UNIT IV Proteomics Techniques

S.Sridhar
Assistant Professor
Department of Biotechnology
Protein level estimation
Protein assays based on these methods are
divided into two categories:
Coomassie Dye (Bradford)-dye based protein
assays
Peptides and the Biuret Reaction-copper based
protein assays





Biuret assay
Peptide Chains
Biuret Complexes
( purple color )
Copper-based protein assays, including the BCA and Lowry methods, depend on the well-
known "biuret reaction", whereby peptides containing three or more amino acid residues
form a colored chelate complex with cupric ions (Cu2+) in an alkaline environment
containing sodium potassium tartrate. This became known as the biuret reaction because it
is chemically similar a complex that forms with the organic compound biuret (NH2-CO-NH-
CO-NH2) and the cupric ion. Biuret, a product of excess urea and heat, reacts with copper
to form a light blue tetradentate complex
Biuret test
Advantages
Reproducible
Very few interfering agents
(ammonium salts being one such agent )
Fewer deviations than with the Lowry or
ultraviolet absorption methods
Disadvantages
Requires large amounts protein (1-20mg)
Low sensitivity

Methodology
Warm up the spectrophotometer 15 min. before use.
Dilute samples to an estimated 1 to 10 mg/ml with buffer. Add 1 ml to each assay tube.
Duplicate samples are recommended, and a range of dilutions should be used if the actual
concentration cannot be estimated.
Prepare a reference tube with 1 ml buffer.
Prepare standards from 10 mg/ml bovine serum albumin, preferably calibrated using
absorbance at 280 nm and the extinction coefficient. Range should be from 1 to 10 mg
protein.
Add 9 ml Biuret reagent to each tube, vortex immediately, and let stand 20 min.
Read at 550 nm.

Folin-Ciocalteu ( Lowry ) Assay
The phenolic group of tyrosine and trytophan
residues ( amino acid) in a protein will produce a blue
purple color complex , with maximum absorption in the
region of 660 nm wavelength, with Folin- Ciocalteau
reagent which consists of sodium tungstate molybdate
and phosphate. Thus the intensity of color depends on
the amount of these aromatic amino acids present
and will thus vary for different proteins.

Folin-Ciocalteu (Lowry ) Assay -
Chemistry
The assay is performed in two distinct steps.
First, protein is reacted with alkaline cupric sulfate in the presence of
tartrate for 10 minutes at room temperature.
During this incubation, a tetradentate copper complex forms from four
peptide bonds and one atom of copper (this is the "biuret reaction").
Second, a phosphomolybdic-phosphotungstic (Folin-phenol reagent)
acid solution is added, producing an intense blue color.
The color enhancement occurs when the tetradentate copper complex
transfers electrons to the phosphomolybdic-phosphotungstic acid
complex.
The blue color continues to intensify during a 30 minute room
temperature incubation. It has been suggested that during the 30
minute incubation, a rearrangement of the initial unstable blue
complex leads to the stable final blue colored complex which has
higher absorbance
Folin-Ciocalteu ( Lowry ) Assay
Folin-Ciocalteu ( Lowry ) Assay
Advantages
Sensitive over a wide range
Can be performed at room temperature
10-20 times more sensitive than UV detection
Can be performed in a microplate format

Disadvantages
Many substances interfere with the assay (Strong acids, ammonium
sulfate )
Takes a considerable amount of time to perform
The assay is photosensitive, so illumination during the assay must be kept
consistent for all samples
Amount of color varies with different proteins

Methodology
1. Add samples containing up to 100 g of protein.

