Presented by
Upasana Ganguly
Proteomics is the large scale analysis ofproteins, particularly their structure andfunction.
Discovery of 2-D gels was a landmark inproteomics which provided the first feasibleway to display thousands of protein spots ona single gel.
In 1990s biological mass spectrometry haddeveloped into a sensitive and robusttechnique which gave proteomics its impetus.
Mass spectroscopy or Mass spectrometry(MS) is an instrumental approach that allowsthe mass measurement of ions derived frommolecules.
Mass spectrometers are capable of forming,separating, detecting molecular ions basedon their mass to charge ratio (m/z).
Among the well known analyticaltechniques, MS holds a special place as itmeasures an intrinsic property of amolecule – its mass.
MS has replaced Edman degradation.
Uses of MS in proteomics are in three majorareas
1. Characterization and quality control ofrecombinant proteins
2. Protein identification
3. Detection and characterization of post-translational modifications.
Ionization source, analyzer, detector areunder high vacuum to allow unhinderedmovement of ions
aspirin
Relative Abundance
120 m/z-for singly charged ion this is the mass
1897 – J.J Thomson constructed the first MassSpectrometer called „parabola spectrograph‟. Hewas awarded Noble prize in 1906.
1918-1919 – More sophisticated Massspectrometers by Arthur Dempster and FrancisAston.
1946 – W.F Stephens proposed the concept of Timeof Flight (TOF)
1950 – Wolfgang Paul developed Quadrupole massanalyzer and later Quadrupole ion trap massanalyzer.
1983 – First Ion trap became availablecommercially.
1988 – Soft Ionization techniques were introduced :MALDI and ESI.
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
MALDI first introduced in 1985 by FranzHillenkamp 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
hn
Laser
UV 337nm
1. Sample (A) is mixed withexcess matrix (M) and driedon a MALDI plate.
2. Laser flash ionizes matrixmolecules.
3. Sample molecules areionized by proton transferfrom matrix:
MH+ + A M + AH+.
AH+
+30 kV
Variable
Ground
Grid Grid
Sample plate
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
Precise ionization process : not known
Signal intensities : depend on incorporationof peptides into crystals
Masses below 500 Daltons are obscured bymatrix related ions
Low resolution
Background interference
Possibility of photodegradation by laserdesorption/ionization
Poor for determining peptide modifications
Electro-spray ionization first conceived in 1960‟sby Malcolm Dole but put into practice in 1980‟s byJohn Fenn (Yale).
Unique feature : both singly and multiplecharged 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
Positive ion mode measures (M + H)+
If the sample has functional groups thatreadily accept H+ (such as amide andamino groups found in peptides andproteins) then positive ion detection isused-PROTEINS
Negative ion mode measures (M - H)-
If a sample has functional groups thatreadily lose a proton (such as carboxylicacids and hydroxyls as found in nucleicacids and sugars) then negative iondetection is used-DNA
Practical mass limit : 70,000 Daltons
Sensitivity : good
Can be easily coupled to LiquidChromatography (LC)
No matrix interference
Multiple charging gives better mass accuracy
Excellent for determining peptidemodifications
Low salt tolerance
Low tolerance for mixtures
Difficulty in cleaning overly contaminatedinstrument
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
Full Width Half Maximum 10% Valley
Mass
analyzers
Mass
separation
Time of Flight
(TOF)
Quadrupole
MS
Ion trap / FT
ICR
Structural
analysis
Tandem Mass
Spectrometry
or MS/MS
Measures m/z values of analytes by pulsingmolecular ions from the ionization sourceinto flight tubes.
All ions are accelerated across the samedistance by the same force, they same sameK.E. As velocity is dependent on K.E, lighterions travel faster.
m/z values are calculated by the time delayfrom formation of ion till it reaches to strikethe detector (Time of flight).
Linear Time Of Flight tube
Reflector Time Of Flight tube
detector
reflector
ion source
ion source
detector
time of flight
time of flight
Peaks are inherently broad in MALDI-TOFspectra (poor mass resolution).
Cause : Ions of the same mass coming fromthe target have different speeds. This is dueto uneven energy- distribution when theions are formed by the laser pulse.
Can be compensated with the use ofreflector TOF analyzer : a single stagegridded ion mirror that subjects the ions toa uniform repulsive electric field to reflectthem.
A quadrupole mass filter consists of fourparallel 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 ofions traveling down the flight path.
For given dc and ac voltages, only ions of acertain mass-to-charge ratio pass throughthe quadrupole filter and all other ions arethrown out of their original path.
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 quadrupolarfield.
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.
Use of Tandem Mass Spectroscopy is to inducefragmentation 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
Product ion nomenclature
Site of backbonecleavage
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
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
Sensitivity of the procedure is determined bysensitivity of the protein purification strategyrather than the sensitivity of the MSinstrument.
Protein purification starts with a whole celllysate and ends with a gel separated proteinband /spot.
MS analysis is usually carried out on peptidesobtained after enzymatic digestion of thesegel separated proteins
Intact proteins are analyzed in special cases.
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.
Trypsin XXX[KR]--[P]XXX
Chymotrypsin XX[FYW]--[P]XXX
Lys C XXXXXK-- XXXXX
Asp N endo XXXXXD-- XXXXX
CNBr XXXXXM--XXXXX
K-Lysine, R-Arginine, F-Phenylalanine, Y-Tyrosine, W-Tryptophan,D-Aspartic Acid, M-Methionine, P-Proline
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
The peptide fragment masses provide afingerprint of the protein of interest.
Masses of measured proteolytic peptide iscompared to masses of predicted proteolyticpeptide from sequence databases.
A theoretical mass spectrum is constructedfor each protein entry from the database.
Scoring: based on mass, pI, digestionconditions, post-translational modifications.
Ranked output
Post-search analysis: Protein identification
Fig. Flow chart for the principle of Protein Mass Finger printing
First used by Gibson and Biemann in 1984.
FRAGFIT was the first computer programdeveloped for this purpose by Henzel et al in1993.
More sensitive comparisons : redundancy of64 codons is reduced to 20 amino acids.
Three types of sequence databases aresearched :
1. non – redundant protein databases
2. EST
3. genome databases.
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