Metabolomics 3 Flashcards
Mass Spec in Drug Discovery
- LIBRARY GENERATION
- verify identity and determine purity
- purify drug candidates - HIGH-THROUGHPUT SCREEENING
- screen combinatorial libraries for activity - ADME (ABSORPTION, DISTRIBUTION, METABOLISM AND EXCRETION)
- determine PK in animal models
- in vitro screen for permeability and metabolism
- metabolite identification
- mass balance - BIOMOLECULES, OMICS
- proteomics
- variant and degradation products (etc AB lacking a light chain)
- QC testing lot-to-lot for recombinant protein therapeutics
- in process monitoring
- metabolomics, pharmaco-metabolomics
Mass Spec in MS Method
LIBRARY GENERATION
- flow injection analysis MS (FIA-MS)
- parallel multiple column with on-line MS
- ultra fast HPLC ESI/TOF
- dual column m/z triggered fraction collection
- also NMR
HIGH TROUGHPUT SCREENING
- affinity capillary electrophoresis MS
- affinity MALDI-TOF
ADME (ABSORPTION, DISTRIBUTION, METABOLISM AND EXCRETION)
- LC-MS/MS, 96 well solid-phase extraction, Ce-MS for direct urine or plasma analysis
- 96 well SPE coupled with LC-MS
- ESI triple quadrupole, ESI/Q-TOF, ESI/ion trap, many NMR methods
- continous flow-isotrope ratio MS
BIOMOLEUCLES, OMICS
- various PC-MS and MS/MS
- MALDI-TOF-MS, LC-ESI-MS
- MALDI
- LC-ESI-Q-TOF-MS
- various assorted MS methods, NMR
The Mass Spec Market
In 2009 global sales, instrument service contracts and ancillary services were approximately $3.3 billion (21). The sector has continued strong growth prospects and is projected to experience annual growth of 8-10% through 2012. At least 27 different companies manufacture and sell a wide range of MS instrumentation.
Aufbau eines Massenspektrometers
Inlet = introduction of sample
Source = Formation of ions
Mass Analyzer = separation of ions
Detector = detection of ions
Vacuum system (combines source, mass analyzer and detector) = minimize ion deactivation
Data system = data analysis
Upfront separation: chromatography
- The separation of components in a mixture that involves passing the mixture dissolved in a “mobile phase” through a stationary phase, which separates the analyte to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases
- Column, thin layer, liquid, gas, affinity, ion exchange, size exclusion, reverse phase, normal phase, gravity, high pressure
HPLC Schematic
- Solvent (Mobile Phase) Reservoir
- Pump/Solvent Manager/Solvent Delivery System
- Sample
- Injector/AutoSampler/Sample Manager
- HPLC Column/Packing Material
- Detector
- Chromatogram/Computer Data Station
- Waste
High Pressure (Performance) Liquid Chromatography HPLC
- Developed in 1970’s
- Uses high pressures (6000 psi) and smaller (5 μm), pressure-stable particles
- Allows compounds to be detected at ppt (parts per trillion) level
- Allows separation of many types of polar and nonpolar compounds
HPLC Modalities
- Reversed phase–for separation of non-polar molecules (non-polar stationary phase, polar mobile phase)
- Normal phase–for separation of non-polar molecules (polar stationary phase, non-polar/organic mobile phase)
- HILIC–hydrophilic interaction liquid chromatography for separation of polar molecules (polar stationary phase, mixed polar/nonpolar mobile phase)
HPLC Separation Efficiency
umso länger die HPLC Röhre umso besser die Auftrennung
Mass Spectrometry - definition
What is Mass Spectrometry?
John B. Fenn, the originator of electrospray ionization for biomolecules and the 2002 Nobel Laureate in Chemistry, probably gave the most apt answer to this question:
Mass spectrometry is the art of measuring atoms and molecules to determine their molecular weight. Such mass or weight information is sometimes sufficient, frequently necessary, and always useful in determining the identity of a species. To practice this art one puts charge on the molecules of interest, i.e., the analyte, then measures how the trajectories of the resulting ions respond in vacuum to various combinations of electric and magnetic fields.
