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Analytical Chemistry of Proteins. How can the structure(s) of a protein be determined?. Analytical Chemistry of Proteins, cont. Preparation of proteins Differentiation/visualization of proteins Determination of structure Chemical analysis 3D methods Chemical synthesis of peptides.

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analytical chemistry of proteins

Analytical Chemistry of Proteins

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

analytical chemistry of proteins cont
Analytical Chemistry of Proteins, cont
  • Preparation of proteins
  • Differentiation/visualization of proteins
  • Determination of structure
    • Chemical analysis
    • 3D methods
    • Chemical synthesis of peptides
preparation of proteins
Preparation of proteins
  • Gross separation (Lysis of cells, etc)
  • Dialysis
  • Chromatography
    • Size Exclusion (Gel filtration)
    • Ion Exchange
    • Affinity
  • Concentration - Ultrafiltration
  • Precipitation
preparation of proteins cont

Preparation of proteins, cont

Protein solution

N2 pressure

Concentration - Ultrafiltration

Excess aqueous phase

Ultrafiltration membrane

preparation of proteins cont1
Preparation of proteins, cont

10 mM salt concn

  • Precipitation

Solubility (mass/vol)

1 mM salt concn

pH

Typical solubility behavior of protein as a function of pH and salt concentration

differentiation visualization of proteins
Differentiation/Visualization of Proteins
  • Electrophoresis
  • Chromatography
  • Ultracentrifugation
  • Detection
differentiation visualization of proteins1
Differentiation/Visualization of Proteins

Electric Field

  • Electrophoresis - movement of particles in an electric field

v = Ez / f

Friction Coeff.

Velocity

f = 6r

Charge

Radius

Viscosity

differentiation visualization of proteins cont
Differentiation/Visualization of Proteins, cont
  • Thus,

v ~ z/r

  • Note that if proteins were spheres of constant charge/mass, v would be a weakmonotonically decreasing function of size (r).

(v ~ z/M1/2)

differentiation visualization of proteins cont1
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 dependenton molecular weight.
differentiation visualization of proteins cont2
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]?
differentiation visualization of proteins cont3
Differentiation/visualization of proteins, cont

Polyacrylamide gel

Extremely hydrophilic polymer

crosslinking provides sieve

(NH4)2S2O8 initiator

differentiation visualization of proteins cont4
SDS Micelle Differentiation/visualization of proteins, cont

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

differentiation visualization of proteins cont5
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 ~2:1 amino acid residue:SDS

differentiation visualization of proteins cont6
Differentiation/visualization of proteins, cont

Visualizing stains for protein electrophoresis

Silver (Ag) - more sensitive, trickier

Coomassie blue - simpler

differentiation visualization of proteins cont7
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
ampholytes
Ampholytes
  • Low molecular weight compounds with both acidic and basic groups
    • -amino acids
    • Others; polyaminopolycarboxylic acids (low polymers
  • pI’s over a particular pH range
immobilines
Immobilines
  • Polyacrylamides with acidic and basic groups
    • Monomers with acidic and basic groups polymerized in situ
    • pH gradient more stable than that with ampholytes
differentiation visualization of proteins cont8
2D ElectrophoresisDifferentiation /visualization of proteins, cont

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

differentiation visualization of proteins cont9
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

differentiation visualization of proteins cont10

EO flow

Differentiation /visualization of proteins, cont

Detection end

  • 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

Pos charged particle

Injection end

neg charged particle

+

Separation by charge

differentiation visualization of proteins cont11
Differentiation /visualization of proteins, cont
  • Chromatography
    • Ion exchange
    • Size exclusion
    • Affinity
    • HPLC (RPLC)

analytical or prep

analytical

differentiation visualization of proteins cont12

More hydrophobic

Differentiation /visualization of proteins, cont

C18 -coated packing (stationary phase)

FLOW

  • HPLC (RPLC, “reverse phase”)

molecules partitioning between stationary and increasingly hydrophobic mobile phase

differentiation visualization of proteins cont13
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)
differentiation visualization of proteins cont14
Differentiation /visualization of proteins, cont
  • Detectors
    • UV/Vis; Beer’s 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
differentiation visualization of proteins cont15
Differentiation /visualization of proteins, cont

 = extinction coef.

c = concentration

l = path length

 = wavelength

I = intensity

  • UV Absorbance abs = cl (= log I0/I)

I

I0

abs

280 nm = max for protein

(due to Y, W)

F at 260 nm, too.

280

, nm

differentiation visualization of proteins cont16
Differentiation /visualization of proteins, cont
  • Fluorescence - more sensitive than abs

fl - max = Stokes shift

fl

max

I

I

I0

log I0/I

I

, nm

differentiation visualization of proteins cont17
Differentiation /visualization of proteins, cont
  • Mass Spectrometry - Charged particle in the gas phase sorted by mass/charge ratio. Provides identification as well as detection
differentiation visualization of proteins cont18
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

differentiation visualization of proteins cont19
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
determination of structure
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
determination of structure cont
Determination of Structure,cont

quantitation, identification

peptide

  • Amino acid composition of a peptide by complete hydrolysis

6N HCl / 110° / 24 h

amino acids

visualizing reagent

ion exchange chromatography

tagged amino acids

determination of structure cont1
Determination of Structure,cont
  • Visualizing reagents
    • ninhydrin + peptide  high absorbance
    • fluorescamine + peptide  high fluorescence
    • o-phthalaldehyde +-mercaptoethanol (OPA) + peptide  high fluorescence
determination of structure cont2
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
determination of structure cont3
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
determination of structure cont4
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.
determination of structure cont5
Determination of Structure,cont

peptide

terminal label

terminal labelled peptide

  • Terminal identification
    • Fluorodinitrobenzene
    • Dabsyl chloride
    • Dansyl chloride

hydrolysis

terminal labelled amino acid

Identification by chromatography

determination of structure cont6
Scheme of Edman Degradation

ΦNCS

H2NCHRCO-AA2- AA3- AA4- AA5…AAn-CO2H

phenyl isothiocyanate

short peptide

labelling

ΦNHCSNHCHRCO-AA2- AA3- AA4- AA5…AAn-CO2H

repeat

phenylthiocarbamoyl derivative of peptide

release

Φ

S

C

N

O

N

H

R

+

H2NCHRCO-AA3- AA4- AA5…AAn-CO2H

peptide shorter by one amino acid

phenylthiohydantoin amino acid

Determination of Structure, cont
determination of structure cont7
C-terminal sequencingDetermination of Structure, cont

coupling

peptide

+

shortened peptide

thiohydantoin amino acid

determination of structure1
Determination of Structure
  • Secondary, Tertiary, Quaternary
    • Circular Dichroism (see Stryer)
    • X-Ray Crystallography
    • NMR
determination of structure cont8
Determination of Structure, cont

diffraction

  • X-Ray Crystallography (Solid phase)

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

determination of structure cont9
Determination of Structure, cont

NMR (Solution phase)

H

H

Protons close to each other in space affect each other’s chemical shifts. Entire 3D structure can be worked out for small enough proteins (<30 kD)

determination of structure2
Determination of Structure
  • Check by resynthesis
    • Merrifield Method
    • Recombinant techniques