Download Mass Spectrometry - Maharana Pratap PG College hardooi

Dr. A. K. YADAV
Assistant Professor-Chemistry
Maharana Pratap Govt. P.G. College, Hardoi
Principles Of Mass
Spectrometry
The mass spectrometer is an
instrument designed to
separate gas phase ions
according to their m/z (mass
to charge ratio) value.
Mass spectrometers are used in industry and academia
for both routine and research purposes. The following
list is just a brief summary of the major mass
spectrometric applications:
☞Chemistry: Structural characterisation of natural and synthetic compounds
☞•Biotechnology: the analysis of proteins, peptides, oligonucleotides
☞•Pharmaceutical: drug discovery, combinatorial chemistry,
pharmacokinetics, drug metabolism
☞•Clinical: neonatal screening, haemoglobin analysis, drug testing
☞•Environmental: PAHs, PCBs, water quality, food contamination
☞•Geological: oil composition, carbon dating
Mass Spectrometry (or MS) is a way to
‘weigh’ individual molecules or individual
atoms. The mass of these tiny particles is
microscopic.
A molecule of water, for example, weighs
about 10-22 grams
( i.e.,0.0000000000000000000001g).
 A mass spectrometer is an instrument that
measures the masses of individual molecules that
have been converted into ions, i.e., molecules that
have been electrically charged.
 Since molecules are so small, it is not convenient to
measure their masses in kilograms, or grams, or
pounds. In fact, the mass of a single hydrogen atom
is approximately 1.66 X 10-24 grams. We therefore
need a more convenient unit for the mass of
individual molecules.
 This unit of mass is often referred to by chemists
and biochemists as the Dalton (Da for short), and is
defined as follows: 1 Da=(1/12) of the mass of a
single atom of the isotope of carbon-12(12C). This
follows the accepted convention of defining the 12C
isotope as having exactly 12 mass units.
Mass-to-Charge Ratio, m/z
• The charge of an ion is defined by z, which is
the fundamental unit of charge on an ion ( 1.6 x 10-19C). Therefore, m/z is the Daltons
per unit charge (Th, Thomson).
• If a molecule weighs 1000 Da, the [M+H]+ ion
of that molecule will appear at 1001 Da, but
the doubly charged ion of the same
compound [M+2H]2+ will appear at m/z 501.
The mass spectrometer also includes
•A vacuum system
•Tools to introduce the sample (LC, GC …)
•Tools to produce the gas phase ions from the sample molecules
•Tools to fragment the ions, in order to obtain structural information, or to
get more selective detection
• An analyzer to separate the ions
•A detection system
•Software and computing
Schematic of Mass Spectrometer System
• An ion source is a device that
vaporises and puts charge on the
molecules.
• Ion sources are available for just about
all atoms and molecules- from very
small and medium sized ones, e.g.,
atmospheric gases, respiratory gases,
drugs, to extremely large molecules
like proteins and DNA.
•
•
Electron Ionization (EI)
Chemical Ionization (CI)
– Desorption Chemical Ionization (DCI)
•
•
Field Desorption (FD)
Field Ionization (FI)
•
•
Fast Atom Bombardment (FAB)
Secondary Ion Mass Spectrometry (SIMS)
 Electrospray Ionization (ESI)
 Atmospheric Pressure Chemical Ionization (APCI)
 Matrix-Assisted Laser Desorption Ionization (MALDI)
A mass spectrometer uses the rules of physics to





determine mass by deflecting these particles in electric
or magnetic fields after the particles have been
electrically charged. The whole process must take
place in a vacuum.
There are several types of mass spectrometers, such
as
Magnetic sector
Quadrupole
Quadrupole ion trap
Fourier-transform ion-cyclotron resonance (FT-ICR)
&
Time-of-flight mass spectrometers.
The mass spectrometer records
the mass of molecules along
with
the
masses
and
abundances of the fragments
that occur when the molecules
break up. Such a record is called
a mass spectrum. It allows
scientists to work out the
structure of a molecule or
identify it by comparison with a
previously
recorded
mass
spectrum.
The ionized CO2 molecule (or
molecular ion) appears at m/z 44.
