3. Proteomics is the large scale analysis of
proteins, particularly their structure and
function.
Discovery of 2-D gels was a landmark in
proteomics which provided the first feasible
way to display thousands of protein spots on
a single gel.
In 1990s biological mass spectrometry had
developed into a sensitive and robust
technique which gave proteomics its impetus.
4.
5. Mass spectroscopy or Mass spectrometry
(MS) is an instrumental approach that allows
the mass measurement of ions derived from
molecules.
Mass spectrometers are capable of forming,
separating, detecting molecular ions based
on their mass to charge ratio (m/z).
Among the well known analytical
techniques, MS holds a special place as it
measures an intrinsic property of a
molecule – its mass.
6. MS has replaced Edman degradation.
Uses of MS in proteomics are in three major
areas
1. Characterization and quality control of
recombinant proteins
2. Protein identification
3. Detection and characterization of post-
translational modifications.
7. Ionization source, analyzer, detector are
under high vacuum to allow unhindered
movement of ions
9. 1897 – J.J Thomson constructed the first Mass
Spectrometer called „parabola spectrograph‟. He
was awarded Noble prize in 1906.
1918-1919 – More sophisticated Mass
spectrometers by Arthur Dempster and Francis
Aston.
1946 – W.F Stephens proposed the concept of Time
of Flight (TOF)
1950 – Wolfgang Paul developed Quadrupole mass
analyzer and later Quadrupole ion trap mass
analyzer.
1983 – First Ion trap became available
commercially.
1988 – Soft Ionization techniques were introduced :
MALDI and ESI.
10. Electron Impact (EI - Hard method)
◦ small molecules, 1-1000 Daltons
Fast Atom Bombardment (FAB – Semi-hard)
◦ peptides, sugars, up to 6000 Daltons
Electrospray Ionization (ESI - Soft)
◦ peptides, proteins, up to 70,000 Daltons
Matrix Assisted Laser Desorption (MALDI-Soft)
◦ peptides, proteins, DNA, up to 300 kD
11.
12. MALDI first introduced in 1985 by Franz
Hillenkamp and Michael Karas (Frankfurt)
Mechanism :Analyte molecules are embedded in a bed of
specific wavelength (UV337nm) absorbing matrix
Dried to produce a co-crystallized mixture
Bombarded with short duration (1-10ns) pulses of UV light from a
nitrogen laser
Ionization of both matrix and analyte molecules via an energy
transfer mechanism from matrix to the embedded analyte
A high potential electric field is applied between the sample probe
and orifice
Accelerates the ions to the mass analyzer
13. hn
Laser
UV
337nm
1. Sample (A) is mixed with
excess matrix (M) and dried
on a MALDI plate.
2. Laser flash ionizes matrix
molecules.
3. Sample molecules are
ionized by proton transfer
from matrix:
MH+ + A M + AH+.
AH+
+30 kV
Variable
Ground
Grid Grid
Sample plate
14.
15. Practical mass limit : ~300,000 Daltons
Sensitivity : low femtomole to low picomole
No fragmentation
Suitable for analysis of complex mixtures
Samples are added directly to the matrix
16. Precise ionization process : not known
Signal intensities : depend on incorporation
of peptides into crystals
Masses below 500 Daltons are obscured by
matrix related ions
Low resolution
Background interference
Possibility of photodegradation by laser
desorption/ionization
Poor for determining peptide modifications
17.
18. Electro-spray ionization first conceived in 1960‟s
by Malcolm Dole but put into practice in 1980‟s by
John Fenn (Yale).
Unique feature : both singly and multiple
charged ions can be formed.
Mechanism :
A solution is introduced through a small diameter needle in the
presence of a strong electric field(3-5kV)
A fine spray of charged droplets is created
Charged droplets are desolvated by the application of a
countercurrent flow of gas and/or heat causing the droplet to
evaporate
Explosion of droplet and expulsion of gas phase ions
19.
20. Positive ion mode measures (M + H)+
If the sample has functional groups that
readily accept H+ (such as amide and
amino groups found in peptides and
proteins) then positive ion detection is
used-PROTEINS
Negative ion mode measures (M - H)-
If a sample has functional groups that
readily lose a proton (such as carboxylic
acids and hydroxyls as found in nucleic
acids and sugars) then negative ion
detection is used-DNA
21. Practical mass limit : 70,000 Daltons
Sensitivity : good
Can be easily coupled to Liquid
Chromatography (LC)
No matrix interference
Multiple charging gives better mass accuracy
Excellent for determining peptide
modifications
22. Low salt tolerance
Low tolerance for mixtures
Difficulty in cleaning overly contaminated
instrument
23.
24. Resolution is defined as the ability to
separate and measure the masses of ions
of similar but not identical molecular mass.
The better the resolution, the better the
instrument and the better the mass
accuracy.
Resolution is represented as:
M
DM
28. Measures m/z values of analytes by pulsing
molecular ions from the ionization source
into flight tubes.