Clearly, the sine qua non of such a method is the conversion of neutral analyte molecules into ions. For small and simple species the ionization is readily carried by gas-phase encounters between the neutral molecules and electrons, photons, or other ions. In recent years, the efforts of many investigators have led to new techniques for producing ions of species too large and complex to be vaporized without substantial, even catastrophic, decomposition.
Mass Spec Principles
- Sample
- Ion Source
- generation of intact molecular ions in the gas phase
(- MALDI - solid phase) - Mass Analyzer
- separation of ions according to their mass to charge ratio (m/z) - Ion Detector
- intensity measurements of different m/z ratios
-> Mass spec requires a high vacuum: 10^-3 - 10^-7 hPa
Molecules can be separated by unique masses
Butorphanol: MW = 327.1
L-DOPA: MW = 197.2
Ethanol: MW = 46.1
Typical mass spectrum
- Monoisotopic mass is the mass determined using the masses of the most abundant isotopes
- Average mass is the abundance weighted mass of all isotopic components
Mass Spec - what is measured
m/z – only the mass/charge ratio can be measured!
x-axis = m/z value
y-axis = relative abundance / intensity
Different ionisation methods
- Electron ionisation (EI – hard method)
-> Small molecules 1-1000 Daltons, structure - Chemical ionisation (CI – semi-hard)
-> Small molecules 1-1000 Daltons, simple spectra - Electrospray ionisation (ESI – soft)
-> Small molecules, peptides, proteins, up to 200,000 Daltons - Matrix-assisted-laser-desorption (MALDI – soft)
-> Peptides, proteins, DNA, up to 500kD
-> Can identify viruses (MALDI-typing)
Electron Impact (EI)
- Sample introduced into instrument by heating it until it evaporates
- Gas phase sample is bombarded with electrons coming from rhenium or tungsten filament (energy = 70 eV)
- Molecule is “shattered” into fragments (70 eV»_space; 5 eV bonds)
- Fragments sent to mass analyzer
- Data bases used to identify molecules
- Most commonly used in GC-MS
Electron Impact (EI) Ionization Source
The method, or mechanism, of electron ejection for positive ion formation proceeds as follows:
- The sample is thermally vaporized.
- Electrons ejected from a heated filament are accelerated through an electric field at 70 V to form a continuous
electron beam.
- The sample molecule is passed through the electron beam.
- The electrons, containing 70 V of kinetic energy (70 electron volts or 70 eV), transfer some of their kinetic energy
to the molecule. This transfer results in ionization (electron ejection) with the ion internally retaining usually no
more than 6 eV excess energy. M + e- (70 eV) → M+ (~5 eV) + 2e- (~65 eV)
- Excess internal energy (6 eV) in the molecule leads to some degree of fragmentation. M+ → molecular ions +
fragment ions + neutral fragments
Electron capture is usually much less efficient than electron ejection, yet it is sometimes used in the following way for high sensitivity work with compounds having a high electron affinity: M + e- → M-.
Disadvantages:
- involatility of large molecules,
- thermal decomposition, and
- excessive fragmentation.
-> EI breaks up molecules in predictable ways
Chemical Impact
- Chemical ionization uses gas phase ion-molecule reactions within the vacuum of the mass spectrometer to produce ions from the sample molecule.
- The chemical ionization process is initiated with a reagent gas such as methane, isobutane, or ammonia, which is ionized by electron impact.
- High gas pressure in the ionization source results in ion-molecule reactions between the reagent gas ions and reagent gas neutrals.
- Some of the products of the ion-molecule reactions can react with the analyte molecules to produce ions.A possible mechanism for ionization in CI occurs as follows:
- M+XH+ →[M+H]+ +X
- M+X+ →[M+X]+
Methan CI ion forming reactions
- methane molecular ion formation
- carbocation formation
- protonated analyte formation
- alternative carbocation formation
- alternative analyte ion formation
- side reaction carbocation formation
- analyte adduct ion formation
Soft ionization methods
MALDI: 337 nm UV laser
ESI: Fluid (no salt) -> gold tip needle
Electrospray Ionisation - ESI
Sprüher ist an eine high voltage power supply angeschlossen aus der die Elektronen in For eines Taylor cones herausgespült werden. An dem Sprüher kommt es zur Oxidation und die Elektronen wandern zur Scheibe, wo sie aufgefangen/detektiert und letztendlich reduziert werden.