Since the ionization process
breaks up or fragments some of
the CO2 molecules, a fraction of
the ions appear in the spectrum
at m/z values less than the m/z
value that corresponds to the
molecular
mass
of
CO2.
Cleavage of a carbon-oxygen
bond in the molecular ion to
produce
ionized
carbon
monoxide or ionized atomic
oxygen result in the fragment
ions at m/z 28 and 16; loss of two
neutral oxygen atoms results in
an additional fragment at m/z 12
for carbon. The molecular ion is
designated as M+ or CO2+ and
the fragment ions are designated
as CO+, O+ and C+.
Typical Mass Spectrum
aspirin
M+.
What Information does Mass
Spectrometry Provide?
☻ For large samples such as biomolecules, molecular weights can be
measured to within an accuracy of 0.01% of the total molecular weight of
the sample i.e. within a 4 Daltons (Da) or atomic mass units (amu) error for
a sample of 40,000 Da. This is sufficient to allow minor mass changes to be
detected, e.g. the substitution of one amino acid for another, or a posttranslational modification.
☻ For small organic molecules the molecular weight can be measured to
within an accuracy of 5 ppm, which is often sufficient to confirm the
molecular formula of a compound, and is also a standard requirement for
publication in a chemical journal.
☻ Structural information can be generated using certain types of mass
spectrometers, usually tandem mass spectrometers, and this is achieved by
fragmenting the sample and analysing the products generated. This
procedure is useful for the structural elucidation of organic compounds, for
peptide or oligonucleotide sequencing, and for monitoring the existence of
previously characterised compounds in complex mixtures with a high
specificity by defining both the molecular weight and a diagnostic fragment
of the molecule simultaneously e.g. for the detection of specific drug
metabolites in biological matrices.
 Look for the molecular ion peak. This peak (if it appears) will be the highest mass
peak in the spectrum, except for isotope peaks.
Nominal MW (meaning=rounded off) will be an even number for compounds
containing only C, H, O, S, Si.
Nominal MW will be an odd number if the compound also contains an odd
number of N (1,3,...).
 Try to calculate the molecular formula:
The isotope peaks can be very useful, and are best explained with an example.
Carbon 12 has an isotope, carbon 13. Their abundances are 12C=100%,
13C=1.1%. This means that for every 100 (12)C atoms there are 1.1 (13)C
atoms. If a compound contains 6 carbons, then each atom has a 1.1% abundance
of(13)C.Therefore, if the molecular ion peak is 100%, then the isotope peak (1
mass unit higher) would be 6x1.1%=6.6%.
Look for A+2 elements: O, Si, S, Cl, Br
Look for A+1 elements: C, N
"A" elements: H, F, P, I
 Calculate the total number of rings plus double bonds:
For the molecular formula: CxHyNzOn
rings + double bonds = x - (1/2)y + (1/2)z + 1
 Postulate the molecular structure consistent with abundance and m/z of
fragments.
The fragments corresponding to the peaks are shown
below along with the structure of the parent
compound, ethyl benzene:
Much of the emphasis in mass spectrometry
in the past century was placed on volatile
atomic and small polyatomic species.
However, over the course of the century,
matter in all forms (solid, liquid, gas) was
subjected to scrutiny by MS and the nature
of the species of interest has come to
include subatomic particles, elements,
inorganic and organic polyatomic species,
clusters, polymers (including biopolymers,
non-covalently bound biocomplexes and
microparticles.
ESI allows for large, non-volatile molecules to
be analyzed directly from the liquid phase
Used for:
Mass determination of biomolecules.
Analysis and sequencing of proteins and
oligonucleotides.
Analyzing drugs, pesticides, and carbohydrates
Long chain fatty acids.
ELECTROSPRAY IONIZATION (ESI)
In ESI MS the ionisation process is carried out at atmospheric pressure
(API), and involves spraying a solution of the sample in a suitable solvent
out of a small needle, to which a high voltage is applied. This process
produces small charged droplets, and the solvent is then evaporated
leaving the sample molecule in the gas phase and ionised. This is then
'swept' into a MS that is held essentially in vacuom and the ions separated
and detected using a Mass Analyzer.
