mass spectrometry
TRANSCRIPT
M.Prasad NaiduMSc Medical Biochemistry,
Ph.D,.
Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles.
It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and other chemical compounds
The sample is ionised to generate parent molecular ions
Further fragmentation will generate fragment ions
The process of ionisation has to be controlled to generate similar ions from all the molecules in the mixture
The ions are separated according to their mass-to-charge ratio in an analyzer by electromagnetic fields
The ions are detected, usually by a quantitative method
The ion signal is processed into mass spectra
The analyser, detector and ionisation source are under high vacuum to allow unhindered movement of ions
Operation is under complete data system control
Samples easier to manipulate if ionised
Separation in analyser according to mass-to-charge ratios (m/z)
Detection of separated ions and their relative abundance
Signals sent to data system and formatted in a m/z spectrum
Prior to sample introduction, 2 things must be achieved:
Sample must be introduced into vacuum Sample must be vaporized
The sample is introduced by placing it on the probe, which is then inserted through a vacuum lock into ionisation region of mass spectrometer
Ionization means placing a charge on a neutral molecule
Methods:
1) Electron ionization 2) Electrospray 3) Matrix-assisted laser
desorption/ ionization(MALDI)
Also known as electron bombardment / electron impact method
The sample is heated to vaporize it
The sample in the gas phase is now delivered into electron ionization region
Here a beam of electrons with energy of 70EV is made to interact with the sample
This interaction causes electron ejection in the sample molecules leading to ionization
Generates ions directly from aqueous or aqueous/organic solutions
The solution is forced through a narrow needle which is kept at a high potential (3.5 kV)
The voltage on the needle causes the spray to be charged as it is nebulized
Thus, very small droplets are created and they are charged on their surfaces
The electric charge density on the surface of the droplet is a function of its size- smaller the droplet, larger is the electric charge density
Thus, as the droplets decrease in size, there is repulsion between mutually charged droplets
At this point, ions begin to leave the droplet
Ions are led into mass analyzer
It nondestructively vaporizes & ionizes both big and small molecules
The analyte is first co-crystallized with an excess of a matrix compound
Matrix compounds are organic acids, which absorb in the UV range
After the co-crystallization, a pulse UV laser beam is focused on the surface of the crystal
The matrix absorbs the radiation & is vaporized
The analyte is also vaporized and carried along with the matrix
The matrix doubles up as a proton acceptor or donor & thus also ionizes the analyte
Different matrices:
2,5 dihydroxy benzoic acid-proteins,peptides & oligonucleotides
Sinapinic acid – proteins & peptides
After ionization, ions that are in the gas phase enter the mass analyzer
It separates ions within a selected range of mass-to-charge (m/z) ratios
To separate ions,different mass analyzers use magnetic fields,electric fields or the time taken by an ion to move over a fixed distance
J.J Thompson,who built the 1st mass spectrometer, used a magnet to measure the m/z value of electrons
It separates ions in a magnetic field according to the momentum & charge of ion
A 1 to 10kV electric field accelerates ions from the source region into the magnetic sector
Once it reaches the magnetic field,the ion beam is bent in an arc by the magnetic field
The radius of the arc(r) depends on: Momentum of the ion Charge of the ion Magnetic field strength
The greater the momentum of the ions, the larger is their arc radius
The separation of ions is thus based upon their momentum
Hence, magnetic analyzers are also called momentum analyzers
In the mass spectrometer, an electric field accelerates ions out of the source region and into the quadrupole analyzer
The quadrapole analyzer consists of 4 rods/electrodes arranged across from each other
The ions are made to travel through the quadrupole
Here, they get filtered according to their m/z ratio
Only one of the separated ion beams is allowed to strike the detector
The separation according to m/z ratio is based upon the radio frequency & direct current voltages applied to these electrodes
These voltages produce an oscillating electric field that transmits ions according to their m/z value by alternatively focusing them in different planes
Used mostly with MALDI
The time-of-flight (TOF) analyzer uses an electric field to accelerate the ions through the same potential, and then measures the time they take to reach the detector
The smaller ions will reach the detector first because they will acheive great velocities
The larger ions will have lesser velocities & reach the detector late
The final element of the mass spectrometer is the detector
The detector generates a signal current from incident ions by generating secondary electrons which are further amplified
Types:
Faradey Cup Electron Multiplier Photomultiplier Conversion Dynode
Concept: A change in charge on a metal plate results in a flow of electrons
The flow creates a current
When a single ion strikes the surface of a dynode in faradey cup, it results in ejection of several electrons
This ejection induces a current in the cup
Uses a series of dynodes maintained at successively higher potentials
Thus,electrons released by the 1st dynode (when ion impinges on it) are dragged to 2nd dynode because it has a higher potential
Highly sensitive
Ions strike a dynode resulting in emission of electrons
These electrons are made to strike a
phosphorous screen The screen releases photons
Photons detected by a photomultipier
Complex mixtures are now analyzed without prior purification by tandem MS
It employs the equivalent of 2 mass spectrometers linked in series
The 1st spectrometer separates individual peptides upon their differences in mass
By adjusting the field strength of 1st magnet, a single peptide can be directed into 2nd mass spectrometer ,where fragments are generated and their mass determined
Applications:
1) Identification & quantification of proteins
2) Drug screening
3) Pesticides & pollutants screening
4) Used to screen blood samples from new borns for the presence & conc of proteins,F.A,other metabolites
5) Screening of inborn errors of metabolism (phenyl ketonuria, ethylmalonic encephalopathy,glutaric acidemia type 1)
Gas chromatography-mass spectrometry
In this technique, a gas chromatograph is used to separate different compounds.