John Bennett Fenn, Nobel Prize 2002
* Under high voltage Taylor cone emits a jet of liquid drops
* The solvent from the droplets evaporates
* Droplets get more and more charged
* When the charge exceeds the Rayleigh limit the droplet explosively dissociates, leaving a stream of charged (positive) ions
- sovent evaporation
- droplet fission at Rayleigh limit
- formation of desolated ions by further droplet fissions and/or ion evaporation
Electronspray Ionization -> Details
- Can be modified to “nanospray” system with flow < 1 μL/min
- Very sensitive technique, requires less than a picomole of material
- Strongly affected by salts & detergents
- Positive ion mode measures (M + H)+ (add formic acid to solvent)
- Negative ion mode measures (M - H)- (add ammonia to solvent)
Electrospray Ionization -> Vor- und Nachteile
Vorteile:
* very soft ionization technique
* low consumption of material
* normal ESI: 20.0 – 1 μL/min
* nano-ESI: 0.01-0.1 μL/min
* online-coupling of HPLC and CE à LC-MS
(-> large masses proteins)
Nachteile:
* multiply charged analytes : [M+nH] +n
* Intolerant to salts
* ESI needs different spray solution for detection in the positive and negative ion mode
Matrix Assisted Laser Desorption/Ionisation -> MALDI
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was first introduced in 1988 by Tanaka, Karas, and Hillenkamp.
Widespread use for peptides, proteins, and most other biomolecules (oligonucleotides, carbohydrates, natural products, and lipids).
The efficient and directed energy transfer provides high ion yields of the intact analyte, and allows for the measurement of compounds with sub-picomole sensitivity.
MALDI matrix — A nonvolatile solid material facilitates the desorption and ionization process by absorbing the laser radiation. As a result, both the matrix and any sample embedded in the matrix are vaporized. The matrix also serves to minimize sample damage from laser radiation by absorbing most of the incident energy.
Matrix Assisted Laser Desorption/ionisation -> MALDI
-> Advantages and Disadvantages
Advantages
* Practical mass range of up to 300,000 Da.
* Species of much greater mass have been observed using a high current detector
* Typical sensitivity on the order of low femtomole to low picomole. Attomole sensitivity is possible
* Soft ionization with little to no fragmentation observed
* Tolerance of salts in millimolar concentrations
* Suitable for the analysis of complex mixtures
Disadvantages
* Matrix background, which can be a problem for compounds below a mass of 700 Da.
* This background interferences is highly dependent on the matrix material
* Possibility of photo-degradation by laser desorption/ionization
* Acidic matrix used in MALDI my cause degradation on some compounds
Matrices for MALDI
1,8,9-Trihydroxyanthracen (dithranol) -> used for: Polymere !!
2,5-Dihydroxy-benzoesäure (DHB) -> used for: Proteine, Peptide, Polymere !!
