Analysis of Polar Metabolites using Mass Spectrometry TransMed Course: Basics in Clinical Proteomics and Metabolomics. Oct 10-19, 2012 dd.mm.yyyy Vidya Velagapudi, Ph.D, Adjunct Professor Head of the Metabolomics Unit, Tech Centre, Institute for Molecular Medicine Finland FIMM, Helsinki, Finland 12/10/12 FIMM - Institiute for Molecular Medicine Finland www.fimm.fi
Outline Polar metabolite extraction Liquid chromatography Mass spectrometry Work flow Small Test www.fimm.fi 2
Automated sample extraction Polar metabolite extraction www.fimm.fi 3
Why LC/MS/MS? Analysis of labile analytes Analysis of more polar compounds without derivatization. Analysis of significantly higher masses Reduction of lengthy clean-up LC-MS (Single quadrupole) LC-MS/MS (Triple quadrupoles) LC-TOF-MS (Time-of-flight) Q-TOF-MS (Quadrupole time-of-flight) LC-Q (Ion traps, linear ion traps) LC-Q-TRAPS (Quadrupole linear ion trap) MALDI-TOF-MS FT-MS (Fourier Transform) www.fimm.fi 4
Response Residual 21121102 Smooth(Mn,2x2) Class123_81cmpds_acidic_level1 100 Chromatogram F8:MRM of 1 channel,es 116.1 > 70.1 1.88 4.162e005 % Proline 1.43 11563 1.58 1.71 0 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 1.65 1.70 1.75 1.80 1.85 1.90 min Compound name: Proline Correlation coefficient: r = 0.997859, r^2 = 0.995722 Calibration curve: 190.945 * x -4438.66 Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 40.0 20.0-0.0 ppb 10000000 7500000 5000000 2500000-0 -0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 55000 ppb www.fimm.fi 5
What is Mass Spectrometry (MS) MS does not measure the mass of a compound Mass spectrometers use the difference in mass-to-charge ratio (m/z) of ionized compounds to separate them from each other. Compounds have distinctive fragmentation patterns that provide structural information to specifically detect each compound very precisely. MS has emerged as an ideal technique for the identification of almost all structurally diverse metabolites. MS/MS data provides tremendous structural information for any metabolites www.fimm.fi 6
System Configuration Liquid Chromatography Very important! Many columns Many solvent systems ESI APCI APPI Triple Quadrupoles Ion-Traps Hybrids Ionisation Source It depends on the exact application. Increasing polarity and molecular weight and thermal instability favors electrospray. Lower polarity and molecular weight favors APCI or APPI www.fimm.fi 7
Electrospray: Overview 5kV Orifice Plate Nebulizing Gas Electrospray is a method of getting the solution phase ions into the gas phase so that they can be sampled by the MS. Droplet Formation Drying Gas Desolvation & Fission Gas Phase Ion Generation To MS 1.Production of charged droplets. 2.Droplet size reduction, and fission. 3.Gas phase ion formation Curtain Plate Curtain Gas www.fimm.fi 8
ESI: Production of Charged Droplet 1. A large voltage ( up to 6kV) is applied between the end of a capillary carrying the LC mobile phase and the mass spectrometer entrance. 2. Ions (of the same polarity) are drawn out toward the counter electrode (curtain plate) pulling the mobile phase along. 3. When the excess charge at the tip of the capillary overcomes surface tension, a droplet is formed. Droplet Formation www.fimm.fi 9
ESI: Droplet size reduction & fission Droplet size reduction occurs by the continual repetition of two processes: 1. Desolvation (evaporation of neutral solvent and volatile buffers) 2. Droplet fission caused by electric repulsion between like charges. Congratulations! You have made an ion. Now what do you do with it? www.fimm.fi 10
Triple Quad Configuration Q0 Q1 Q2 Q3 RF only Scanning RF/DC RF only Collision Cell Scanning RF/DC Q1 and Q3 are standard mass filter quadrupoles. The can scan masses sequentially (e.g. 50 to 500 amu) The can be used to select a single mass. Q2 is an RF only quadrupole that is in a gas filled chamber. Q2 is the collision cell where mass fragmentation occurs. Q2 does not filter ions. It accepts all ion sent to it by Q1 and passes all ions formed by collision to Q3 to be sorted. www.fimm.fi 11
Collision Cell LINAC (linear accelerator) Collision Cell Filled with N 2 gas at roughly 3x10-5 torr. Drives ions out, reducing cross-talk The analyte molecules undergo collision activated disassociation by energetic collision with the N 2 molecules. The N 2 also acts to cool fragments, facilitating transport to the detector. Q0 Q1 Q2 Q3 RF only Scanning RF/DC RF only Collision Cell Scanning RF/DC www.fimm.fi 12
Triple Quads In scanning mode 99% ions lost between the rods. Poorer full scan sensitivity In SIM mode 100% of selected ion reaches detector. Makes them highly sensitive and great for quantitation! Mass resolution typically limited to unit (/- 0.2 amu) Fragmentation is controlled by the energy ions have when they enter the collision cell. Higher energy >> greater fragmentation. www.fimm.fi 13
Multiple Reaction Monitoring (MRM) Q0 Q1 Q2 Q3 Exit lens Ion accumulation Precursor ion selection Fragmentation Daughter ion selection Q1 Selects an [MH] Q2 fragments the selected ion. Q3 monitors only one daughter ion Only the daughter ion reaches the detector. Sensitivity of MRM is a function of how much of the daughter ion is produced. The parent ion fragmentation to daughter ion is commonly referred to as a transition www.fimm.fi 14
Electron multipliers, Dynolyte photomultiplier, Microchannel plates. Detectors Electron multiplier A conversion dynode is used to convert either negative or positive ions into electrons. These electrons are amplified by a cascade effect in a horn shape device, to produce a current. This device, also called channeltron, is widely used in quadrupole and ion trap instruments. www.fimm.fi 15
MS provides info about The elemental composition of samples of matter The structures of organic, inorganic and biological molecules The qualitative and quantitative composition of complex mixtures Isotopic ratios of atoms and samples Structure and composition of solid surfaces www.fimm.fi 16
Analysis of the extract MRM UPLC/3Q RPLC/HILIC Extracted Metabolites ISTDs Calibration curves Daughter ions chromatogram Extracted sample A E /A I Quantification Concentration Retention time www.fimm.fi 17