Characterization of Proteins
Determination of the Sizes of Proteins
•
•
•
•
Sedimentation analysis
Centrifugation
Gel filtration
SDS PAGE
Differential Precipitation
• Precipitation:
• The process of formation of a solid that was previously held in solution. – NH4 SO4 – Polyethylene glycol are added to a protein solution
• A precipitate forms and separated from the solution after centrifugation.
• If the concentrations of NH4 SO4 or polyethylene glycol are increased, more precipitate forms. 3
Centrifugation
•
Zonal centrifugation: Mixture to be separated is layered on top of a gradient (e.g.
sucrose or ficoll) increasing concentration down the tube ‐ can be continuous or discontinuous (layers)
‐ provides gravitational stability as different species move down tube at different rates forming separate bands. – Species are separated by differences in SEDIMENTATION COEFFICIENT (S) = Rate of movement down tube/Centrifugal force
– S is increased for particle of LARGER MASS
(because sedimenting force a M(1‐vr)
– S is also increased for MORE COMPACT STRUCTURES of equal particle mass (frictional coefficient is less)
Zonal ultracentrifugation
Centrifugation
•
Isopycnic (equal density) centrifugation: Molecules separated on EQUILIBRIUM POSITION, NOT by RATES of sedimentation.
Each molecule floats or sinks to position where density equals density of solution (e.g. CsCl gradient for nucleic acid separation). Gel Filtration Chromatography
7
Size‐Exclusion Chromatography
•
•
•
•
•
•
Separation of proteins based on kinetics of moving through the available space Larger proteins have less space than smaller molecules
Proteins larger than matrix elute in void volume (1 exchange of volume outside beads)
Proteins smaller than matrix partition in and out of beads
Pore size in beads is not uniform
Also some surface interaction with beads
Gel Filtration Elution Volumes as a Function
of Molecular Weight
Gel Electrophoresis
SDS Gel Electrophoresis
• Tris‐glycine buffer
• 10% SDS
Protein Size Determination by SDS Polyacrylamide
Gel Electrophoresis
- electrode
+ electrode
Protein Detection in Electrophoresis Gel
• Protein detection
– Coomassie blue
– Sypro: green, ruby, Red, orange
– Cybergreen
– Silver staining
coomassie brilliant blue A595
Detection of Amino Acids, Peptides, and
Proteins
•
•
•
•
•
•
•
UV
Biuret Reaction
Ninhydrin Reagent Fluorescarmine
Ortho‐phthalaldehyde
Coomassie Brilliant Blue
Silver Staining
Absorption spectra of Trp & Tyr
Beer’s law: A = εcl. Used to estimate protein concentration
Biuret Reaction
• Primarily measure peptide bonds
• Cupric acid react with the peptide groups of proteins and form a cupric ion complex • Generate purplish‐violet color with Amax at 540 nm
• Lowry method
• Low sensitivity
Ninhydrin Reaction
• The most widely used reagents for amino acids, peptides, and proteins
• Reacts with amino groups
• The major step is oxidative deamination of an amino acid to CO2, NH3, and an aldehyde conatining one less carbon atom than the original amino acid
• Ninhydrin is simultaneously reduced to ninhydrindantin
• Ninhydrindantin reacts with NH3 and produce purple color (Amax 570 nm)
• Destroy amino acid or peptide and the material detected cannot be used for further characterization
Fluorescarmine
•
•
•
•
React with amino group
Produce fluorescent compounds
Sensitive
The amino group reacted can be recovered by hydrolysis and used for further analysis
Ortho‐phthalaldehyde
• React with amino group in the presence of mercaptoethanol
• Forms a fluorescent reaction product
• Sensitive
• As Ninhydrin and fluorescarmin, it is not specific for protein
Circular Dichroism Spectroscopy of Polypeptides
Adapted from
T. E. Creighton,
Proteins
W.H.Freeman,
1984
Protein Characterization
•
Characterization of proteins and peptides involves three different processes:
1. Determining the Amino Acid Composition
• Involves finding out the amino acids that make up the protein and their number.
2. Determining the Amino Acid Sequence
• Involves finding out the sequence of amino acids of the proteins in their order.