3,5-Dimethoxy-4-hydroxyzimtsäure (Sinapinsäure) -> used for: Proteine, Polymere
alpha-cyano-4-hydroxyzimtsäure -> used for: Peptide, (Polymere)
4-Hydroxypicolinsäure -> used for: Oligonucleotide
Trans-Indol-3-acrylsäure (IAA) -> used for: Polymere
Vitamin A-Säure -> used for: polymere
Sample Prep for MALDI
- feste Proben (Protein oder Matrix)
- lösen
- auftropfen der Lösung
- 0.5-10µL jeder Lösung
- trocknen
- Massenspektrometer
Important Ion Sources
-> Electron Impact (EI)
SAMPLE
- gaseous
PRESSURE MBAR
- 10^-4 - 10^-6
MODE OF ACTION
- 70 eV electrons hit the neutral molecules in the gas phase
CHARACTERSTICS
- molecules <1000 u, GC-MS mainly fragment ions
Important Ion Sources
-> Chemical impact (CI)
SAMPLE
- gaseous
PRESSUR MBAR
- 1
MODE OF ACTION
- low energy ion hit the neutral molecules in the gas-Hase
CHARACTERSTICS
- molecules < 1000u, GC-MS mainly molecular ions
Important Ion Sources
-> electrspray ionizatioon (ESI)
SAMPLE
- fluid
PRESSURE MBAR
- atmospheric pressure
MODE OF ACTION
- spray production in an electric field
- desolation of highly charged droplets
CHARACTERSITICS
- soft ionization/desolvation
- multiple charged molecular ions
- LC-MS, CE-MS
Important Ion Sources
-> Matrix assisted laser desorption/ionization
SAMPLE
- solid
PRESSURE MBAR
- 10^-3 - 10^-6
MODE OF ACTION
- matrix enhanced absorption of energetic photons decompose the solid matrix-sample mixture
CHARACTERSTICS
- soft desorption/ioniuzation -> mainly singly charged ions
- high mass range imaging
Important Ion Sources
-> older or not so frequently used ion sources
SAMPLE
- FD: field desorption
- PD: plasma desorption
- FAB: fast atom bombardment
MODE OF ACTION
- ICP-MS: inductively coupled plasma mass spec. SIMS: secondary ion mass spectrometry
- LD: laser desorption mass spectrometry
Quadrupol MS
- 4 parallel hyperbolic metal rods to which both a dc voltage and an oscillating radiofrequency voltage
- is applied.
- Two opposite poles are positively charged and the other two negatively charged, and their polarities change throughout the experiment.
- The applied voltages determine the trajectory of the ions down the flight path between the four poles.
- As ions from the ionization source enter the RF field along the z-axis of the electrodes, they oscillate along the z-axis.
- Only those with a specific mass-to-charge ratio will resonate along the field and have a stable path through
to the detector. - Other non-resonant ions will be deflected (unstable path) and collide with the electrodes and be lost (they
are filtered out). - By rapidly varying the voltages, ions of one mass after another will take the stable path and be collected by the
detector. - Either ω is varied while holding U and V constant or U and V are varied, keeping U/V constant.
Advantages:
- Path independent of kin energy of ions -> high transmission rate
- Fast scans possible (20s scans per second over 800 mass units)
- High dynamic range
- Mass resolution of about 1500
TOF MS
- Ions formed are accelerated as a pulsed packet using repeller or accelerator plates, which supply a potential of thousands to ten-thousands of volts.
- The ion packets are pulsed into the flight tube at rates greater than 20,000 times per second, with the goal of supplying a constant kinetic energy to each ion.
- Ions of different m/z will travel at different velocities and arrive at the detector at the end of the tube at
different times (smaller ions will travel faster than larger ions).
Orbitrap
- Mass resolution: >60000 at m/z 400 at 1 sec cycle
- Up to 200,000 (FWHM)
- Mass accuracy: <5ppm external calibration
- Mass accuracy: <2ppm internal calibration
- Mass range: 50,000
- Sensitivity: sub-fm !!
- Speed: 3 HR scans/sec
Characteristic frequencies:
- Freq of rotation: omega phi
- Freq of radial oscillations: omega r
- Freq of axial oscillations: omega z
omega z = Wurzel aus (k/(m/z))
FT-ICR
- Determine m/z by measuring the frequency at which the ion processes in a magnetic field
- Frequencies, which are typically in the 100 KHz to MHz regime, can be very accurately measured with modern electronics making it possible to determine the mass of an ion to within ±0.000005 amu or 5 ppm.
- Much higher resolving power than TOF
- Masses > 100kD
!! - does not require destruction of the ions and allows us to do MSn, ion-molecule reactions, and isotope exchange reactions !!
- Convoluted frequency spectrum -> FT
- Deconvoluted frequency spectrum -> MC
- Mass spectrum
Ion Trap MS
Using a three-electrode (entrance, ring, and endcap) arrangement, the applied fields allow for ions to be trapped for extended periods of time and manipulated during the course of an extended time domain.
As a consequence, consecutive tandem mass spectrometry (excitation, fragmentation, and detection of ions) experiments can be performed.
