to perform high-throughput metabolic profiling analysis on human blood plasma by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS). In the development of the protocol, UPLC columns with three different stationary phases (C8, C18, and phenyl) were used in identical experimental runs consisting of a total of 60 injections of extracted male and female plasma samples. The C8 column was determined to be the best for metabolic profiling analysis on plasma. The acquired UPLC/MS data of extracted male and female plasma samples was subjected to principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS–DA). Furthermore, a strategy for compound identification was applied here, demonstrating the strength of high-mass-accuracy time-of-flight (TOF)/MS analysis in metabolic profiling. An article providing the full scientific background of the protocol is available at: http://www.ncbi.nlm.nih.gov/pubmed/17964273
This protocol provide guidance from blood sampling to data analysis according to instructions in an external article.
- Distilled water from a Millipore Milli-Q system (Billerica, MA, USA). HPLC-grade methanol and acetonitrile purchased from J. T. Baker Phillipsburg, NJ, USA). 3-Aminofluoranthene purchased from Aldrich (Milwaukee, WI, USA). 1-Aminopyrene 1-palmitoyl-glycero-3-phosphocholine caffeine cytosine formic acid (LC/MS grade) nalidixic acid theophylline HPLC-grade leucine enkephalin purchased from Fluka (Milwaukee, WI, USA) Amitriptyline hydrochloride β-Asp-Leu isoleucine γ-Glu-Leu hippuric acid leucine 1-oleoyl-glycero-3-phosphocholine phenylalanine 1-stearoyl-glycero-phosphocholine tryptophan and tyrosine purchased from Sigma (St. Louis, MO, USA).
- MM301 vibration mill (Retsch, Haan, Germany) 0.22-μm ultrafiltration tube (Millipore) SpeedVac concentrator (Savant Instrument, Framingdale, NY, USA) Chromatography was performed on a Waters Acquity UPLC system, equipped with a column oven, coupled to a Micromass LCT Premier TOF mass spectrometer, equipped with an electrospray source operating in positive ion mode with lockspray interface for accurate mass measurements. MATLAB software (version 7.0, MathWorks, Natick, MA, USA) SIMCA-P+ 11 (Umetrics, Umeå, Sweden)
IntroductionThis protocol is based on an evaluation using 20 healthy donors (10 male and 10 female). All instructions regarding pooling are based on the assumption that 20 donors are used.Chromatography settings
Chromatography was performed on a Waters Acquity UPLC system, equipped with a column oven, coupled to a Micromass LCT Premier TOF mass spectrometer, equipped with an electrospray source operating in positive ion mode with lockspray interface for accurate mass measurements. The source temperature was 120 °C with a cone gas flow of 10 L/h, a desolvation temperature of 300 °C, and a nebulization gas flow of 600 L/h. The capillary voltage was set at 3 kV for positive ion mode, with a cone voltage of 0 V, a data acquisition rate of 0.1 s, and an interscan delay of 0.1 s, with dynamic range enhancement (DRE) mode activated. Leucine enkephalin was employed as the lockmass compound, infused straight into the MS at a concentration of 400 pg/μl (in 50:50 acetonitrile/water) at a flow rate of 20 μl/min. The normal lockmass in the DRE mode was the positive ion second 13C peak of leucine enkephalin at 558.2829, and the extended lockmass peak was the normal positive ion peak observed at 556.2771. All mass spectral data were acquired in the centroid mode, 50 to 850 m/z, with a data threshold value set to 3. A 2-μl aliquot of extracted plasma sample was injected onto a 2.1 × 100-mm, 1.7-μm UPLC column (C18, C8, and phenyl UPLC columns all employed in separate experiments). The gradient elution buffers were A (water and 0.1% formic acid) and B (acetonitrile and 0.1% formic acid), and the flow rate was 500 μl min–1. The column was eluted with a linear gradient, and four separate gradient methods were employed. Gradient 1 consisted of 1 to 20% B over 0 to 4 min, 20 to 40% B over 4 to 6 min, and 40 to 95% B over 6 to 9 min, with the composition being held at 95% B for 4.5 min, returned to 1% B for 14.5 min, and kept at 1% B for a further 4.5 min before the next injection. Gradient 2 consisted of 5% B to 95% B in 10 min and thereafter being held at 95% B at 5.5 min. Gradient 3 consisted of 5% B to 95% B in 30 min and thereafter being held at 95% B at 5.5 min. Gradient 4 consisted of 1 to 20% B over 0 to 4 min, 20 to 40% B over 4 to 6 min, and 40 to 95% B over 6 to 9 min, with the composition being held at 95% B for 8.5 min, returned to 1% B for 18.5 min, and kept at 1% B for a further 4.5 min before the next injection.
