Analytical Chemistry of Proteins

1
Analytical Chemistry of Proteins

• How can the structure(s) of a protein be
  determined?

2
Analytical Chemistry of Proteins, cont

• Preparation of proteins
• Differentiation/visualization of proteins
• Determination of structure
• Chemical analysis
• 3D methods
• Chemical synthesis of peptides

3
Preparation of proteins

• Gross separation (Lysis of cells, etc)
• Dialysis
• Chromatography
• Size Exclusion (Gel filtration)
• Ion Exchange
• Affinity
• Concentration - Ultrafiltration
• Precipitation

4
Preparation of proteins, cont

• Concentration - Ultrafiltration

Protein solution
N2 pressure
Excess aqueous phase
Ultrafiltration membrane
5
Preparation of proteins, cont

• Precipitation

10 mM salt concn
Solubility (mass/vol)
1 mM salt concn
pH
Typical solubility behavior of protein as a
function of pH and salt concentration
6
Differentiation/Visualization of Proteins

• Electrophoresis
• Chromatography
• Ultracentrifugation
• Detection

7
Differentiation/Visualization of Proteins

• Electrophoresis - movement of particles in an
  electric field

Electric Field
v Ez / f
Friction Coeff.
Velocity
f 6??r
Charge
Radius
Viscosity
8
Differentiation/Visualization of Proteins, cont

• Thus,
• v z/r
• Note that if proteins were spheres of constant
  charge/mass, v would be a weak monotonically
  decreasing function of size (r).
• (v z/M1/2 )

9
Differentiation/visualization of proteins, cont

• SDS PAGE (Sodium Sodecyl Sulfate PolyAcrylamide
  Gel Electrophoresis)
• Protein is highly negative, moves to anode.
  Different proteins move through matrix at rates
  strongly dependent on molecular weight.

10
Differentiation/visualization of proteins,cont

• Why, in SDS PAGE is the velocity
• a strong function?
• remarkably regular exceptions glycoproteins
  (less polar but still hydrophilic), membrane
  (highly hydrophobic) proteins?

11
Differentiation/visualization of proteins, cont

• Polyacrylamide gel
• Extremely hydrophilic polymer
• crosslinking provides sieve

(NH4)2S2O8 initiator
12
Differentiation/visualization of proteins, cont

• SDS Micelle

SDS Micelles
SO3?
SO3?
? O3S
highly charged exterior
SO3?
? O3S
SO3?
? O3S
SO3?
? O3S
SO3?
? O3S
? O3S
SO3?
? O3S
SO3?
SO3?
? O3S
hydrophobic interior
SO3?
(actually a sphere)
? O3S
SO3?
? O3S
13
Differentiation/visualization of proteins, cont

• In SDS, protein may form string of pearls

dithiothreitol or other thiol breaks S-S links
Assembly highly negatively charged























Globules 21 amino acid residueSDS

14
Differentiation/visualization of proteins, cont

• Visualizing stains for protein electrophoresis

Silver (Ag) - more sensitive, trickier
Coomassie blue - simpler
15
Fig 5-20a, b
16
Differentiation /visualization of proteins, cont

• 2D electrophoresis The Proteome
• Isoelectric focusing
• pH gradients
• ampholytes
• Immobilines (substituted polyacrylamide)
• pI of protein position of zero charge in
  gradient
• Follow by SDS PAGE dimension

17
Ampholytes

• Low molecular weight compounds with both acidic
  and basic groups
• ?-amino acids
• Others polyaminopolycarboxylic acids (low
  polymers
• pIs over a particular pH range

18
Immobilines

• Polyacrylamides with acidic and basic groups
• Monomers with acidic and basic groups polymerized
  in situ
• pH gradient more stable than that with ampholytes

19
Fig 5-21
20
Differentiation /visualization of proteins, cont

• 2D Electrophoresis

protein stops at pI in IEF dimension
low pH
high pH
high MW
protein moves according to MW in SDSPAGE dimension
low MW
From Gel Electrophoresis of Proteins ed. Hames
and Rickwood,IRL Press, 1981
21
Differentiation /visualization of proteins, cont

• Capillary electrophoresis - primarily analytical
• No supporting matrix FSCE
• Can separate proteins of different charge
• Protease digests - glycopeptides identified
• SDS PAGE CE - similar to non-capillary, but
  higher resolution
• Mainly for ssDNA (single stranded DNA) or short
  stretches of DNA

See R. R.Holloway, Hewlett Packard Journal ,
June 1996
22
Differentiation /visualization of proteins, cont

• FSCE - Free Solution Capillary Electrophoresis
• Narrow capillary allows very high fields (1000
  V/cm), high resolution
• cathodic EO flow in silica
• Usually inject at anode