Collision activated dissociation in an ion trap involves isolating the ion of interest and subjecting it to multiple collisions over an extended timeframe (∼10 msec) to induce fragmentation.
Tandem mass spectrometery: MS/MS, MSn: repeated isolation of ions following multiple fragmentation events.
-> controlled frequentation
MS-MS using an ion trap
- Accumulation
- isolation
- Excitation
- Fragmentation
- Fragment Accumulation
- Ejection
-> Detection
Overview Mass Analysers
-> E-, B- or EB-, BE-MS (electr. (E) and magnetic (B) Sector fields
MODE OF ACTION
- radius of ion in the field depend on energy and moment of ions
m/z RANGE
- <2 10^3
RESOLUTION
2 10^4
ACCURACY
- 3 ppm
COSTS
- high
Overview Mass Analysers
-> TOF-MS (time of flight instruments)
MODE OF ACTION
- time of flight of ions with the same energy is dependent of the m/z ration
m/z RANGE
- >4 20^5
RESOLUTION
- 10^3 - 10 ^4
ACCURACY
- 20 ppm - 200 ppm
COSTS
- low - middle
Overview Mass Analysers
-> Quadrupol mass filter (Q-MS)
MODE OF ACTION
- due to varying electrical fields ions of a special m/z ratio can pass the quadrupole
m/z RANGE
< 2 10^3
RESOLUTION
< 10^3
ACCURACY
> 300 ppm
COSTS
- low
Overview Mass Analysers
-> Ion Traps (Traps)
MODE OF ACTION
- 3D trap for ions from which they can be released selectivity according to their m/z value
m/z RATIO
- < 6 10 ^3
RESOLUTION
- <7 10^3
ACCURACY
- > 150 ppm
COSTS
- low
Overview Mass Analysers
-> Fourier transform MS (FT-MS)
-> FT ICR MS
-> Orbitrap
MODE OF ACTION
- frequency of a periodic motion of ions in an high magnetic (FT-ICR) or electric field (Orbitrap) depends on m/z ratio
m/z RANGE
- 50-10^4
- 50-4 10^3
RESOLUTION
- 10^6
- 10^5
ACCURACY
< 1 ppm
< 2 ppm
COSTS
- high
Overview Mass Analysers
-> MS/MS (Tandem-MS)
-> Traps, Triple Q, TOF-TOF, Hybrid-MS: Q-TOF, Q-FT-MS
MODE OF ACTION
1. MS 1 selects an ion of interest
2. fragment this precursor ion
3. MS 2 analyses the fragment ions
COSTS
- middle to high
Fragmentation Patterns
Principles:
* Fragmentation of a molecular ion, M ,produces a radical and a cation.
* Only the cation is detected by MS.
Fragmentation of M
- A great deal of the chemistry of ion fragmentation can be understood in terms of the formation and relative stabilities of carbocations in solution.
- Where fragmentation occurs to form new cations, the mode that gives the most stable cation is favored.
- The probability of fragmentation to form new carbocations increases in the order.
Fragmentation Patterns
Alkanes: Molecular ion peaks are present, possibly with low intensity. The fragmentation pattern contains clusters of peaks 14 mass units apart, representing loss of (CH2)nCH3.
Example: Hexane (C6H14) with MW = 86.18
- Fragmentation tends to occur in the middle of unbranched chains rather than at the ends.
- The difference in energy among allylic, benzylic, 3°, 2°, 1°, and methyl cations is much greater than the difference among comparable radicals.
- Where alternative modes of fragmentation are possible, the more stable carbocation tends to form in preference to the more stable radical.
Fragmentation Patterns: Alkenes
- Alkenes characteristically
- show a strong molecular ion peak
- cleave to form resonance-stabilized allylic cations.
Fragmentation Patterns: Amides
Amides: Primary amides show a base peak due to the McLafferty rearrangement. Example: 3-Methylbutyramide (C5H11NO) with MW = 101.15
gamma-hydrogen rearrangement
- in the radical cations with unsaturated functional groups, like ketones, aldehydes, carboxylic acids, esters, amides, olefins, phenylalkanes (C=O, C=N, S=O, C=C)
- 6-membered cyclic transition state, #-hydrogen is transferred to a couble-bound atom
- Shift of the double bond + production of a neutral particle
Fragmentation Patterns: Alcohols
Alcohols: Molecular ion is small or non-existent. Cleavage of the C-C bond next to the oxygen. Loss of H2O may occur as in the spectra below. Example: 3-Pentanol, MW=88.15
- One of the most common fragmentation patterns of alcohols is loss of H2O to give a peak which corresponds to M-18.
