Identification of Proteins in Renaissance Paintings by Proteomics

1
Identification of Proteins in Renaissance
Paintings by Proteomics

• Caroline Tokarski, Elisabeth Martin, Christian
  Rolando, Cécile Cren-Olivé
• Université des Sciences et Technologies de Lille,
  Centre de Recherche et de Restauration des Musées
  de France
• Anal Chem. 2006,vol. 78, 1494-1502

2
Objectives

• Detection and analysis of proteins in Renaissance
  paintings challenges being
• Protein extraction without hydrolysis
• Microgram samples

3
Background

• Paint pigment binder
• Previous unsuccessful methods for isolation and
  detection of proteins
• Organic dyes, immunodetection
• Pyrolysis TLC, GC, HPLC
• UV-vis, IR

4
New Methodology

• Small-scale purification
• Enzymatic hydrolysis
• Nanochromatography MS/MS
• Protein databank interrogation

5
Methodological Development

• Two paint models (variable protein oil
  pigment)
• Ovalbumin protein
• Egg white and yolk

6
Model Preparation

• Thin film formation
• Aging process
• Purposeful lack of special precautions

7
Sample extraction

• Microsample excision (200 ?g)
• Pulverization
• Aqueous dissolution (1 trifluoroacetic acid)
• Dialysis
• Sonication
• Centrifugation supernatant collection

8
Ovalbumin (pre-extraction)

• MALDI-TOF (linear mode)
• Commercial ovalbumin plus residual eggwhite
  proteins
• Ovomucoid
• Lysozyme
• Ovotransferrin

9
Ovalbumin Model (post-extraction)

• MALDI-TOF (linear mode)
• Ovalbumin, ovomucoid, lysozyme

10
Enzymatic Hydrolysis

• Egg-based model and Renaissance samples
• Denaturation
• Reduction
• Alkylation
• Trypsin cleavage

11
Egg-based Model

• MALDI-TOF (reflector mode)
• Peptide mass fingerprint
• MS-FIT (Protein Prospector)
• MASCOT (Matrix Science)

12
Fingerprint Search Results
13
Peptide Identification

• Nanochromatography
• ESI-Qq-TOF MS (nanoelectrospray)

14
Egg-based Model

• Triply charged ion m/z 522.28
• Ovotransferrin peptide fragments

15
Protein Identification Results
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Application of Methodology to Renaissance
Painting I

• Benedetto Bonfiglis Triptych
• The Virgin and Child, St. John the Baptist, St.
  Sebastian

17
MS/MS spectrum of triply charged ion at m/z
551.61 from hydrolyzed extract
18
MS/MS spectrum of doubly charged ion at m/z
714.82 from the hydrolyzed extract
19
MS/MS spectrum of monocharged ion at m/z 402.28
from the hydrolyzed extract
20
Proteins Identified
21
Application of Methodology to Renaissance
Painting II

• Niccolo di Pietro Gerini - The Virgin and Child

22
MS/MS spectrum of triply charged ion at m/z
519.23 from hydrolyzed extract of Gerini Painting
23
Proteins Identified
24
Conclusion

• Successful methodology for extraction and
  identification of binder proteins for the first
  time
• Determination of tempera grassa
• Applications to archaeology