2. Bring all tubes to 1 mL total volume with water.

3. Prepare the Assay Mix and diluted Folin-Ciocalteu reagent.

4. To each tube add 5 mL of assay mix and thoroughly vortex.

5. Incubate tubes at room temperature for 10 min.

6. Add 0.5 mL of diluted Folin-Ciocalteu reagent. Vortex immediately.

7. Incubate at room temperature for 30 min.

8. Vortex the tubes, zero the spectrophotometer with the blank and
measure absorbance at 500-750 nm.

Bicinchoninic Acid ( BCA ) Assay
The BCA Protein Assay was introduced by
Smith, et al. in 1985.
Best colorimetric detection method and
quantitation of total protein.
The BCA Protein Assay is compatible with
samples that contain up to 5% surfactants
(detergents).
The BCA Assay responds more uniformly to
different proteins than the Bradford method.
Bicinchoic acid method
The BCA Protein Assay combines the protein-induced biuret reaction with
the highly sensitive and selective colorimetric detection of the resulting
cuprous cation (Cu
+
) by bicinchoninic acid (BCA).
Thus, two steps are involved.
First is the biuret reaction, whose faint blue color results from the reduction of
cupric ion to cuprous ion.
Second is the chelation of BCA with the cuprous ion, resulting in an intense
purple color.
The purple colored reaction product is formed by the chelation of two
molecules of BCA with one cuprous ion.
The BCA/copper complex is water-soluble and exhibits a strong linear
absorbance at 562 nm with increasing protein concentrations.
The purple color can be measured at any wavelength between 550 nm and
570 nm.
The BCA reagent is approximately 100 times more sensitive than the biuret
reagent.
BCA method
The reaction that leads to BCA color formation as a result of the
reduction of Cu2+ is especially influenced by the presence of three
particular amino acid residues in proteins: cysteine/cystine, tyrosine
and tryptophan.
BCA method
Advantages
Very sensitive and rapid if you use elevated
temperatures
Compatible with many detergents
Working reagent is stable
Very little variation in response between different
proteins
Broad linear working range

Disadvantages
The reaction does not go to completion when
performed at room temperature

Methodology
1. Prepare the required amount of protein determination
reagent by adding 1 volume copper sulfate solution to 50
volumes of bicinchoninic acid solution.

2. Set up test tubes containing samples and known amounts
of bovine serum albumin in the range of 0 to 100
micrograms. Each tube should contain 0.1 mL total volume.

3. Add 2.0 mL of the protein determination reagent to each
tube and vortex.

4. Incubate the tubes at 60
o
C for 15 min.

5. Cool the tubes to room temperature and determine the
absorbance at 562 nm.

Bradford assay Dye based assay
In the acidic environment of the reagent, protein binds to the Coomassie dye. This results in a
spectral shift from the reddish/brown form of the dye (absorbance maximum at 465 nm) to
the blue form of the dye (absorbance maximum at 610 nm). The difference between the two
forms of the dye is greatest at 595 nm, so that is the optimal wavelength to measure the blue
color from the Coomassie dye-protein complex.
CBBG primarily responds to arginine residues
CBBG binds to these residues in the anionic form
Absorbance maximum at 595 nm (blue)
The free dye in solution is in the cationic form,
Absorbance maximum at 470 nm (red).

Dye based protein assay
Advantages
Fast and inexpensive
Highly specific for protein
Very sensitive [1-20 g (micro assay) 20-200 g (macro assay)]
Compatible with a wide range of substances
Extinction co-efficient for the dye-protein complex is stable
over 10 orders of magnitude (assessed in albumin)
Dye reagent is complex is stable for approximately one hour

Disadvantages
Non-linear standard curve over wide ranges
Response to different proteins can vary widely, choice of
standard is very important

Methodology
1. Warm up the spectrophotometer for 15 min. before use

2. Dilute samples with buffer to an estimated concentration of 1 to 20
micrograms/milliliter

3. Prepare standards containing a range of 1 to 20 micrograms protein
(albumin or gamma globulin are recommended) to a volume of 200
l (to a volume of 100 l if you are adding 1 M NaOH)

4. Prepare unknowns to estimated amounts of 1 to 20 micrograms
protein per tube to 200 l (100 l if you are using 1 M NaOH)

5. Add 100 l 1 M NaOH to each sample and vortex.

6. Add 800 l dye reagent and incubate 5 min.

7. Measure the absorbance at 595 nm.

Amino Acid Analysis
Acid-hydrolysis of the peptide (6 M HCl, 24 hr) gives a mixture
of amino acids.
The mixture is separated by ion-exchange chromatography.





Amino acids are detected using ninhydrin.
Automated method; requires only 10
-5
to 10
-7
g of peptide.
Partial Hydrolysis of Peptides and
Proteins
Acid-hydrolysis of the peptide cleaves all of
the peptide bonds.
Cleaving some, but not all, of the peptide
bonds gives smaller fragments.
These smaller fragments are then separated
and the amino acids present in each
fragment determined.
Enzyme-catalyzed cleavage is the preferred
method for partial hydrolysis.
Carboxypeptidase
protein
H
3
NCHC
O
R
+
NHCHCO
O
R

C
O
Carboxypeptidase is a proteolytic enzyme
(catalyzes the hydrolysis of proteins).

