Orbitrap™ Technology HRAMS Potentiates Metabolomic Research Sensitive but Stable High Resolution Mass Spectrometry by Tosapol Anukunwithaya, PhD MS Product Specialist FUNDAMENTAL OF MASS SPECTROMETER What is Mass Spectrometer? The basis in mass spectrometry (MS) is the production of ions, that are subsequently separated or filtered according to their mass-to-charge (m/z) ratio, and detected. Mass Spectra – m/z m/z = ( molecular weight + charge ) / charge 512.287 = (1022.6 + 2) / 2 1023.566 = (1022.6 + 1) / 1 Information Rich Data Mass Spectrometry – Block Diagram Liquid Ionization Source Chromatography - Columns • ESI - Solvent systems • APCI • APPI ION SOURCE IONIZATION TECHNIQUES Ionization • Ion sources : Converts sample molecules (neutral) into charged molecules or molecular ions. • Type of ionization techniques o Atmospheric Pressure Ionization (API) - Electrospray Ionization (ESI) - Atmospheric Pressure Chemical Ionization (APCI) - Atmospheric Pressure Photo Ionization (APPI) o Matrix Assisted Laser Desorption Ionization (MALDI) Atmospheric Pressure Ionization (API) ESI APCI Electrospray Ionization (ESI) Atmospheric Pressure Chemical Ionization (APCI) Atmospheric Pressure Ionization (API) MASS ANALYZERS Mass Spectrometry – Block Diagram Liquid Ionization Source Mass Analyzer Detector Chromatography •Ion-Traps •Triple Quadrapoles •Time-of-flight •Orbitrap Mass Analyzers Separate Ions • Operate under high vacuum environment (prevent ions from bumping into gas molecules). • Actually measure mass-to-charge ratio of ions (m/z). • Key specifications are resolution, mass measurement accuracy, and sensitivity. • Several kinds exist: for ion traps, quadrupole, time-of-flight and orbitrap are most used. Mass Analyzer : Ion Trap • Consists of ring electrode and two end caps • Principle very similar to quadrupole • Ions stored by RF & DC fields • Scanning field can eject ions of specific m/z Mass Analyzer : Quadrupole Uses a combination of RF and DC voltages to operate as a mass filter. • Has four parallel metal rods. • Lets one mass pass through at a time. • Can scan through all masses or sit at one fixed mass. Quadrupole → Triple Quadrupoles (QqQ) Mass Analyzer : Triple Quadrupoles (QqQ) HyperQuad quadrupole mass Active collision cell (Q2) filter (Q3) Ion beam guide with neutral blocker HyperQuad quadrupole mass Dual-mode discrete-dynode filter (Q1) detector Asymetric RF drive Electrodynamic ion funnel (EDIF) High-capacity transfer tube (HCTT) Mass Analyzer : Triple Quadrupoles (QqQ) Mass Analyzer : Triple Quadrupoles (QqQ) Full Scan Mode Purpose: Survey scan of a chromatographic peak Q1 Scanning RF Only Q3 RF Only Full scan Q1: Mass Analyzer : Triple Quadrupoles (QqQ) R T : 0 .0 0 - 2 0 .0 2 1 .2 4 N L : 1 0 0 7.35E 7 9 5 TIC M S H S -he lin- 9 0 1024-1 8 5 8 0 4 .6 0 7 5 7 0 6 5 6 0 5 5 Chromatogram 5 0 4 5 4 0 RelativeAbundance 3 5 2 .6 2 3 0 3 .7 9 2 5 3 .1 0 2 0 5 .3 8 1 5 7 .7 8 1 0 7 .5 8 5 8 .2 3 8 .9 0 9 .8 1 1 2 .7 3 1 4 .0 5 1 5 .0 9 1 5 .9 5 16.931 8 .3 4 18.94 0 0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 Tim e (m in) One Click 100 240 Spectrum H + OH Base peak NH tBu + HO at m/z 240 (MH ) % HO 241 0 60 80 100 120 140 160 180 200 220 240 260 280 300 Mass Analyzer : Triple Quadrupoles (QqQ) SIM is in essence a full scan acquisition on a relatively narrow mass window (defined as center mass / scan width) Fixed m/z Pass All Pass All Advantages Disadvantages Targeted analyte monitoring Can suffer from interferences High duty cycle Not as sensitive or selective as SRM Mass Analyzer : Triple Quadrupoles (QqQ) RT: 0.00 - 75.04 SM: 7G 52.33 NL: 2.91E8 100 Base Peak F: + c NSI Full ms [ 90 400.00-1800.00] MS data14 80 70 60 50 40 Full Scan 47.88 RelativeAbundance 30 31.30 55.14 20 34.47 50.24 10 1.00 6.50 8.0911.51 17.22 18.87 23.56 24.15 39.42 42.17 44.24 56.03 63.65 65.28 70.26 72.63 0 31.30 NL: 7.97E7 100 Base Peak m/z= 1030.90-1031.90 F: 90 + c NSI Full ms [ 400.00-1800.00] 80 MS data14 70 60 50 SIM 40 RelativeAbundance 30 20 10 38.39 39.85 3.233.45 10.3614.03 19.66 21.90 27.26 30.99 40.53 47.88 52.4455.53 59.41 64.64 67.24 73.57 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time (min) Mass Analyzer : Triple Quadrupoles (QqQ) Fixed m/z Pass All Fixed m/z Q1 Q2 Q3 Advantages Disadvantages