3. Determining the Molecular mass of the Protein
21
Determination of Amino Acid Composition
• The peptide is first hydrolyzed into its constituent amino acids
by heating it in 6M HCl at 110ºC for 24‐72 hrs. The R groups remain intact, except for:
• Trp – indole ring damaged
• Asn, Gln – converted to Asp, Glu
• Gly does not react
22
Amino acid analysis
• The amino acids can be derivatized with ninhydrin or
o‐phthalaldehyde to make fluorescent derivatives that are easy to detect. • These are chromatographed by reverse‐phase HPLC (high‐
pressure liquid chromatography). – The characteristic retention times are used to identify the amino acids. – The fluorescence level can be quantified to determine the amount of that amino acid.
Amino acid analysis.
Protein Sequencing Strategy
1) Purify protein
2) Cleave disulfides – react with:
A) reducing agent followed by alkylating agent
1) DTT 2) β‐ME
B) performic acid
Determination of Primary Structure 1
• Hydrolyze peptides with hot 6M HCl.
– Identify AA and % of each.
– Usually done by chromatography
• Identify the N‐term and C‐term AAs
– C‐term via carboxypeptidase
– N‐term via Sanger’s Reagent (DNFB) • Fluoro‐2,4‐dinitrobenzene and dansyl chloride • Phenylisothiocyanate (Edman degradation)
5P2-27
Determination of Primary Structure 2
• Selectively fragment large proteins into smaller ones.
– Tripsin: cleave to leave Arg or Lys as C‐term AA
– Chymotrypsin: cleave to leave Tyr, Trp or Phe as C‐term AA
– Cyanogen bromide cleaves at internal Met leaving Met as C‐term homoserine lactone
5P2-28
Determine AA sequence of peptides
• Edman’s reagent:
– First, phenylisothiocyanate reacts with the terminal amino group to form a phenylthiocarbamoyl derivative.
– This residue cyclizes under acidic conditions to give a phenylthiohydantoin (PTH)‐amino acid and a peptide shortened by one amino acid residue.
– This PTH‐amino acid is identified by HPLC.
– The amino acid composition of the shortened peptide can be compared with the original peptide.
5P2-29
30
Sanger’s reagent
• Use fluoro‐2,4‐dinitrobenzene and dansyl chloride
• For N‐terminus amino acid
• Fluoro‐2,4‐dinitrobenzene reacts with an amino group and forms dinitrophenyl peptide
• Dansyl chloride reacts with dinitrophenyl peptide and form dansyl peptide
• Acid hydrolysis and analysis using HPLC
Standard Run on 19 PTH AAs
Residue 1 = Leu
Residue 2 = Ile
Protein Sequencing Strategy
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•
•
Reassemble sequence through overlaps of peptides created by different means
Map disulfides by cleaving protein into peptides before disulfide bond cleavage. After purification of disulfide‐linked peptides and cleavage of their disulfide bonds, sequencing of the peptides should reveal which cysteines are linked in disulfide bonds.
Det. Primary Structure
• A twelve AA peptide was hydrolyzed.
• Trypsin hydrolysis: – Leu‐Ser‐Tyr‐Gly‐Ile‐Arg
One is
C-term
– Thr‐Ala‐Met‐Phe‐Val‐Lys
• Chymotrypsin hydrolysis
Lys is internal!
– Val‐Lys‐Leu‐Ser‐Tyr
– Gly‐Ile‐Arg
– Thr‐Ala‐Met‐Phe
• Deduce the AA sequence
5P2-37
Det. Primary Structure
Keeping in mind the N-term AA and overlaping the
sequences properly gives:
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•
•
•
•
Tr Leu‐Ser‐Tyr‐Gly‐Ile‐Arg
Ct Gly‐Ile‐Arg
Ct Val‐Lys‐Leu‐Ser‐Tyr
Tr Thr‐Ala‐Met‐Phe‐Val‐Lys
Ct Thr‐Ala‐Met‐Phe
• The complete sequence is:
•
Thr‐Ala‐Met‐Phe‐Val‐Lys‐Leu‐Ser‐Tyr‐Gly‐Ile‐Arg
5P2-38
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
Isoelectric point (pI)
• Separation by charge:
4
Stable pH gradient
5
Low pH:
Protein is
positively
charged
6
7
8
9
10
High pH:
protein is
negatively
charged
At the isolectric
point the protein
has no net
charge and
therefore no
longer migrates
in the electric
field.
