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 Relative Abundance Relative 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 Relative Abundance Relative 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 Relative Abundance Relative 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 Relative 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 AbundanceRelative 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 AbundanceRelative 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 Relative 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
Relative Abundance Relative 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. Mass Resolution
100 Low Resolution 90 80 70 R = 786.6 = 1,000 60 50 Δm 0.786 40 = 0.786 30 20 10 m = measured mass 786 788 790 792 794 Δm = peak width measured at 50% peak intensity 100 (Full Width Half Maximum) 90 80 High Resolution 70 60 R = 786.6 = 100,000 50 Δm 40 = 0.007 30 0.007 20 10
786 788 790 792 794 Mass Accuracy
• Mass error reporting in ppm (relative mass error):
ퟔ
• Exact Mass is the mass of an ion with a given empirical formula calculated using the exact mass of the most abundant isotope of each element C = 12.0000 Example M = 249; C20H9+ 249.0070 H = 1.0078 C19H7N+ 249.0580 N = 14.0031 C13H19N3O2+ 249.1479 O = 15.9949 S = 31.9721 Mass Resolution and Accuracy
• Main advantage: the possibility to determine the elemental composition of individual molecular or fragment ions, a powerful tool for the structural elucidation or confirmation.
Mass Accuracy Number of Hits*
+ [M+H] 381.0782 ± 200 ppm 265
± 100 ppm 133
± 30 ppm 39 CxNxOxHx ± 10 ppm 14
± 5 ppm 5
± 3 ppm 4
± 1ppm 1 C12H22O11 Mass Resolution and Accuracy
• Isobaric compounds separation Mass Resolution and Accuracy
• Removing interferences: High resolution is very important for samples with complex matrix (e.g. biological, food), since they will contain a significant number of background ions.
• High resolution is needed for: • Separating co-eluting molecules with close masses • Mass accuracy • Good quantitation results Sensitivity and Resolution : No Compromise Stability : Robust and Reproducible HRAMS
Ultimate 3000 HPG UHPLC coupled to Q Exactive
Full Scan MS: R=70K, d5-Hippuric acid, theoretical m/z 185.0969 Stability : Robust and Reproducible HRAMS
• Peak intensity (area) variation during 900 injections (blue) with according mass accuracy (peak apex scan, red)
RSD = 11.9% Orbitrap Applications Universe
Environmental testing in Water MS Technology in Pharma Research
Drug Drug Discovery Preclinical Evaluation Formulation Natural Product Determination of in vitro Testing Metabolism Study • Unknown active compound • Permeability test • Target/ Non- compound concentration target identification • Solubility test in vivo Testing metabolite • Plant metabolic • Stability test • Toxicological identification pathway study • Pharmacokinetic Metabolomics Synthetic Compounds study • Molecular mass confirmation
High Resolution High Sensitivity High Resolution ORBITRAP™ TECHNOLOGY HRAMS POTENTIATES METABOLOMIC RESEARCH Metabolomics
• Metabolomics is the scientific study of chemical processes involving metabolites, the small molecule intermediates and products of metabolism.