Targeted analyte monitoring No structural information High duty cycle “Simultaneous” monitoring of multiple transitions Mass Analyzer : Triple Quadrupoles (QqQ) m/z 400 m/z 250 m/z 400 m/z 150 m/z 250 m/z 400 m/z 300 m/z 400 m/z 100 Q1: Precursor Ion Q2: Fragmentation Q3: Product Ion Fixed m/z: 400 Fixed m/z: 250 m/z 400 m/z 250 m/z 300 m/z 400 Mass Analyzer : Triple Quadrupoles (QqQ) RT: 17.13 - 27.39 RT: 23.76 NL: 6.37E4 100 m/z= 236.50-237.50 F: + c SIM 24.01 ms [236.50-237.50, 80 271.50-272.50, 306.50-307.50] MS probe20f_sim 60 23.55 24.11 25.52 SIM 23.28 24.55 25.35 22.52 22.97 25.65 40 22.17 25.87 21.67 26.45 26.78 21.09 21.30 20 20.27 20.40 Relative Abundance 0 25.37 NL: 2.67E4 100 m/z= 271.50-272.50 F: + c SIM ms [236.50-237.50, 80 271.50-272.50, 306.50-307.50] MS probe20f_sim 60 23.53 RT: 23.76 27.35 40 22.93 20.19 23.43 26.62 21.2522.41 24.55 24.67 25.48 26.26 20 19.61 20.57 22.17 24.02 Relative Relative Abundance 0 NL: 9.94E3 0 RT: 23.77 NL: 6.10E5 100 m/z= 207.50-208.50 F: + c EI SRM ms2 237.000 80 [207.999-208.001] MS Genesis Probe20F 60 SRM 40 20 Relative Relative Abundance 0 RT: 23.77 NL: 1.06E6 100 m/z= 236.50-237.50 F: + c EI SRM ms2 272.000 80 [236.999-237.001] MS Genesis Probe20F 60 Superior Selectivity 40 20 Free from sample matrix Relative Abundance 0 18 19 20 21 22 23 24 25 26 27 Time (min) Mass Analyzer : Triple Quadrupoles (QqQ) RT: 2.28 - 5.89 SM: 15G x300 RT: 5.37 NL: 8.14E7 SN: 1093 m/z= 191.50-192.50 100 MS Genesis Full-MS2 80 60 40 Full MS Scan 20 0 RT: 5.37 NL: 1.38E8 SN: 27528 m/z= 191.50-192.50 F: 100 + c EI Q1MS [191.945-191.995] MS 80 Genesis SIM-1_111128173605 60 Noise Level 40 SIM Scan 20 RelativeAbundance 0 RT: 5.37 NL: 3.27E7 SN: 201353020 TIC F: + c EI SRM ms2 100 191.950 [126.935-126.985] MS 80 Genesis Full-SRM-Survey-1 60 40 SRM Scan 20 0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Time (min) Mass Analyzer : Triple Quadrupoles (QqQ) http://www.youtube.com/watch?v=LFB14D8pkoc HIGH RESOLUTION MASS ANALYZERS Mass Analyzer : Time-of-flight (TOF) • A package of ions is accelerated by a potential V into a field free flight tube. • The time tflight needed for ions to reach a detector placed at distance d is measure. • Small ions have higher velocity than large mass ions therefore their time of flight in the tube are different and related to m/z ratio Mass Analyzer : Time-of-flight (TOF) • Resolution limited by: – Length of TOF flight tube – Kinetic energy distribution – Propagation delay in detector • Sensitivity limited by: – Ion stability – Ion transfer efficiency ORBITRAP MASS ANALYZER Alexander Makarov Mass Spectrometry – Block Diagram Liquid Ionization Source Mass Analyzer Detector Chromatography •Ion-Traps •Triple Quadrapoles •Time-of-flight •Orbitrap Mass Spectrometry – Block Diagram Liquid Ionization Source Mass Analyzer Detector Chromatography • Orbitrap Mass Analyzer : Orbitrap™ Technology Mass Analyzer : Orbitrap™ Technology • An ion packet of a selected m/z enters the field. • Increasing voltage squeezes ions. • Voltage stabilises and ion trajectories are also stabilized. • Angular spreading forms a rotatating ring. (r,φ) (r,z) Mass Analyzer : Orbitrap™ Technology • Ions trapped in an electrostatic field. • Central electrode kept on high voltage. • Outer electrode is split and able to pick up an image current induced by ion packets moving inside the trap. r Characteristic frequencies: z • Frequency of rotation ωφ • Frequency of radial oscillations ωr • Frequency of axial oscillations ωz φ k U (r, z) z 2 r 2 / 2 R 2 ln(r / R ) 2 m m A. Makarov, Anal. Chem 2000, 1156-1162 Mass Analyzer : Orbitrap™ Technology Image Current Fourier Transform k z m / q Frequency Domain Mass Spectrum Mass Analyzer : Orbitrap™ Technology Mass Analyzer : Orbitrap™ Technology http://planetorbitrap.com/q-exactive-plus#.WmoCMeRG3IX Orbitrap™ Technology HRAMS Potentiates Metabolomic Research Sensitive but Stable High Resolution Mass Spectrometry by Tosapol Anukunwithaya, PhD MS Product Specialist Orbitrap™ Technology HRAMS • HRAMS = High Resolution Accurate Mass Spectrometry • Mass Resolution = ability of a mass spectrometer to distinguish between ions of nearly equal m/z ratios (isobars). • Mass Accuracy = the precision of which the mass is measured by the mass spectrometer.