2D‐gel technique example
Advantages vs. Disadvantages
• Good resolution of
proteins
• Detection of
posttranslational
modifications
• Not for hydrophobic
proteins
• Limited by pH range
• Not easy for low abundant
proteins
• Analysis and quantification
are difficult
2D - LC/MS/MS
(trypsin)
Study protein
complexes without
gel electrophoresis
Complex mixture is
simplified prior to
MS/MS by 2D LC
Peptides all bind
to cation
exchange column
Successive elution
with increasing salt
gradients separates
peptides by charge
Peptides are
separated by
hydrophobicity on
reverse phase
column
A simple definition of a Mass Spectrometer • A Mass Spectrometer is an analytical instrument that can separate charged molecules according to their mass–to–charge ratio. • The mass spectrometer can answer the questions “what is in the sample” (qualitative structural information) and “how much is present” (quantitative determination) for a very wide range of samples at high sensitivity Mass Spectrometer
• Works by ionizing molecules and then sorting and identifying the ions according to their mass‐to‐
charge (m/z) ratios.
• Two key components – ion source, which generates the ions
– mass analyzer, which sorts the ions
Mass Spectrometer Schematic
Turbo pumps
Diffusion pumps
Rough pumps
Rotary pumps
High Vacuum System
Inlet
Sample Plate
Target
HPLC
GC
Solids probe
Ion
Source
MALDI
ESI
IonSpray
FAB
LSIMS
EI/CI
Mass
Filter
TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS
Detector
Microch plate
Electron Mult.
Hybrid Detec.
Data
System
PC’s
UNIX
Mac
Ion Sources
• Electrospray ionization (ESI)
• Atmospheric pressure chemical ionization
(APCI)
• Atmospheric pressure photoionization (APPI)
• Matrix Assisted Laser Desorption Ionization (MALDI)
Ion Sources make ions from sample molecules
(Ions are easier to detect than neutral molecules.)
Electrospray ionization:
Pressure = 1 atm
Inner tube diam. = 100 um
Partial
vacuum
Sample Inlet Nozzle
(Lower Voltage)
N2
Sample in solution
N2 gas
+
+ ++
++
++++
++
+
+ ++
++
+
++
+
++
+
+
++
+
++
+
++
+
++
+ +
+
+
+
+
+
+
+
MH+
MH2+
MH3+
High voltage applied
to metal sheath (~4 kV)
Charged droplets
Desorption of ions from solution
• As the droplets shrink, the charge concentration in the
droplets increases.
• The repulsive force between ions with like charges
exceeds the cohesive forces and ions are ejected (desorbed)
into the gas phase.
• Useful for analyzing large biomolecules
APCI Ion Source
• The LC eluent is sprayed
through a heated (250°C –
400°C) vaporizer at
atmospheric pressure.
• The gas-phase solvent
molecules are ionized by
electrons discharged from a
corona needle.
• The solvent ions then transfer
charge to the analyte
molecules through chemical
reactions (chemical
ionization).
• The analyte ions pass through
a capillary sampling orifice
into the mass analyzer.
•
APPI Ion Source
• Avaporizer converts
the LC eluent to the
gas phase.
• A discharge lamp
generates photons in a
narrow range of
ionization energies.
• The energy ionizes
analyte molecules
while minimizing the
ionization of solvent
molecules.
• The resulting ions pass
through a capillary
sampling orifice into
the mass analyzer.
MALDI Ionization
+
+ +-+
+
+ ++ + --+
-+
+
+
+
++
Matrix
Laser
Analyte
• 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”)
Mass Analyzers
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Quadrupole
Time‐of‐flight
Ion trap
Fourier transform‐ion cyclotrone resonance (FT‐ICR)
• Mass Spectrometers separate ions according to their mass‐to‐charge (m/z) ratios Quadrupole Mass Analyzer
• Uses a combination
of radio frequency
and DC
• voltages to operate
as a mass filter.