Carboxypeptidase
protein
H
3
NCHC
O
R
+
NHCHCO
O
R

C
O
Carboxypeptidase is a proteolytic enzyme
(catalyzes the hydrolysis of proteins).

Carboxypeptidase is selective for cleaving
the peptide bond to the C-terminal amino acid.
Trypsin
Trypsin is selective for cleaving the peptide bond
to the carboxyl group of lysine or arginine.
NHCHC
O
R'
NHCHC
O
R"
NHCHC
O
R
lysine or arginine
Chymotrypsin
Chymotrypsin is selective for cleaving the peptide
bond to the carboxyl group of amino acids with
an aromatic side chain.
NHCHC
O
R'
NHCHC
O
R"
NHCHC
O
R
phenylalanine, tyrosine, tryptophan

End Group Analysis
End Group Analysis
Amino sequence is ambiguous unless we know whether to
read it left-to-right or right-to-left.
We need to know what the N-terminal and C-terminal
amino acids are.
The C-terminal amino acid can be determined by
carboxypeptidase-catalyzed hydrolysis.
Several chemical methods have been developed for
identifying the N-terminus. They depend on the fact that
the amino N at the terminus is more nucleophilic than any
of the amide nitrogens.
Sanger's Method
The key reagent in Sanger's method for
identifying the N-terminus is 1-fluoro-2,4-
dinitrobenzene.
1-Fluoro-2,4-dinitrobenzene is very reactive
toward nucleophilic aromatic substitution.

F
O
2
N
NO
2
Sanger's Method
1-Fluoro-2,4-dinitrobenzene reacts with the
amino nitrogen of the N-terminal amino acid.

F
O
2
N
NO
2
NHCH
2
C NHCHCO
CH
3
NHCHC
CH
2
C
6
H
5
H
2
NCHC
O
O
O O
CH(CH
3
)
2

+
Sanger's Method
1-Fluoro-2,4-dinitrobenzene reacts with the
amino nitrogen of the N-terminal amino acid.

F
O
2
N
NO
2
NHCH
2
C NHCHCO
CH
3
NHCHC
CH
2
C
6
H
5
H
2
NCHC
O
O
O O
CH(CH
3
)
2

+

O
2
N
NO
2
NHCH
2
C NHCHCO
CH
3
NHCHC
CH
2
C
6
H
5
NHCHC
O
O
O O
CH(CH
3
)
2

Sanger's Method
Acid hydrolysis cleaves all of the peptide bonds
leaving a mixture of amino acids, only one of
which (the N-terminus) bears a 2,4-DNP group.

O
2
N
NO
2
NHCH
2
C NHCHCO
CH
3
NHCHC
CH
2
C
6
H
5
NHCHC
O
O
O O
CH(CH
3
)
2

Sanger's Method
Acid hydrolysis cleaves all of the peptide bonds
leaving a mixture of amino acids, only one of
which (the N-terminus) bears a 2,4-DNP group.

O
2
N
NO
2
NHCH
2
C NHCHCO
CH
3
NHCHC
CH
2
C
6
H
5
NHCHC
O
O
O O
CH(CH
3
)
2

H
3
O
+
O
O
2
N
NO
2
NHCHCOH
CH(CH
3
)
2
Sanger's Method
Acid hydrolysis cleaves all of the peptide bonds
leaving a mixture of amino acids, only one of
which (the N-terminus) bears a 2,4-DNP group.