Electrospray Ion Generation
Droplets are generated when a high
voltage is applied to a liquid
stream. In electrospray larger
droplets explode into smaller
droplets and so on until the analyte
enters the gas phase as an ion.
In this example a single peptide is
ionized to produce a population of
charged and uncharged
peptides. The number of positive
charges that a molecule can
support is generally related to the
number of basic sites on the
molecule.
In positive ion mode the analyte is sprayed at
low pH to encourage positive ion formation. In
negative ion the analysis is normally carried out
well above a molecules isoelectric point to
deprotonate the molecule. The basic principle of
all mass spectrometers is that a molecule must
be charged (ionized) before the mass
spectrometer can influence it in an electric field.
Note: Most peptides obtained from a trypsin
digest have two potential sites for protonation,
the amino terminus and the basic C-terminal
residue, lysine or arginine.
A population of variably charged
ions are generated in the
electrospray process. In this
example the population will
contain peptides that have 1, 2
and 0 sites of protonation.
The resulting spectrum contains singly and
doubly charged species. The intensity of
the peaks is a reflection of the population
generated in the electrospray
process. Some researchers have used
peak abundance information in protein
folding studies. The principles that they
apply are; the more tightly folded a protein
the more difficult it will be to protonate and
it then follows that as a protein unfolds the
peak distribution may change across a
spectrum possibly favoring the more highly
charged species
Characteristics of ESI mass spectra
• Small organic molecules (< 1000 Da) normally give singly charged
ions.
• Samples (M) with molecular weights greater than ca. 1200 Da give
rise to multiply charged molecular-related ions such as (M+nH)n+ in
positive ionisation mode and (M-nH)n- in negative ionisation mode.
• Proteins have many suitable sites for protonation as all of the
backbone amide nitrogen atoms could be protonated theoretically,
as well as certain amino acid side chains such as lysine and arginine
which contain primary amine functionalities.
• As ESI is a very soft ionization technique even non-covalently
bound molecules can be detected intact.
The upper trace depicts the negative ESI-MS m/z spectrum of a 23mer RNA sample, with multiply charged ions from n =8 (m/z 950.5)
to n = 15 (m/z 505.6). The lower trace shows the molecular mass
profile produced from the m/z spectrum by Maximum Entropy data
handling techniques. The mass accuracy was within 0.5 Da.
Electrospray-Summary
Sample introduction
• Flow injection
• LC/MS
• Typical flow rates are less than 1 microliter per minute up to about a milliliter per
minute
Benefits
• Good for charged, polar or basic compounds
• Permits the detection of high-mass compounds at mass-to-charge ratios that are
easily determined by most mass spectrometers (m/z typically less than 2000 to 3000).
• Best method for analyzing multiply charged compounds
• Very low chemical background leads to excellent detection limits
• Can control presence or absence of fragmentation by controlling the interface lens
potentials
• Compatible with MS/MS methods
Limitations
• Multiply charged species require interpretation and mathematical transformation (can
sometimes be difficult)
• Complementary to APCI. Not good for uncharged, non-basic, low-polarity compounds
(e.g.steroids)
• Very sensitive to contaminants such as alkali metal salts or basic compounds
Mass range
Low-high Typically less than 200,000 Da.
ESI features
 Multiply charged analytes
 Good for on-line coupling with liquid
phase separations
 Ion suppression
 Requires good desolvation
(presence of organic solvent)
 Intolerant to salts
Hyphenated MS
• GC-MS - Gas Chromatography MS
– separates volatile compounds in gas column and ID’s
by mass
• LC-MS - Liquid Chromatography MS
– separates delicate compounds in HPLC column and
ID’s by mass
• MS-MS - Tandem Mass Spectrometry
– separates compound fragments by magnetic field and
ID’s by mass
Chromatograph-Mass Spectrometer
A very important combination in mass
spectrometry involves using a
chromatograph (a device for separating
complicated mixtures as they pass through a
column- GC or LC) before the mass
spectrometer. In this way very small traces of
molecules can be detected in complicated
samples containing thousands of different
types of molecules. e.g. protein molecules
from a sample of cells or a trace of an
anabolic steroid in an olympic athlete’s body.