This stream of separated compounds is fed online into the ion source, a metallic filament to which voltage is applied.
This filament emits electrons which ionize the compounds.
The ions can then further fragment, yielding predictable patterns.
Intact ions and fragments pass into the mass spectrometer's analyzer and are eventually detected
Liquid chromatography-mass spectrometry
Separates compounds chromatographically before they are introduced to the ion source and mass spectrometer.
It differs from GC/MS in that the mobile phase is liquid, usually a mixture of water and organic solvents
Most commonly, an electrospray ionization source is used in LC/MS. There are also some newly developed ionization techniques like laser spray
PROTEIN CHARACTERIZATION: Proteins are 1st digested into smaller
peptides using different proteases A collection of these smaller peptides is
then introduced into the mass analyzer
ANALYSIS OF BIOLOGICAL NONCOVALENT COMPLEXES
Electrospray ionization gets these noncovalent complexes into gaseous phase & MS can be used to observe these complexes
Eg: Hb complex , DNA duplex , cell surface carbohydrates, whole viruses
CHARACTERIZAION OF SMALL BIOMOLECULES ASSOCIATED WITH DIFFERENT BIOLOGICAL STATES
MS successfully discovered that cis-9,10-octadecenoamide was present in the sleep state & was absent during the wake state
APPLICATIONS IN VIROLOGY: Identification of a virus in a given sample by
analyzing the mass of the capsid proteins or DNA/RNA through MS
SEQUENCING PEPTIDES & OLIGONUCLEOTIDES MALDI has been used recently to sequence
proteins & oligonucleotides
Isotope ratio MS: isotope dating and tracking
Mass spectrometry is also used to determine the isotopic composition of elements within a sample
Trace gas analysis
selected ion flow tube (SIFT-MS), andproton transfer reaction (PTR-MS), are variants of chemical ionization dedicated for trace gas analysis of air, breath or liquid headspace
Use well defined reaction time allowing calculations of analyte concentrations from the known reaction kinetics without the need for internal standard or calibration.
Atom probe An atom probe is an instrument that combines
time-of-flight mass spectrometry and field ion microscopy (FIM) to map the location of individual atoms.
Pharmacokinetics Pharmacokinetics is often studied using MS
because of the complex nature of the matrix (often blood or urine) and the need for high sensitivity to observe low dose and long time point data.
The most common instrumentation used in this application is LC-MS with a triple quadrupole MS
Glycan analysis
Mass spectrometry (MS), with its low sample requirement and high sensitivity, has been the predominantly used in glycobiology for characterization and elucidation of glycan structures.
Mass spectrometry provides a complementary method to HPLC for the analysis of glycans
Space exploration As a standard method for analysis, mass
spectrometers have reached other planets and moons.
Two were taken to Mars by the Viking program.
In early 2005 the Cassini-Huygens mission delivered a specialized GC-MS instrument aboard the Huygens probe through the atmosphere of Titan, the largest moon of the planet Saturn.
This instrument analyzed atmospheric samples and was able to vaporize and analyze samples of Titan's frozen, hydrocarbon covered surface once the probe had landed.
Advantages
Provides molecular weights of peptides and proteins with high accuracy (0.1-0.01%)
Highly sensitive; requires fmol-pmol quantities of sample
Sample purity not important
Can be coupled with on-line separation methods such as HPLC and capillary electrophoresis for the analysis of mixtures
Disadvantages
Noncovalent complexes are often disrupted
Cannot distinguish stereoisomers
Expensive instrumentation