O
2
N
NO
2
NHCH
2
C NHCHCO
CH
3
NHCHC
CH
2
C
6
H
5
NHCHC
O
O
O O
CH(CH
3
)
2

H
3
O
+
H
3
NCHCO

CH
3
+
H
3
NCH
2
CO

O
O
O
O
2
N
NO
2
NHCHCOH
CH(CH
3
)
2
+
O
H
3
NCHCO

CH
2
C
6
H
5
+
+
+
+

The Edman Degradation
and Automated
Sequencing of Peptides
Edman
degradation
Cyclic degradation of peptides
based on the reaction of
phenylisothiocyanate with the
free amino group of the N-
terminal residue such that
amino acids are removed one
at a time and identified as
their phenylthiohydantoin
derivatives:
Three steps
Coupling step
Cleavage step
Conversion step
2D Gel Electrophoresis
Introduction
- Sample preparation
- First dimension: Isoelectric focusing
- Second dimension: SDS-PAGE
- Detection of protein spots: staining
- Imaging analysis & 2D Gel databases
- Spot handling: excision, in-gel digestion
2D Gel Electrophoresis
The goal of two-dimensional electrophoresis is to separate
and display all gene products present.
It is the only method currently available which is capable of
simultaneously separating thousands of proteins.
The first dimension of 2-DE - isoelectric focusing (IEF).
pH gradient support
pI
2D Gel Electrophoresis
The second dimension of 2-DE - sodium dodecyl
sulfate PAGE (SDS-PAGE).
SDS as surfactant.
Molecular mass.
High resolution from independent protein parameters.
In the early 1970s, first use of 2-DE to separate serum
proteins.
Drawbacks
Poor reproducibility
Limited sample loading
Progress
Chemical or Mass spectrometric analysis
Immobilized pH gradient (IPG) gels
More sensitive detection procedures
Computer software
Sample preparation
Sample preparation
A critical step in 2-DE.
Solubilization, denaturation, reduction & removal of non-protein
sample components.
Standard sample solubilization solution:
Sample preparation
Optional modifications for more proteins displayed, shorter focusing time, and
more sharply focused spots:
Chaotropes: 8M Urea, 2M Thiourea & 7M Urea - Disrupt of hydrogen and
hydrophobic bonds
Surfactants: 4% CHAPS, 2% CHAPS & 2% Sulfobetaine 3-10
Disrupt membranes
Break hydrophobic interactions
Solubilise lipids
Release membrane bound proteins
Reducing agents: 100mM DTT, 2mM TBP (Tributyl phosphine) -and/or 65mM DTT -
- Break disulfide bridge (within or between protein)

Buffer preparation- composition
interfering substances
Ampholytes (up to 2%)
Help protein solubilisation
Scavenge cyanate ions
Precipitate nucleic acids (during centrifugation)
Prevent interaction immobilines/protein
Should represent the pH range desired

Interfering substances
Lipids (detergents)
Proteases (inhibitor cocktails)
Nucleic acids (ultracentrifugation, nucleases)
Polysaccharides (ultracentrifugation)
Salts (dialyse; less than 20 mM)

Sample preparation
Samples
Soluble samples
Serum, Plasma, CSF, Urine and aqueous extracts of cells/tissues. Samples
with low protein concentration or high salt concentrations are desalted
by dialysis/ liquid chromatography .
Tissue samples
Disrupted in presence of solubilization buffer break tissues using liquid
nitrogen temperature.
Small tissues wrapped in aluminium foil crushed
Large tissues homogenization in sol. Buffer
Cells
Harvested washed with PBS-solubilized in sample buffe
Sample preparation
Protein of medium abundance, occurring in about 1,000
copies per cell, or less than two picomoles (100 ng) in one
liter of cell culture.
Preparation method:
- Differential extraction
- Removal of nucleic acids
- Subcellular fractionation
- Chromatography
- Immunoprecipitation
- Affinity-based selection
- Others
Sample fractionation
Immobilized pH gradient (IPG)

The first dimension: IEF with IPG

pH gradient
Ampholytes: (carrier ampholytes) tube gels - mixture of
amphoteric species with a range of pI values
Immobilines: (~ ampholytes) IPG strips - covalently bound to
acrylamide gel immobilised pH gradient (IPG): gel plastic backed
Immobilines are series of eight acrylamide derivatives forms a series
of buffers with different pK values throughtout 3-10 range
The narrower the pH range of IPG, the more protein should be loaded.
Sample loading on IPG strips
IPG are supplied dry and needed to be rehydrated to its original thickness
(0.5 mm) prior to IEF.
Three methods
- Cup loading of sample after IPG rehydration (current & absorption)
- Passive in-gel rehydration of sample (absorption)
- Active in-gel rehydration of sample (current & absorption)
Advantages:
Mechanically strong
pH gradient cannot drift
Load larger amount of sample (dehydrated strip)
Disadvantages
membrane/hydrophobic proteins poorly represented on 2D
some larger proteins lost (size exclusion)
IEF run
pI
+
+ -
-
- - - -
+ + + + + + +
-
pH 4 pH 7
Cathode
Anode
Place wet wicks (DH
2
O) under each end of strip
Program power supply
Number of gels: (1-10)
Max voltage: 5000 V
Vhold: 125 V
Duration: 24 hrs 00 min
Max current: 80 mA/strip
Volt hours: 80,000 Vh
Set chiller temperature for 20C (setting ~2.5)
After IEF run
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
50mM of Tris pH 8.8 containing
2%w/v SDS
1% w/v DTT
6M urea reduce electroendosmosis
30% glycerol reduce electroendosmosis
0.002% bromophenol blue
Followed by incubation for 15mts at RT rocking condition 5%
w/v iodoacetamide in place of DTT.
After equilibration, the gel are drained out for 1 minute using
filter paper to remove excess liquid before applying for 2-
Dimension.