- Another common pattern is loss of an alkyl group from the carbon bearing the OH to give a resonance-stabilized oxonium ion and an alkyl radical.
Fragmentation Patterns: Aldehydes and Ketones
Aldehydes and Ketones: Cleavage of bonds next to the carboxyl group results in the loss of hydrogen (molecular ion less 1) or the loss of CHO (molecular ion less 29) Example: 3-Phenyl-2-propenal (C9H8O) with MW = 132.16
* Characteristic fragmentation patterns are
* alpha-cleavage
* McLafferty rearrangement
Ketones: Major fragmentation peaks result from cleavage of the C-C bonds adjacent to the carbonyl. Example: 4-Heptanone (C7H14O) with MW = 114.19
Fragmentation Patterns: Amines
Amines: Molecular ion peak is an odd number. Alpha-cleavage dominates aliphatic amines. Example: n-Butylamine (C4H11N) with MW = 73.13
Secondary amine shown below. Again, the molecular ion peak is an odd number. The base peak is from the C-C cleavage adjacent to the C-N bond. Example: n-Methylbenzylamine (C8H11N) with MW = 121.18
Fragmentation Patterns: Aromatic compounds
Aromatic compounds: Strong molecular peak owing to stable structure Example: Naphthalene (C10H8) with MW = 128.17
Fragmentation Patterns: Esters and Ethers
Esters: Fragmentation tends to occur alpha to the oxygen atom (C-C bond next to the oxygen). Example: Ethyl acetate (C4H8O2) with MW = 88.11
Ethers: Fragmentation tends to occur alpha to the oxygen atom (C-C bond next to the oxygen). Example: Ethyl methyl ether (C3H8O) with MW = 60.10
Fragmentation Patterns: Halides
Halides: The presence of chlorine or bromine atoms is usually recognizable from isotopic peaks. Examples: 1-Bromopropane (C3H7Br) with MW = 123.00
Fragmentation Patterns: Retro-Diels-Alder Reaction
- [4+2] cycloelimination.
- Formation of a diene and dienophile from a cyclohexene
Electron Multiplier
Made up of a series (12 to 24) of aluminum oxide (Al2O3) dynodes maintained at ever increasing potentials. Ions strike the first dynode surface causing an emission of electrons. These electrons are then attracted to the next dynode held at a higher potential and therefore more secondary electrons are generated. Ultimately, as numerous dynodes are involved, a cascade of electrons is formed that results in an overall current gain on the order of one million or higher.
Advantages
- Robust
- Fast response
- Sensitive (≈gains of 106)
Disadvantages
- Shorter lifetime than scintillation counting (~3 years)
Faraday Cup
A Faraday cup (Figure 2.19) involves an ion striking the dynode (BeO, GaP, or CsSb) surface which causes secondary electrons to be ejected. This temporary electron emission induces a positive charge on the detector and therefore a current of electrons flowing toward the detector. This detector is not particularly sensitive, offering limited amplification of signal, yet it is tolerant of relatively high pressure.