• Has four parallel
metal rods.
• Lets one mass pass
through at a time.
Can scan through
all masses or sit at
one fixed mass.
Principle of Time of Flight Analyzer
• A uniform electromagnetic force is applied to all ions at the same time, causing them
to accelerate down a flight tube.
• Lighter ions travel faster and arrive at the detector first, so the mass-to-charge ratios
of the ions are determined by their arrival times.
• Time-of-flight mass analyzers have a wide mass range and can be very accurate in
their mass measurements.
Ion Trap • Consists of a circular ring electrode plus two end caps that together form a chamber. • Ions entering the chamber are “trapped” there by electromagnetic fields.
• Scanning field can eject ions of specific m/z • Advantages – MS/MS/MS….. – High sensitivity full scan MS/MS Fourier transform‐ion cyclotron resonance (FT‐ICR) Mass Analyzer
• Ions entering a chamber are trapped in
circular orbits by powerful electrical and
magnetic fields.
• When excited by a radio-frequency (RF)
electrical field, the ions generate a timedependent current.
• This current is converted by Fourier
transform into orbital frequencies of the
ions which correspond to their mass-tocharge ratios.
• Can perform multiple stages of mass
spectrometry without additional mass
analyzers.
• Have a wide mass range and excellent
mass resolution.
• Very expensive mass analyzers.
What is MS/MS?
MS/MS means using two mass analyzers (combined in
one instrument) to select an analyte (ion) from a mixture,
then generate fragments from it to give structural
information.
Mixture of
ions
Ion
source
Single
ion
MS-1
Fragments
MS-2
What is MS/MS?
Peptide
mixture
1 peptide
selected for
MS/MS
+
+
MS/MS
+
Have only masses to
start
+
+
The masses of all the
pieces give an MS/MS
spectrum
Interpretation of an MSMS spectrum to derive structural
information is analogous to solving a puzzle
+
+
+
+
+
Use the fragment ion masses as specific pieces of the
puzzle to help piece the intact molecule back together
Tandem Mass Spectrometry
• Different MS‐MS configurations
– Quadrupole‐quadrupole (low energy)
– Magnetic sector‐quadrupole (high energy)
– Quadrupole‐time‐of‐flight (low energy)
– Time‐of‐flight‐time‐of‐flight (low energy)
• Fragmentation experiments may also be performed on single analyzer instruments such as ion trap instruments and TOF instruments equipped with post‐source decay
Tandem MS
Fragmentation in MS/MS produces sequence‐specific fragment ions
Mass Spectra
57 Da =K
‘G’
D
D
V
99 Da = ‘V’
L
L
H2O
G
D
K
V
G
0
• The peaks in the mass spectrum:
– Prefix and Suffix Fragments.
– Fragments with neutral losses (‐H2O, ‐NH3)
– Noise and missing peaks.
mas
s
Protein Identification with MS/MS
G
V
D
K
Peptide
Identification:
Intensity
MS/MS
L
00
mas
s
Peptide sequencing by MS/MS
(Standing 2003 Curr. Opin. Struct. Biol. 13, 595-601)
Protein Identification by Tandem Mass Spectrometry
MS/MS instrument
S#: 1708 RT: 54.47 AV: 1 NL: 5.27E6
T: + c d Full ms2 638.00 [ 165.00 - 1925.00]
850.3
100
95
687.3
90
85
588.1
80
75
70
65
R e la tiv e A b u n d a n ce
S
e
q
u
e
n
c
e
60
55
851.4
425.0
50
45
949.4
40
326.0
35
Database search
•Sequest
de Novo interpretation
•Sherenga
524.9
30
25
20
589.2
226.9
1048.6
397.1
1049.6
489.1
15
10
629.0
5
0
200
400
600
800
1000
m/z
1200
1400
1600
1800
2000
Advantages vs. Disadvantages
• Determination
of MW and aa.
Sequence
• Detection of
posttranslational
modifications
• High-throughput
capability
• High capital costs
• Requires sequence
databases for analysis