Clinical Applications of Mass Spectrometry - RCPA

Mass spectrometry
1.Principles and
2.Clinical Applications
Prof F G Bowling
Director of Biochemical Diseases
Mater Children’s Hospital
Brisbane, AUSTRALIA

Overview
‣ Mass spectrometry is an analytical technique that identifies the chemical composition of a
compound or sample based on the mass-to-charge ratio of charged particles.
‣ A sample undergoes chemical fragmentation, thereby forming charged particles (ions). The ratio of
charge to mass of the particles is calculated by passing them through electric and magnetic fields in
a mass spectrometer.
‣ The design of a mass spectrometer has three essential modules:
• an ion source, which transforms the molecules in a sample into ionized fragments;
• a mass analyzer, which sorts the ions by their masses by applying electric and magnetic fields; and
• a detector, which measures the value of some indicator quantity and thus provides data for calculating the
abundances of each ion fragment present.
‣ The technique has both qualitative and quantitative uses, such as
• identifying unknown compounds,
• determining the isotopic composition of elements in a compound,
• determining the structure of a compound by observing its fragmentation, quantifying the amount of a
compound in a sample,
• studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in a vacuum), and
• determining other physical, chemical, or biological properties of compounds
‣ The technology is frequently used as the definitive method for molecules analysed routinely by
other methods in Pathology Laboratories, but
‣ Because of advances in technology it is now replacing many of those older methods in routine use.
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1.Principles
Mass spectrometry
‣ method for determining the mass of molecules
by producing and analysing charged species
‣ measures the mass to charge ratio (m/z) of ions.
‣ USES:
• analyser:
•To identify unknown molecules,
•To elucidate structure & chemical
properties
• detector:
•To quantify known materials
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‣ Mass Spectrometer
• INLET SYSTEM
MS Instrumentation
• ION SOURCE: form gas-phase ions from sample,
• ANALYSER: separate ions based on their mass-to-charge
ratio (m/z),
• DETECTOR: measure the abundance of the ions according
to m/z.
• DATA OUTPUT: mass spectrum
Relative abundance
m/z
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Ionisation
‣ Molecules gain or lose electrons such that they acquire a
positive or negative charge
‣ ionisation process can produce
• molecular ion: same molecular weight and elemental
composition of the starting analyte
• fragment ion: corresponds to smaller piece of the analyte
molecule
‣ Common molecular ion products of ionisation
• Molecular ions M + or M -
• Protonated molecules [M + H] +
• Simple adduct ions [M + Na] +
‣ Different ionisation techniques depending on chemical &
physical properties of molecule of interest
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Electron Ionisation (EI)
‣ Suitable for volatile organic compounds
‣ Gaseous sample molecules bombarded with
beam of energetic electrons
‣ Results in loss of electrons from sample,
formation of radical cation
• M + e - M +· + 2e -
‣ EI mass spectra contain intense fragment ion
peaks and much less intense molecular ion peaks
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Chemical Ionisation (CI)
‣ suitable for volatile, more polar compounds
‣ excess of reagent gas is firstly ionised by EI, then
introduced to sample
‣ ion molecule reactions occur between ionised
reagent gas molecules and volatile analyte
neutral molecules to produce analyte ions.
‣ Pseudo-molecular ion MH + (positive ion mode)
or [M-H] - (negative ion mode) + few fragment
ions produced
‣ main reagent gases used: Ammonia, Methane,
and Isobutane
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Fast Atom Bombardment (FAB)
‣ Used for large compounds with low volatility (eg peptides,
proteins, carbohydrates)
‣ sample mixed with a non-volatile matrix and attached to end of
probe
‣ bombarded with fast beam of
atoms (neutral inert gas Ar/Xe)
‣ Sample molecules ejected from
matrix, ionised and extracted
into the mass analyser
‣ produces [M+H] + & [M+Na] +
ions
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Electrospray Ionisation (ESI)
‣ Soft ionisation technique suitable for proteins, peptides,
oligonucleotides
‣ Type of atmospheric pressure ionisation
‣ Generates mainly multiply charged ions directly from solution
‣ Liquid (solvent + analyte mixture) passed through fine stainless
steel capillary into chamber at atm pressure in presence of
strong electrostatic field & heated drying gas
‣ Emerging solution dispersed into fine spray of charged
droplets all at same polarity
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Electrospray Ionisation (ESI)
‣ Solvent evaporates away, shrinking droplet size, increasing
charge concentration at droplet’s surface
‣ Droplets explode once charge repulsion overcomes surface
tension
‣ shrinking and explosion continues until individually charged
‘naked’ analyte ions formed.
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Matrix Assisted Laser Desorption/Ionisation
(MALDI)
‣ analyte co-crystallised with excess of matrix compound on
MALDI target
‣ Inserted into MS under high vacuum
‣ Pulsed laser beam (nitrogen UV laser) directed at
matrix/sample spot
‣ Laser energy absorbed by matrix, transferred to analyte
‣ Matrix/analyte desorbed from
surface & ionised
‣ Results in mainly singly charged
ions [M+H] + or [M+Na] + in
positive ion mode
‣ Electric field applied between
sample and extraction grid to
accelerate ions
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Matrix Assisted Laser Desorption/Ionisation
MATRICES
‣ Nature of matrix highly important in MALDI experiment
‣ Functions of matrix
• Dilute sample and isolate sample molecules from each other
• Absorb laser light energy and indirectly cause analyte to vaporize.