Detection end

Pos charged particle
Injection end
neg charged particle

Separation by charge
23
Differentiation /visualization of proteins, cont

• Chromatography
• Ion exchange
• Size exclusion
• Affinity
• HPLC (RPLC)

analytical or prep
analytical
24
Differentiation /visualization of proteins, cont

• HPLC (RPLC, reverse phase)

C18 -coated packing (stationary phase)
FLOW
molecules partitioning between stationary and
increasingly hydrophobic mobile phase
25
Differentiation /visualization of proteins, cont

• Ultracentrifugation
• Velocity ultracentrifugation - characterization
• Equilibrium ultracentrifugation - accurate MW
• Zonal ultracentrifugation - separation by buoyant
  density in a density gradient (e.g., sucrose)

26
Differentiation /visualization of proteins, cont

• Detectors
• UV/Vis Beers law absorbance most common
• proteins absorb at 280 nm (W, Y), 200-210 nm
  (peptide bond)
• Fluorescence most sensitive, requires
  fluorophore
• MS Electrospray, MALDI can give accurate MW,
  other structural information

27
Differentiation /visualization of proteins, cont

• UV Absorbance abs ?cl ( log I0/I)

? extinction coef. c concentration l path
length ? wavelength I intensity
I0
I
abs
280 nm ?max for protein (due to Y, W) F at
260 nm, too.
280
?, nm
28
Differentiation /visualization of proteins, cont

• Fluorescence - more sensitive than abs

?fl - ?max Stokes shift
?fl
?max
I
I0
I
log I0/I
I
?, nm
29
Differentiation /visualization of proteins, cont

• Mass Spectrometry - Charged particle in the gas
  phase sorted by mass/charge ratio. Provides
  identification as well as detection

30
Differentiation /visualization of proteins, cont

• Electrospray - Protein solution in (usually) acid
  aerosolized droplets desolvate to multiply
  charged ions

most abundant ion
computation
deduced mass distribution
m/z
m
31
Differentiation /visualization of proteins, cont

• MALDI - Matrix-assisted Laser Desorption and
  Ionization. Protein dissolved in matrix- an
  organic fluorophore. Laser blasts puff of
  material, protein is usually charge 1 or 2
• sometimes easier than electrospray
• simple interpretation of spectra
• physics not well understood yet

32
Determination of Structure

• Primary structure - divide and conquer
• Chemical generation of shorter segments
• complete hydrolysis - amino acid analysis
• chemical/enzymatic cleavage
• treatment of S-S links
• terminal identification
• sequencing of segments
• N terminal - Edman degradation
• C terminal

33
Determination of Structure,cont

• Amino acid composition of a peptide by complete
  hydrolysis

quantitation, identification
peptide
6N HCl / 110 / 24 h
amino acids
visualizing reagent
ion exchange chromatography
tagged amino acids
34
Determination of Structure,cont

• Visualizing reagents
• ninhydrin peptide ? high absorbance
• fluorescamine peptide ? high fluorescence
• o-phthalaldehyde ?-mercaptoethanol (OPA)
  peptide ? high fluorescence

35
Determination of Structure,cont

• Enzymatic cleavage
• The specificity of many proteases (examples in
  text) is known, and can be used to help determine
  structure
• trypsin - cleaves peptide bond on the C terminal
  side of K, R

36
Determination of Structure,cont

• Chemical cleavage
• Specific reagents (examples in text) can be used
  for cleavage at specific places in a peptide
  chain
• cyanogen bromide - cleaves peptide bond on the C
  terminal side of methionine

37
Determination of Structure,cont

• S-S links between chains must be broken to do an
  amino acid analysis or sequence
• oxidation produces -SO3 s, can reveal which
  peptides are linked (diagonal electrophoresis)
• reduction/stabilization preparation for
  sequencing or analysis.

38
Determination of Structure,cont

• Terminal identification
• Fluorodinitrobenzene
• Dabsyl chloride
• Dansyl chloride

peptide
terminal label
terminal labelled peptide
hydrolysis
terminal labelled amino acid
Identification by chromatography
39
Determination of Structure, cont

• Scheme of Edman Degradation

40
Determination of Structure, cont

• C-terminal sequencing

coupling
peptide

thiohydantoin amino acid
shortened peptide
41
Determination of Structure

• Secondary, Tertiary, Quaternary
• Circular Dichroism (see Stryer)
• X-Ray Crystallography
• NMR

42
Determination of Structure, cont

• X-Ray Crystallography (Solid phase)

diffraction
X-Ray source
protein crystal
Regular lattice of electrons in crystal diffracts
into deconvolutable pattern. Nobel prize for
Perutz and Kendrew for the structure of
myoglobin 1000 structures have now been done
photographic plate
43
Determination of Structure, cont

• NMR (Solution phase)

Protons close to each other in space affect each
others chemical shifts. Entire 3D structure can
be worked out for small enough proteins (lt30 kD)
44
Determination of Structure

• Check by resynthesis
• Merrifield Method
• Recombinant techniques