(R-S-S-R R-SH + R-SH)
Strip equilibration
+ -
+ -
+ -
+ -
(in chemical hood)
SDS-PAGE run
-
+
Conditions
Number of gel: (1-10)
Max voltage: 500 V
Max power: 1600 mW/gel
4C (setting 10)
~19 hrs
10% Duracryl
TM
gel
(22 cm x 23 cm x 1 mm)
MW
4 7
+ -
Tris/acetate
25 mM Tris-base
pH 8.3 (acetic acid)
200 mM Tris-base
200 mM Tricine
0.4% SDS
0.45 mm filtered!
Tris/Tricine /SDS
Staining
Coomassie staining
moderate sensitivity (36-47 ng)
non specific
not quantitative

Silver staining
sensitive (0.5-1.2 ng)
time consuming
non specific
negative stain some spots

Fluorescent dye (SYPRO ruby)
sensitive (1-2 ng)
specific, quantitative
end point stain
Staining protocol
Fixative (30 min)

SYPRO ruby (12 hrs)

Washing (30 min)

2% glycerol storage solution

Store gels at 4C
(40% Methanol - 10% acetic acid)
(10% Methanol - 6% acetic acid)
P
r
o
t
e
c
t

f
r
o
m

l
i
g
h
t

Imaging
UV detection (300 nm)
Blue light (470 nm) 5 min
Fuji Imager
4 7
pI
5 6
kDa
116
97
81
66
55
45
30
21
14
M
W

Image analysis
Samples ran in triplicate


Build average gel (software)


Differential expression analysis
1 2 3
Avg
Metabolite labelling
ICAT and its modification
Acrylamide labeling
O
18
labeling during proteolysis
Guanidination of lysine residues

Detecting metabolites
Types of non-radioactive metabolites
Organic dyes
Coomassie Brilliant Blue
Amido black
Silver staining
Acidic /silver nitrate
Alkaline/silver diamine
Reverse stain
Colloidal dispersion stain
Organic fluorophore stain
Metal chelate stain

Organic dye- CBB
Capable of detecting 30-100ng of protein, but less sensitive
than silver staining
Colloidal CBB staining used for background free staining acid
violet 17, serva violet 49 etc
Mechanism

Equilibrium b/w colloidal dye
and freely dispersed dye
Freely dispersed dye
Penetrate the gel matrix
and preferentially stain
the proteins, while
colloidal dye is excluded
Prevent background staining
Can detect 8-10ng protein
CBB+TCA/HclO4/H3PO4 +
EtOH/MeOH
Irreversible, acid catalysed
esterification of glutaric acid
side chain to carboxylic
groups
Silver staining
100 times more sensitive than CBB
Demerits
Ca
2+
binding proteins/glycoproteins poorly stain
Pre-staining is required with cationic dyes
Merits -Detects ng of proteins
Two types of silver staining
Alkaline/silver diamine
Acidic/silver nitrate
Reverse stain
Developed specifically to improve protein
recovery from polyacrylamide gels
Suitable for visualization of proteins, passive
elution of intact proteins from gels and their
analysis by MS
Types
Metal salt reverse stains
Zinc imidazole stain
Colloidal dispersion stain
These stains contain fine particles with high affnity for protein
stains.
Devised for detection of proteins electroblotted, dot-blotted
or slot blotted to nitrocelluose or PVDF.
Not applied for detection of proteins in gels.
Colloidal platinum or palladium particles with an organic shell
affixed to their surfaces have been added to protein mixtures,
subjected PAGE and visualized with a silver enhancement
step.
Types
India ink stain
Colloidal metal stain
Organic fluorophore stain
Types
Covalently bound fluorophore stain
Fluorescamine
Fluorescein isothiocyanate
Monobromobimane
Dansyl chloride
Non-covalently bound fluorophore stain
1-anilino-8-napthalene sulfonate
bis (8-toluidino-1-napthalene sulfonate)
SYPRO 8 and SYPRO Red