- Collect fresh EDTA anticoagulated blood healthy blood donor. Prepare blood plasma by centrifuging at 2053g for 10 min at 4 °C. Prepare total pooled samples by mixing equal amounts of all 20 samples into one vessel and split this new pooled mixture into 100-μl aliquots. Prepare female pooled samples and split these into 100-μl aliquots. Prepare male pooled samples and split these into 100-μl aliquots. Prepare mixed pooled samples consisting of different mixes of male and female pooled samples (40 μl of male pooled sample + 60 μl of female pooled sample = sample 4 male:6 female; 50 μl of male pooled sample + 50 μl of female pooled sample = sample 1 male:1 female; 60 μl of male pooled sample + 40 μl of female pooled sample = sample 6 male:4 female). Store all samples in 100-μl aliquots at –80 °C.
- Thaw frozen plasma aliquots on ice. Use a solution of 80 % methanol as extraction mix for plasma 1. Add 400 μl of extraction mix to each plasma aliquot and vigorously extract at a frequency of 30 Hz for 2 min using an MM301 vibration mill (Retsch, Haan, Germany). 2 Leave the samples on ice for 2h. Centrifuge samples at 2053 g for 10 min at 4 °C. Remove the supernatant (400 μl) and placed it in a 0.22-μm ultrafiltration tube (Millipore) to remove any undissolved particles. Centrifuge the ultrafiltation tubes at 1105g for 2 min and remove the filter from the tube. Keep the resulting filtrate and evaporate to dryness in a SpeedVac concentrator (Savant Instrument, Framingdale, NY, USA). Use the following two stepts to reconstitute the sample in 50 μl of the methanol or acetronitrile mix used in extraction (with the assumption that plasma = 100% aqueous). First add the methanol or acetonitrile and briefly vortex the sample. Then add water and further vortex the sample. Transfer the resulting extracted plasma samples to vials for UPLC/MS analysis.
- Create the blank sample by mixing an 80 % methanol solution and subject it to the same ultrafiltration as the samples to ensure that any contamination peaks included with the filtration step also are added to the blank. Create a standard mix containing the following: 10 μM 1-aminopyrene, 10 μM 3-aminofluoranthene, 10 μM amitriptyline hydrochloride, 100 μM caffeine, 100 μM cytosine, 100 μM nalidixic acid, and 100 μM theophylline in 50% acetonitrile solution.
- Set the chromatopgraph according to the settings given in the introduction or equivalent in the system being used. Divide the plasma samples into batches with each batch consisting of 1 x all samples that are to be injected in a random order within each batch. To each batch add a blank and a standard mix sample as every sevent injection into the machine.
- Convert the MassLynx raw data into netCDF format using the Mass Lynz DataBridgeprogram. Read the netCDF files into MATLAB software. Process the data using peak detection and alignment. Perform data reduction and compression on the data. Instructions for using SIMCA-P+11 is available a footnote to this protocol 3
- 80 % methanol is flammable and unsuited for human consumption. Acetonitrile is mildly toxic.
- 1. Solutions of methanol (100, 90, 80, 70, and 50%) and acetonitrile (100, 90, 80, 70, and 50%) in water was used during testing, refer to the full article for optimal mixtures and 80 % methanol was shown to provide the best results
- 2. With the assumption that plasma = 100% aqueous, the extract mix solutions for both methanol and acetonitrile were considered as 72 % in percentage solvent.
- 3. The data reduction step was achieved by dividing the chromatographic dimension into a number of narrow, equally sized time windows (two data points each). The data in each time window held a three-dimensional data structure defined by the sample dimension, the chromatographic time dimension, and the mass spectral dimension. Window-wise summing of these three-dimensional data cubes in the chromatographic dimension produced a two-dimensional data table defined by the sample dimension and the summed spectral dimension. The data table for each sample was further compressed to a small number of latent variables by means of alternating regression (AR). This compression was also performed window-wise to obtain a data table suited for sample comparisons by means of multivariate analysis (MVA), with AR being the preferred choice for the latent variable compression step due to there being no orthogonality constraint in AR. This is considered to be reasonable when the mass spectra for the different compounds do not need to be orthogonal to each other. The AR procedure was performed for each time window individually. By combining the concentration profiles created from the AR from all time windows, a final reduced data table (X) was obtained and was subject to further MVA. Peak integration was performed with MassLynx software using APEX Track integration. P. Jonsson, S.J. Bruce, T. Moritz, J. Trygg, M. Sjöstrom, R.S. Plumb, J. Granger, E. Maibaum, J.K. Nicholson, E. Holmes, H. Antti Extraction, interpretation, and validation of information for comparing samples in metabolic LC/MS data sets