• Ionise analyte by serving as proton donor or receptor, in both positive
and negative ionisation modes, respectively.
‣ Mostly crystalline compounds
‣ Examples of common matrices:
Sinapinic acid
2,5-dihydroxybenzoic
acid
3-Hydroxypicolinic
acid
6-aza-2-thiathymine
large proteins
glycoproteins
proteins
carbohydrates
oligonucleotides
glycoproteins
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Surface-Enhanced Laser Desorption
Ionisation (SELDI)
‣ Target modified to capture molecule of interest within sample
mixture attach antibodies, lectins, DNA
‣ Functions as solid-phase extraction technique
‣ Unbound fragments washed away prior to ionisation
‣ Matrix solution added to enhance laser energy transfer &
analyte ionisation
‣ Laser desorption/ionisation followed by TOF MS
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Mass analysers
‣ Separates the ions generated in ion source
according to mass/charge ratio
‣ Can combine 2 or more analysers in tandem MS
systems
‣ Different types:
• Magnetic sector
• Time-of-flight
• Quadrupole
• Ion trap
• Fourier-transform ion cyclotron resonance
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Time of Flight (TOF) analysers
‣ Simplest type of mass analyser
‣ virtually unlimited mass range
‣ higher masses lower resolution
‣ potential applied across ion source to extract and accelerate
ions into field-free ‘drift’ zone
‣ ideally ions of same mass/charge will have same kinetic energy
‣ time of arrival at detector relates to mass of ion
‣ the lower the ion's mass, the greater the velocity and shorter its
flight time.
Source, s
Field-free drift zone, d
Detector
Es = Vs/ls
length = ls
Vs
length = ld
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Reflectron TOF
‣ Reflecting & decelerating fields at end of flight tube
‣ Compensate for difference in flight times of same m/z ions with
slightly different kinetic energies
‣ Higher kinetic energy ions penetrate decelerating field further
than ions with lower kinetic energy
‣ Slow ions ‘catch up’ to fast ions of same m/z
‣ Decrease spread of ion flight times improve resolution
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Delayed extraction TOF
‣ Delayed extraction in linear mode also used to improve
resolution
‣ Delayed-extraction grid located between the sample target and
the drift-tube entrance grid.
‣ Ions allowed to spread out before the voltage is applied to
accelerate them into the flight tube.
‣ Time delay typically 5 to 20 microseconds
‣ When the delayed extraction voltage is finally turned on, the
lower-velocity ions experience a greater accelerating voltage
than the higher-velocity ions
‣ This reduces the energy spread between molecules that have
the same m/z ratio and therefore reduces peak broadening.
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Quadrupole (Q) analyser
‣ Four parallel rods with fixed DC & alternating Rf voltages
arranged in a square
‣ Analyte ions directed down centre of square
‣ By varying strengths and frequencies of magnetic fields, change
defined m/z value that will pass through
‣ Upper limit of mass range is
m/z 2200-3000
‣ May operate in two modes:
• Scanning mode
monitoring range of m/z ratios
• Selected ion monitoring mode
monitoring selected m/z ratios
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Ion trap analyser
‣ Consist of 3 electrodes forming a chamber
• Ring electrode
• Entrance endcap electrode
• Exit endcap electrode
‣ Ions entering chamber ‘trapped’ by electromagnetic fields
‣ Various voltages applied to trap and eject ions according
to m/z values
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Triple Quad (Tandem) MS (MS/MS)
‣ Powerful way to obtain structural information
‣ Common example: triple quadrupole
‣ First quadrupole used to select precursor ion
‣ Second quadrupole (Rf only) used as a collision cell for
fragmentation of precursor ion (collision induced
dissociation; collision with inert gas)
‣ Third quadrupole generates spectrum of resulting product
ions
‣ May use TOF analyser in place of third quadrupole
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2. Clinical Applications
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Quantitation of small molecules
‣ Molecules
• Aminoacids
• Intermediate metabolites
• (Organic acids and
Acylcarnitines)
• Neurotransmitters
• Steroids
‣ Technology
• GC-MS
• LC-MS
• Triple Quad
• Ion Trap
• TOF
• Cytokines
• Xenobiotic (metabolites)
• Vitamins
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Quantition of peptides
‣ Molecule
• Glycoprotein
hormones
• Immunoglobulins
• Protein digests
‣ Technology
• LC -- Ion Trap
• LC – Triple Quad
• LC -- TOF
• (MALDI-TOF)
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Identification / Quantitation proteins
‣ Molecule
• Glycosylated
Haemoglobin
• Enzymes
• Troponin and other
structural
‣ Technology
• Peptide digest
• LC or Gel fractionation
• Ion Trap
• MALDI
• TOF
‣ Analysis
• Protein databases
• eg MASCOT
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Identification Micro-organisms
‣ Molecule
• Membrane proteins
• Cell wall glycoproteins
• Surface lipoproteins
• Cytosolic enzymes
‣ Clinical
• Bacteria
• Viruses
• Protozoa
‣ Technology
• MALDI-TOF
‣ Comment
• Proteomic analysis
performed on isolated
organisms
• Biological fluid
techniques in
development
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Tissue Imaging
‣ Molecule
• Pathological markers
• Tissue specific proteins
• Tumour markers
• Acute response
• Apoptosis
‣ Technology
• MALDI Imaging of
• fresh tissue sections
• fixed tissue sections
• including slides
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