Metal chelate stains
Types
Colorimetric metal chelate stains
Pink bathophenanthroline disulfonate/ferrous complex
Amido black, CBB are less commonly used for
electroblotted proteins
Ferrozine/ferrous and pyrogallol/molybdate stains
Luminescent metal chelate stain
SYPRO Rose stain
SYPRO ruby
Mass Spectroscopy
MS is limited to the mass measurement of those molecules that can be
ionized and for which the ions can be transferred to a vacuum.
In mass spectrometry, a substance is bombarded with an electron beam
having sufficient energy to fragment the molecule. The positive fragments
which are produced (cations and radical cations) are accelerated in a
vacuum through a magnetic field and are sorted on the basis of mass-to-
charge ratio.
Protein identification
2D-GE + MALDI-MS
Peptide Mass Fingerprinting (PMF)

2D-GE + MS-MS
MS Peptide Sequencing/Fragment Ion Searching

Multidimensional LC + MS-MS
ICAT Methods (isotope labelling)
MudPIT (Multidimensional Protein Ident. Tech.)

1D-GE + LC + MS-MS

De Novo Peptide Sequencing


Components and variants of MS
MS consists of three components
An ionization source
A mass analyzer
A detector
Variants
MALDI-TOF
ESI-Triple Quadrupole Mass Spectrometer
ESI Ion Trap Mass Spectrometer
How does a mass spectrometer work?
Ionization
method
MALDI
Electrospray
(Proteins must be charged
and dry)
Mass analyzer
MALDI-TOF
MW
Triple Quadrapole
AA seq
MALDI-QqTOF
AA seq and MW
QqTOF
AA seq and protein modif.

Create ions Separate ions
Detect ions
Mass spectrum
Database
analysis
Generalized Protein Identification by MS
Artificial spectra
built
Artificially
trypsinated
Database of
sequences
(i.e. SwissProt)
Spot removed
from gel
Fragmented
using trypsin
Spectrum of
fragments
generated
MATCH
L
i
b
r
a
r
y

Methods for
protein
identification
Mass spectrometers
Linear Time Of Flight tube
Reflector Time Of Flight tube
detector
reflector
ion source
ion source
detector
time of flight
time of flight
Time of flight (TOF) (MALDI)
Measures the time required for ions to fly down the length of a
chamber.
Often combined with MALDI (MALDI-TOF) Detections from multiple
laser bursts are averaged. Multiple laser

Tandem MS- MS/MS
-separation and identification of compounds in complex mixtures
- induce fragmentation and mass analyze the fragment ions.
- Uses two or more mass analyzers/filters separated by a
collision cell filled with Argon or Xenon

Different MS-MS configurations
Quadrupole-quadrupole (low energy)
Magnetic sector-quadrupole (high)
Quadrupole-time-of-flight (low energy)
Time-of-flight-time-of-flight (low energy)
Mass Spectrometer Schematic
Inlet
Ion
Source
Mass
Filter
Detector
Data
System
High Vacuum System
Turbo pumps
Diffusion pumps
Rough pumps
Rotary pumps
Sample Plate
Target
HPLC
GC
Solids probe
MALDI
ESI
IonSpray
FAB
LSIMS
EI/CI
TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS
Microch plate
Electron Mult.
Hybrid Detec.
PCs
UNIX
Mac
Different Ionization Methods
Electron Impact (EI - Hard method)
small molecules, 1-1000 Daltons, structure

Fast Atom Bombardment (FAB Semi-hard)
peptides, sugars, up to 6000 Daltons

Electrospray Ionization (ESI - Soft)
peptides, proteins, up to 200,000 Daltons

Matrix Assisted Laser Desorption (MALDI-Soft)
peptides, proteins, DNA, up to 500 kD
MALDI-MS
Molecules are ionized by MALDI and their m/z ratio measured in a time-
of-flight mass analyzer.
Analysis involves the following
Analytes are mixed with a matrix compound (organic compound) placed on a
metallic slide or probe-evaporated to form analyte:matrix cocrystal.
The probe is inserted into the vacuum chamber
High voltage is applied and a short laser pulse is directed at the dried sample.
Desorption is initiated by absorption of energy at the wavelength of laser by
the matrix crystals, resulting in emission of this absorbed energy as heat-
results in sublimation of the matrix crystals and expansion of the matrix and
analyte molecules into the gas phase of the MS.
Ionization occurs by protonation/deprotonation, cation
attachment/detachment, or oxidation/reduction in the gas phase ions-
accerelated towards a series of lens- direct the ions into the field-free drift of
the TOF analyser detected.
At constant kinetic energy is inversely related to their m/z ratio-arrival time of
the ions at the linear detector at the other end- detected.
MALDI Ionization
+
+
+
+
-
-
-
+
+
+
+
-
-
-
- +
+
Analyte
Matrix
Laser
+
+
+
Absorption of UV radiation by
chromophoric matrix and
ionization of matrix