• Specifically, metabolomics is the "systematic study of the unique chemical fingerprints that specific cellular processes leave behind", the study of their small-molecule metabolite profiles. Metabolomics
Khoomrung et al. (2017) Challenges of Metabolomics
• Diversity in structures and physical chemical properties – Require multiple technologies to capture a metabolome
• Many isomeric and isobaric species – Require high resolving power for correct ID
• Very low to very high concentrations – Require High sensitivity and wide dynamic range
• No single database to identify all unknown metabolites – Require extensive library or fragment ion prediction based on compound structure Diverse Physical Properties among Metabolites
Metabolites Chromatography MS ionization triglycerides Hydrophobic + cholesterol esters + diacylglycerols + sphingomyelines + phosphatidylcholines + phosphatidylethanolamines C18 + other phospholipids +& - fatty acids -
eicosanoids & metabolites Polarity bile acids HILIC - bilirubin - amino acids, amines + or - +
organic acids IC sugars - Other polar: - GC e.g. purines & pyrimidines Hydrophilic - Challenges of Metabolomics
• Diversity in structures and physical chemical properties – Require multiple technologies to capture a metabolome
• Many isomeric and isobaric species – Require high resolving power for correct ID
• Very low to very high concentrations – Require High sensitivity and wide dynamic range
• No single database to identify all unknown metabolites – Require extensive library or fragment ion prediction based on compound structure Resolution: QE HF, QE vs. State-of-Art QTOFs
Q Exactive HF Q Exactive QTOF brand S QTOF brand A QTOF brand B
350000
300000 Superior resolution ideal for small molecule analysis 250000
200000
150000
100000 Resolution Resolution (FWHM) 50000
0 100 200 300 400 500 600 700 800 900 1000 m/z Sensitivity : 5 Orders of Dynamic Range Challenges of Metabolomics
• Diversity in structures and physical chemical properties – Require multiple technologies to capture a metabolome
• Many isomeric and isobaric species – Require high resolving power for correct ID
• Very low to very high concentrations – Require High sensitivity and wide dynamic range
• No single database to identify all unknown metabolites – Require extensive library or fragment ion prediction based on compound structure Untargeted Metabolomics Workflow
Collect Plasma, Urine, Sample Preparation Collect High Resolution Data Processing Lists of Feces, Tissues, etc. LC/MS Profiles Compounds
t-MS/MS
Metabolic Pathway
Pathways Metabolites Trends Statistical Analysis Enhance your Research with Compound Discoverer
https://goo.gl/YvAS2j Compound Discoverer : Retrospective for Unknowns
Complete small molecule structure identification in a Next Generation platform.
• Unknown Analysis • Identification • Statistics Compound Discoverer : Flexible Workflows
• Use common, predefined • New in version 3.0: workflows or create your own – Metabolika™ pathways • Integrate your own nodes – Stable isotope labeling – mzLogic™ annotation of unknown compounds – Interactive heat map with hierarchical clustering – mzVault 2.1 – create spectral libraries from CD results Compound Discoverer : Identifying Unknowns Compound Discoverer : Database Hub Compound Discoverer : Metabolic Pathway Compound Discoverer : Statistics
• Differential Analysis : t-test, ANOVA /w post-hoc tests, ratios & fold changes • p-value adjustment for FDR • Principal Component Analysis (PCA) • Normalization • Descriptive Statistics Summary
• Introduction of mass spectrometry
• Key Features of Orbitrap™ Technology HRAMS – High Resolution and Accurate – Sensitive but Stable
• Compound Discoverer – Powerful workflow to identify non-target compounds for metabolomic research Reference Sites for Orbitrap Analyzer (1)
No. Company Department Model Application
1 Faculty of Medicine, Siriraj Hospital Medical Proteomics Unit LTQ XL Orbitrap Education
2 Chulabhorn Research Institute (CRI) Biotechnology Laboratory Orbitrap ELITE Research
3 Charoen Pokphand Foods (CPF) Quality Control Department Q Exactive R & D
4 Department of Livestock Development, Bureau of Quality Control of Q Exactive Food and Ministry of Agriculture and Cooperatives Livestock Product Agriculture
5 Faculty of Medicine, Siriraj Hospital Department of Pharmacology Q Exactive Education
6 Faculty of Medicine, Chulalongkorn University Center of Excellence in Systems Q Exactive Plus Research Biology
7 National Center for Genetic Engineering and Biomolecular Analysis and Orbitrap Fusion Research Biotechnology (BIOTEC), NSTDA Application Laboratory (SBMA)
Updated: January 2019 Reference Sites for Orbitrap Analyzer (2)
No. Company Department Model Application
8 Synchrotron Light Research Institute (SLRI) Applied Research for Industry Q Exactive Plus Research Division Service
9 Chulabhorn Graduate Institute (CGI) Chemical Biology Q Exactive Focus Research
10 Kokoku Innovative Technology Center of Rubber Innovation Q Exactive Focus Research
11 National Center for Genetic Engineering and Plant Physiology and Q Exactive Focus Research Biotechnology (BIOTEC), NSTDA Biochemistry Laboratory
12 Institute of Forensic Medicine (IFM), Police Department of Toxicology Q Exactive Focus Forensic Hospital Toxicology
13 National Center for Genetic Engineering and National Omics Center Q Exactive HF-X Research Biotechnology (BIOTEC), NSTDA
Updated: January 2019 Thank You for Your Attention