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 Protein solution N2 pressure Concentration - Ultrafiltration Excess aqueous phase Ultrafiltration membrane

5. 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

6. Differentiation/Visualization of Proteins • Electrophoresis • Chromatography • Ultracentrifugation • Detection

7. 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

8. 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)

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 dependenton 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. 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

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 ~2:1 amino acid residue:SDS –

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 • pI’s 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. 2D Electrophoresis Differentiation /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

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. 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

23. Differentiation /visualization of proteins, cont • Chromatography • Ion exchange • Size exclusion • Affinity • HPLC (RPLC) analytical or prep analytical

24. 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

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; 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

27. 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

28. Differentiation /visualization of proteins, cont • Fluorescence - more sensitive than abs fl - max = Stokes shift fl max I I I0 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 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

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 peptide terminal label terminal labelled peptide • Terminal identification • Fluorodinitrobenzene • Dabsyl chloride • Dansyl chloride hydrolysis terminal labelled amino acid Identification by chromatography

39. 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

40. C-terminal sequencing Determination of Structure, cont coupling peptide + shortened peptide thiohydantoin amino acid

41. Determination of Structure • Secondary, Tertiary, Quaternary • Circular Dichroism (see Stryer) • X-Ray Crystallography • NMR

42. 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

43. 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)

44. Determination of Structure • Check by resynthesis • Merrifield Method • Recombinant techniques