Dissociation of matrix, phase
change to super-compressed gas,
charge transfer to analyte
molecule

Expansion of matrix at
supersonic velocity, analyte
trapped in expanding matrix
plume (explosion/popping)
+
+
+
+
+
+
+
+
+
+
+
+
+
pulsed
UV or IR laser
(3-4 ns)
detector
vacuum
strong
electric
field
Time Of Flight tube
peptide mixture
embedded in
light absorbing
chemicals (matrix)
cloud of
protonated
peptide molecules
acc
V
Linear Time Of Flight tube
Reflector Time Of Flight tube
detector
reflector
ion source
ion source
detector
time of flight
time of flight
Mass
Filter
Mass Spectrometer Schematic
Inlet
Ion
Source
Detector
Data
System
High Vacuum System
Turbo pumps
Diffusion pumps
Rough pumps
Rotary pumps
Sample Plate
Target
HPLC
GC
Solids probe
MALDI
ESI
IonSpray
FAB
LSIMS
EI/CI
TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS
Microch plate
Electron Mult.
Hybrid Detec.
PCs
UNIX
Mac
Different Mass Analyzers
Magnetic Sector Analyzer (MSA)
High resolution, exact mass, original MA

Quadrupole Analyzer (Q)
Low (1 amu) resolution, fast, cheap

Time-of-Flight Analyzer (TOF)
No upper m/z limit, high throughput

Ion Trap Mass Analyzer (QSTAR)
Good resolution, all-in-one mass analyzer

Ion Cyclotron Resonance (FT-ICR)
Highest resolution, exact mass, costly
Different Mass Analyzers
Magnetic Sector Analyzer (MSA)
High resolution, exact mass, original MA

Quadrupole Analyzer (Q)
Low (1 amu) resolution, fast, cheap

Time-of-Flight Analyzer (TOF)
No upper m/z limit, high throughput

Ion Trap Mass Analyzer (QSTAR)
Good resolution, all-in-one mass analyzer

Ion Cyclotron Resonance (FT-ICR)
Highest resolution, exact mass, costly
Different types of MS
ESI-QTOF
Electrospray ionization source + quadrupole mass filter + time-of-flight mass analyzer

MALDI-QTOF
Matrix-assisted laser desorption ionization + quadrupole + time-of-flight mass
analyzer
GC-MS - Gas Chromatography MS
separates volatile compounds in gas column and IDs by mass

LC-MS - Liquid Chromatography MS
separates delicate compounds in HPLC column and IDs by mass

MS-MS - Tandem Mass Spectrometry
separates compound fragments by magnetic field and IDs by mass

LC/LC-MS/MS-Tandem LC and Tandem MS
Separates by HPLC, IDs by mass and AA sequence

TOF
NANO
SPRAY
TIP
ION
SOURCE
HEXAPOLE
COLLISION
CELL
HEXAPOLE
HEXAPOLE
QUADRUPOLE
MCP
DETECTOR
REFLECTRON
SKIMMER
PUSHER
Tandem MS-MS
Tandem mass spectrometry (MS-MS) is used to produce
structural information about a compound by fragmenting
specific sample ions inside the mass spectrometer and
identifying the resulting fragment ions. This information can
then be pieced together to generate structural information
regarding the intact molecule. Tandem mass spectrometry
also enables specific compounds to be detected in complex
mixtures on account of their specific and characteristic
fragmentation patterns.

Mass
Filter
Mass Spectrometer Schematic
Inlet
Ion
Source
Detector
Data
System
High Vacuum System
Turbo pumps
Diffusion pumps
Rough pumps
Rotary pumps
Sample Plate
Target
HPLC
GC
Solids probe
MALDI
ESI
IonSpray
FAB
LSIMS
EI/CI
TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS
Microch plate
Electron Mult.
Hybrid Detec.
PCs
UNIX
Mac