Analyse de la structure proteique - Prof. Alain Astier Honoray Head of Pharmacy department ; GHU Henri Mondor, ea grita
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Analyse de la structure proteique
Prof. Alain Astier
Honoray Head of Pharmacy department ; GHU Henri Mondor,
School of Medicine , University Paris Est Créteil, France.
Les médicaments biologiques ont révolutionné le traitement et
la prévention de nombreuses maladies invalidantes et mortelles
Inflammatory digestive
Cancer Arthritis diseases
Kidney Disease Growth Disorders Psoriasis
Cawson MR, et al. BMC Musculoskelet Disorders 2014;15:26; Cohen LB, et al. Transl Res 2014;163:533–56; Dutka P. Nephrol Nurs J 2012;39:447–57;
Hwang IT. Korean J Pediatr 2014;57:379–83; Nam JL, et al. Ann Rheum Dis 2014;73:516–28;Waller, CF. Commun Oncol 2012;9:198–205.
Structure
Glycoproteines
Protein instability
Physical instability :
Aggregation
Denaturation
Adsorption on surfaces
Chemical instability:
Desamidation
Disulfide bond breakage and formation
Oxydation
Hydrolysis
Isomerization O
O
Non-disulfide crosslinking O O
Deglycosylation NH2 N OH N
Asparagine
Maillard reaction
Aspartate
12-08-2018 7
Analyse de la structure primaire • Essentielle – Nouvelle proteine: caractérization – Biosimilaire: preuve d’identité – Etude stabilité chimique • Nombreuses méthodes
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
9
• The peptide is first hydrolyzed into its constituent amino acids by
heating it in 6M HCl at 110ºC for 24 hrs.
• The amino acids are then separated by HPLC.
10Determination of Amino Acid
Composition
• The peptide is first hydrolyzed into its constituent amino acids by heating it in 6M HCl at
110ºC for 24 hrs.
• The amino acids are then separated by HPLC.
- Phase inverse (dérivatisation pré-colonne: PhénylthiohydantoinePTH, dansyl, o-
phtaldéhyde OPA; UV et SM)
- Échange d’ion (analyseur AAs: polystène: dérivarisation post-colonne: ninhydrine, dansyl,OPA,PTH; UV et
SM)
Cation-exchange, trione postcolonne
UV-Vis Detector, 570 nm for primary amino acids,
440 nm for secondary amino acidsModifications structure I
Dégradation
• Peptide mapping
• Chromatographie ionique
• Phase inverse (C4-C18)
• Nombre de SHDetermination of Amino Acid Sequence
• Edman degradation.
• It sequentially removes one residue at a time from the amino end of the
peptide.
– First Phenylisothiocyanate reacts with the terminal amino group to form a
phenylthiocarbamoyl derivative.
– This residue cyclizes under acidic conditions to give a PTH amino acid and a
peptide shortened by one amino acid residue.
– This PTH-amino acid is identified by HPLC.
– Automated repeated Edman degradation by a sequenator that can analyze
sequences of about 50 amino acids long.
– The amino acid composition of the shortened peptide can be compared with the
original peptide.
13EDMAN
DEGRADATION
14Determination of Amino Acid Sequence
• Longer polypeptide chains are broken into shorter ones for
analysis by specifically cleaving them with enzymes that cleave
at specific points.
• Some examples are :
Enzyme Cleavage Point
Trypsin Lys, Arg (C)
Chymotrypsin Phe,Trp, Typ (C)
Pepsin Phe, Trp, Tyr (N)
Cyanogen bromide Met (C)
15Primary structure peptide map
Chromatographie ionique • Dégradation (ASN –> ASP, déglycosylation…)
Structure secondaire • Dichroïsme circulaire (UV lointain) • FTIR (dérivée) • Raman • RMN 2D
Spectrometrie UV 1- UV lointain 180 nm à 250 nm (DC) - Effet de l’environnement sur la structure d’une molécule - Détermination du contenu en structure secondaire de protéines - Effet de ligands sur la structure - Etudes d’interactions protéine-protéine et acides nucléiques- protéines - Etudes dynamiques (dénaturation, renaturation) 2) UV proche 250 nm à 350 nm : - Empreinte digitale de la structure tertiaire - Effet de ligands sur la structure - Effet de l’environnement sur la structure d’une molécule
Analyse spectre FT-IR bande amide I et amide II en dérivée seconde
Analyse par Raman Pas d’effet de l’eau = travail en solution
RMN Interactions entre atomes , chaines Spectres complexes
Analyse de la structure tertiaire • Spectrométrie UV dérivée • Spectrométrie de fluorescence • Diffraction rayons X • RMN
Méthodes spectrales • Simples • Analyses AAs aromatiques (Tyr, Phe,Tryp) • Plus dans études de stabilité ou biosimilaires = comparaison entres conditions de stress ou molécules.
Spectrométrie UV dérivée
• Absorbance entre 260 -
300 nm = aromatique
– Phe 252-258 nm
– Tyr : 275 nm
– Tyr/tryp: 285 nm
– Tryp: 292 nm
• Dépend environnement
hydrophobe
• Si modifications
structure III = shiftsFluorescence
• Tryp
– Exc 290 nm
– Em 337 nm (folded)
– Em 352 nm (unfolded)
• Fluo intrinséque
Tryp ds BSA
• Fluo extrinsèque
Tryp pur
(fluorophore)Courbes de dénaturation Action dénaturant (chaotrope) : chlorure de guanidine FUC = [(FC- F337) / F352-F337)]
Thermodynamic stability curve
Estimation of the global stability; to test “memory effect”
First stress DG2 -> intermediate “excited “state = destabilisation
Second stress DG3 < DG2 -> degradation
Energie
Transtition state 1
Transtition state 2
DG1 Transtition state 3
DG2
DG3
E1 (initial) E1*
DH°= 490 kJ mol-1
DS = 1.43 kJ mol-1
E2 (degraded)
Reactional pathway 29
12-08-2018Autres méthodes • Diffraction X • Diffraction SAXS ''small angle X-ray scattering« : solution; facteur de forme • Diffusion neutrons • RMN hauts champs et 2D
Etude de l’agrégation • Submicronique et micronique – SEC – SEC-MALLS (multiangle laser light scattering ) – Fluorescence; UV – DLS – Flow microscopy – FFFF (flow field-flow fractionation) – FTIR – Diffraction laser petits angles – Vitesse sédimentation (SV: ultracentifugation)
Oligomerisation: SEC
Particle size analysis by diffraction laser spectroscopy
A
B
12-08-2018 Prof. Astier, Sofia, March 2013 34Figure n°10: Aspect en contraste de phase des rétentats des dilutions filtrées à 0,22 µm. A) particule globulaire ; B) particule filamento-fibrillaire 12-08-2018 Prof. Astier, Dresden, October 2013 35
Conclusion
• Nombreuses méthodes complémentaires et
orthogonales
• Instabilité physique = problème crucial
• Dépend question posée:
– Nouvelle structure
– Etudes comparatives
• Biosimilaires
• StabilitéYou can also read