Advantages
- Good for checking iion transmission and low sensitivity measurements
Disadvantages
- Low amplification (≈10)
Isotopic Distributions
1H = 99.9%
12C = 98.9%
2H = 0.02%
13C = 1.1%
35Cl = 68.1%
37Cl = 31.9%
Typical mass spectrum
- Characterized by sharp, narrow peaks
- X-axis:m/z ratio of a given ion (for singly charged ions this corresponds to the mass of the ion)
- Heightofpeak:
- relative abundance of a given ion (not reliable for quantitation)
- ion’s ability to desorb or “fly” (some fly better than others)
Resolution and Resolving power
- Width of peak indicates the resolution of the MS instrument
- The better the resolution or resolving power, the better the instrument and the better the mass accuracy
- Resolving power is defined as: M/ΔM
- M is the mass number of the observed mass (ΔM) is the difference between two masses that can be separated
Low resolution instrument = Ion trap
High resolution instrument = TOF
GC-MS
- Stationary phase: long capillary tubes, often glass, loaded with organic molecules
- Sample in the gas-phase, He gas flow
- Many solid samples can be derivatised into gases by derivatisation (e.g. with TMS)
- High resolution chromatograms
- More reproducible than LC-MS
- Gas: He, N2, H2
- Sample injector
- T regulated oven/ Column: packed or open tubular (capillary)
- Mass spectrometer detector
Metabolite ID by GC-MS
- GC-MS is often best for identification of amino acids, organic acids, sugars, fatty acids and molecules with MW<500
- GC has higher resolution and better reproducibility than LC
- EI-MS is more standardized than soft ionization methods, so EI spectra are more comparable
- Most common route is to use AMDIS + NIST database
Derivatization
- The goal of derivatization is usually to make the analyte more volatile
- Commonly derivatization is done before the analyte is injected into the column (pre-column)
- The polar nature of many metabolites requires derivatization prior to GC-MS analysis
- Derivatization can also be used to improve chromatographic separation and to make an analyte less
reactive - In the case of amino acids derivatization replaces the OH, NH2 and SH functional groups with a non-
polar moiety - Silylation is a very common derivatization technique, and is useful for a wide variety of compounds.
The main disadvantage of this method is its sensitivity to moisture. The presence of moisture results
in poor reaction yield and instability of the derivatized analytes. - The most common approach is trimethylsilylation which can be achieved using bromotrimethylsilane
(TMBS) or chloretrimethylsilane TMCS
Derivatization using a more complex agent
- N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) is today often used for the derivatization of amino acids
- MTBSTFA derivatives are more stable and less moisture sensitive than those formed using lower molecular weight reagents such as N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)
- MTBSTFA, forms tert-butyl dimethylsilyl (TBDMS) derivatives when reacted with polar functional
groups containing an active hydrogen - TBDMS derivatives are found in common GC-MS databases
- Replacement of an active hydrogen with a TBDMS group adds 114 to the molecular weight
Derivatization using MTBSTFA
- Majority of the amino acids produced one derivative, with active hydrogens on hydroxyl, amine, and thiol groups (in the case of cysteine) replaced by TBDMS.
- Some amino acids produced multiple derivatives, specifically asparagine, glutamine, and tryptophan. In the case of these amino acids, a modification in the reaction conditions, such as, lowering the temperature or changing the reaction time can prevent this.
- For example, increasing the reaction time from 2 to 4 hours results in an increased response of the fully derivatized form of tryptophan.
- TBDMS derivatives are more stable than traditional TMS derivatives,
- but their higher molecular weights result in longer elution times during GC analysis.
- Mass of valine: Average mol. weight: 117.1463
- Replacement of an active hydrogen with a TBDMS group adds 114 to the molecular weight
- 117+114=231, 117+2*114=345
- Electron impact spectra of these
derivatives contains typical fragments corresponding to the molecular weight of the derivative less - CH3 (M-15): 330
- C4H9 (M-57): 288
- C4H9 + CO (M-85): 160
- CO-O-TBDMS (M-159): 186.
Solid-Phase Derivatization
Strong cation exchange resin (for amino acids and amines) + Strong anion exchange resin (for amino acids and organic acids)
-> wide targeted metabolomics by SPAD (solid phase analytical derivatization)
- addition of sample (50 µL of sample solution)
- Dehydration (100µL of acetonitrile)
- Methoxymation (5µL of >20% methoxyamine solution, 3 min)
- Trimethyisilyation (25µL of MSTFA, 10 min)
= Methoxymation and Trimethyisilylation are dervatization on solid phase - elution (25µL of hexane)
NIST 20 MS Database
- 350,643 EI spectra of 306,869 cmpds
- 163,532 ion trap MS for 12,992 compounds
- 411,294 Qtof & QqQ spectra for 12,728 compounds
- 447,285 RI values for 139,498 cmpds
NIST types of compounds and spectra
- 75% (+), 25% (-)
- 32% MS2 of in-source
- 8% of MSn
- 6,000 human metabolites
AMDIS
Automated Mass spectral Deconvolution and Identification System
LC-MS
High performance liquid chromatography (HPLC) device (Solvents: mobile phase and Samples: Multiple component and Mixtures)
- HPLC column
- LC-MS Interface + ion source
- mass spectrometer
-detection
- chromatogram + mass spectrum analysis
LC-MS Data
- y-axis = intensity
- x1-axis = retention time (liquid chromatography for separation)
- x2-axis m/z, mass to charge ratio, mass spectrometry for mass analysis
Peak detection
Identifies and quantifies peaks along the RT-axis that represents signals from individual compound ions.