All ions are accelerated across the same
distance by the same force, they same same
K.E. As velocity is dependent on K.E, lighter
ions travel faster.
m/z values are calculated by the time delay
from formation of ion till it reaches to strike
the detector (Time of flight).
29. Linear Time Of Flight tube
Reflector Time Of Flight tube
detector
reflector
ion source
ion source
detector
time of flight
time of flight
30. Peaks are inherently broad in MALDI-TOF
spectra (poor mass resolution).
Cause : Ions of the same mass coming from
the target have different speeds. This is due
to uneven energy- distribution when the
ions are formed by the laser pulse.
Can be compensated with the use of
reflector TOF analyzer : a single stage
gridded ion mirror that subjects the ions to
a uniform repulsive electric field to reflect
them.
31. A quadrupole mass filter consists of four
parallel metal rods with different charges.
Two opposite rods have an applied +
potential and the other two rods have a –
potential.
The applied voltages affect the trajectory of
ions traveling down the flight path.
For given dc and ac voltages, only ions of a
certain mass-to-charge ratio pass through
the quadrupole filter and all other ions are
thrown out of their original path.
32.
33. Represents a 3-D quadrupole mass analyzer.
Consists of a ring electrode and two end-cap
electrodes.
Small holes in end-cap electrodes allow
passage of ions into and out of the trap.
RF voltage is applied to ring electrode while
end cap electrodes are held at ground.
Oscillating potential difference between ring
and end cap electrodes forms a quadrupolar
field.
34.
35. Similar to Quadrupole Ion trap MS.
Reactions are carried out in a cell bound by
electrodes (trapping,excite and detect plates).
Uses powerful magnet (5-10 Tesla) to create a
miniature cyclotron.
m/z value is directly related to its cyclotron
frequency.
36.
37. Use of Tandem Mass Spectroscopy is to induce
fragmentation and obtain structural information.
Series of events:
1. Mass selection of a precursor ion
2. Intermediate reaction event
3. Analysis of product ions or fragment ions
Methods to induce fragmentation include:
1. CID or CAD
2. SID
3. Photodissociation
4. BIRD
5. ECD
38. Product ion nomenclature
Site of backbone
cleavage
If charge is retained
on amino-terminal
fragment
If charge is retained
on carboxy -terminal
fragment
αC-C bond a x
C-N amide bond b y
N-αC bond c z
39.
40. GC-MS - Gas Chromatography MS
◦ separates volatile compounds in gas column and
identifies by mass
LC-MS - Liquid Chromatography MS
◦ separates delicate compounds in HPLC column and
identifies by mass
MS-MS - Tandem Mass Spectrometry
◦ separates compound fragments by magnetic field
and identifies by mass
41.
42. Sensitivity of the procedure is determined by
sensitivity of the protein purification strategy
rather than the sensitivity of the MS
instrument.
Protein purification starts with a whole cell
lysate and ends with a gel separated protein
band /spot.
MS analysis is usually carried out on peptides
obtained after enzymatic digestion of these
gel separated proteins
Intact proteins are analyzed in special cases.
43. Best to minimize the number of separations
Concentration of protein : 5-50 ng
Minimize keratin contamination
Detergents and salts are incompatible.
Dialysis is required.
Purity of sample.
45. Robust, stable enzyme
Works over a range of pH values & Temp.
Quite specific and consistent in cleavage
Cuts frequently to produce “ideal” MW peptides
Inexpensive, easily available/purified
46. The peptide fragment masses provide a
fingerprint of the protein of interest.
Masses of measured proteolytic peptide is
compared to masses of predicted proteolytic
peptide from sequence databases.
A theoretical mass spectrum is constructed
for each protein entry from the database.
Scoring: based on mass, pI, digestion
conditions, post-translational modifications.
Ranked output
Post-search analysis: Protein identification
47. Fig. Flow chart for the principle of Protein Mass Finger printing
48.
49. First used by Gibson and Biemann in 1984.
FRAGFIT was the first computer program
developed for this purpose by Henzel et al in
1993.
More sensitive comparisons : redundancy of
64 codons is reduced to 20 amino acids.
Three types of sequence databases are
searched :
1. non – redundant protein databases
2. EST
3. genome databases.
50. Application URL
Eidgenossische Technische
Hochschule (MassSearch)
http://cbrg.inf.ethz.ch
European Molecular Biology
Laboratory (PeptideSearch)
http://www.mann.emblheidelberg.
de
Swiss Institute of Bioinformatics
(ExPASy)
http://www.expasy.ch/tools
Matrix Science (Mascot) http://www.matrixscience.com
Rockefeller University (PepFrag,
ProFound)
http://prowl.rockefeller.edu
Human Genome Research Center
(MOWSE)
http://www.seqnet.dl.ac.uk
University of California (MS-Tag,
MS-Fit, MS-Seq)
http://prospector.ucsf.edu
Institute for Systems Biology
(COMET)
http://www.systemsbiology.org
University of Washington (SEQUEST) http://thompson.mbt.washington.
edu/sequest