<
Mass
spectrometers
are
commonly
combined
with
separation devices such as
gas chromatographs (GC) and
liquid chromatographs (LC).
The GC or LC separates the
components in a mixture, and
the
components
are
introduced, one by one, into
the mass spectrometer. MS/MS
is an analogous technique
where
the
first-stage
separation device is another
mass spectrometer.
• MALDI is a method that allows vaporization &
ionization of a non-volatile sample from
solid phase directly into the gas phase.
– biopolymers and oligomers
proteins, peptides, oligonucleotides,
oligosaccharides
– synthetic polymers
– inorganics, such a fullerenes
– environmental compounds
kerogens, coal tars, humic acids, fulvic acids
A laser pulse is used for excitation
– UV lasers cause electronic
excitation
– IR lasers cause vibrational
excitation
Matrix molecules transfer energy
– low analyte fragmentation
– matrices are selectable
TOF-MS is used for analysis
• A relatively new ionization technique
• This desorption technique first introduced in 1988 by
Hillenkamp and co-workers is very similar to FAB,
but it utilizes photons instead of particles to desorb
analyte molecular ions, [M+H]+, from a crystalline
matrix.
• The primary role of the matrix is to absorb the
incident radiation which results in rapid heating of
the crystal lattice on a time scale (femtoseconds)
that is faster than thermal equilibration of the matrixanalyte lattice.
• This process results in desorption or transfer to the
gas phase of matrix and intact analyte ions.
Principle of MALDI
Pulsed laser: UV l 337 nm, 100 µJ energy in 3 ns
pulse, 10 Hz repetition rate
Ion extraction energy: Few keV to 25 kEV
Time scales: Flight times > 100 µs, peak width: 10 ns
High vacuum: 10-6 m bar
Ion production in the MALDI source depends on
the generation of a suitable composite material,
consisting of the matrix and analyte. The
prototypical MALDI recipe (i.e., dried-droplet
method) is really very simple: a solution of the
matrix compound is prepared and thoroughly
mixed with analyte solution. A droplet of the
mixture is then dried on the sample target,
resulting in a solid deposit of analyte-doped matrix
crystals. The dried sample spot is finally
introduced into the mass spectrometer for laser
desorption/ionization. The procedure is as simple
as it sounds.
• Simply mix 10 nanomoles of an
aqueous matrix solution with 0.1
picomoles of analyte solution
• Deposit the smallest possible aliquot
on a clean metallic support
• This droplet is either air dried or gently
blown dried by means of hairblowers or
radiative heating
The properties of the matrix material used in the
MALDI method are critical. Only a select group of
compounds is useful for the selective desorption of
proteins and polypeptides. A review of all the matrix
materials available for peptides and proteins shows
that there are certain characteristics the compounds
must share to be analytically useful. Despite its
importance, very little is known about what makes a
matrix material "successful" for MALDI. The few
materials that do work well are used heavily by all
MALDI practitioners and new molecules are
constantly being evaluated as potential matrix
candidates.
Matrix Materials
Application
Matrix
a-cyano-4-hydroxycinnamic acid
(CHCA)
Peptides (<10 KDa), lipids,
carbohydrates
sinapinic acid (SA), or trans-3,5Peptides and large proteins (10dimethoxy-4-hydroxycinnamic acid 150KDa), glycoproteins, membrane
proteins
gentisic acid, or 2,5-dihydroxy
benzoic acid (DHB)
peptides, proteins, carbohydrates,
glycoproteins, glycolipids,
polymers, lipids, organic
molecules
trans-3-indoleacrylic acid (IAA)
synthetic polymers
3-hydroxypicolinic acid (HPA)
Oligonulceotides > 3.5KDa
2,4,6-trihydroxyacetophenone
(THAP)
Oligonucleotides < 3.5KDa
dithranol (DIT)
Polymers and fullerene
compounds
Deposit Matrix/Sample Spot on MALDI Target (Dried Droplet Method)
Take 1 μ L of the mixture, place it on one of the spots on the sample card and
allow the sample to completely dry. Do not disturb the MALDI target during the
drying process. Once the sample is dry, you can immediately introduce the card
into the MALDI spectrometer. Sample preparation is completed!
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