Output: A list of ions for each sample (unique m/z and RT)
LC-Processing
- Identify, remove (or consolidate) adducts and multiply charged species
- Identify, remove (or consolidate) fragments (neutral losses, breakdown products, rearrangements)
- Identify, remove (or consolidate) isotope peaks
- Remove noise peaks (from sample blanks or peaks that do not appear in >2/3 technical replicates or peaks that do not show dilution trends in 4 dilution replicates)
LC-Processing
- Raw +ve mode spectrum * 15,000 features
- Remove adducts * 12,000 features
- Remove multiple charges * 10,000 features
- Remove neutral losses * 8,000 features
- Remove isotope peaks * 3,000 features
- Remove noise peaks * 2,500 features
- Final spectrum * 2,500 M+H peaks
- Repeat for –ve mode * 1,500 M-H peaks
MS Quantification
- Most MS-based metabolomics studies are not absolutely quantitative
- Absolute quantitation requires spiked addition of 2H, 13C or 15N isotopic standards
- Also requires that the labeled compound is of the same chemical type or very nearly the same chemical type as target compound
- Single Reaction Monitoring (SRM) or Multiple Reaction Monitoring (MRM) is used to ensure correct compound ID
BioCrates IDQ Kit
40 acylcarnitines, 10 biogenic amines, 20 amino acids, 15 LysoPCs, 77 PCs, 15 SMs, glucose = 180 compounds
LC-MS Software
- XCMS Online
- METLINE Gen2
MassBank
MassBank is an ecosystem of databases and tools for mass spectrometry reference spectra. It is provided as open source.
MassBank is developed in different research groups at the Leibniz Institute of Plant Biochemistry, the Helmholtz Centre for Environmental Research and the University of Luxembourg. MassBank is supported by the German Network for Bioinformatics Infrastructure.
4 tiers proposed for cognitive computing
Purpose: Identification of new drugs by drug-repurposing
- Initial metabolite annotation and prioritization -> list of metabolites for follow-up
- Find literature evidence of activity of metabolites and metabolic pathways in governing the biological condition on a systems biology level
- Identify candidate biomarkers
- Find metabolic conditions or drug-repurposing targets that two diseases have in common
Metabolome-level disease comparisons for drug repurposing
- Upon manual inspection for logic testing and analysis, we observed that vitamin D has been studied for both diseases, but far more articles were found for the CKD– vitamin D relationship than for an AMI–vitamin D relationship (260 versus 24).
- Of the 24 articles on AMI and vitamin D, many cite the known relationship between CKD and vitamin D as the nexus for further studying vitamin D and AMI69.
- These results suggest that CKD and AMI could be metabolically linked on pathways involving vitamin D because vitamin D is synthesized in the kidneys and has been suggested as cardio-protectant.
- In addition, vitamin D or drugs targeting vitamin D pathways might be useful in treating both disorders.
iknife
Beim iKnife handelt es sich nicht, wie man vermuten könnte, um das neueste Gadget eines bekannten Computerherstellers, sondern um ein intelligentes Messer (intelligent knife), das Ärzten bei Tumoroperationen die Unterscheidung von erkranktem und gesundem Gewebe erleichtern soll.
Während der Chirurg den Elektrokauter betätigte, analysierte im Hintergrund das REIMS-Gerät die Zusammensetzung der abgesaugten Dämpfe. Nach jeweils drei Sekunden lag das Ergebnis vor.
