Identification of proteins in works of art: combined use of Maldi mass fingerprinting and ELISA

Daniel P. Kirby*1, Julie Arslanoglu2, Adriana Rizzo2 and Julia Schultz3

1Harvard Art Museum; 2The Metropolitan Museum of Art; 3University of Applied Sciences and Arts of Hildesheim (HAWK), Germany

IntroductionProteins are present in artworks

and historic artifacts in many

forms—as paint binders,

adhesives, and coatings—to name

a few, and their identification is

important for knowledge of artists’

techniques and to guide

conservation treatments. This

presentation will describe a simple

procedure for producing peptide

mass fingerprints from micro-

samples and validate its use for

conservation science by showing

results from a number of historical

and modern works and by

corroborating the obtained data

with ELISA analyses in some

cases.

Methods / Experimental•Maldi mass fingerprinting-samples as small as 100µm largest

dimension have been successfully

analyzed

-single Eppendorf tube

-solubilize protein in 50:50

trifluoroethanola : 50mM ammonium

bicarbonate with heat and ultrasonic

-speedvap to near dryness

-resuspend in 50mM ammonium

bicarbonate

-reduce disulfides with TCEP·HCl

(Tris(2-carboxyethyl)phosphine

hydrochloride)

-endcap with iodoacetamide

-digest with trypsin

-Maldi with α-cyano-4-hydroxycinnamic

acid matrix

• ELISAELISA experiments reported here were

carried out with a horseradish

peroxidase (HRP) reporting system

developed at HAWK and further

optimized at The Metropolitan Museum

of Artb.

AcknowledgementsStraus Center Staff

-K. Kennelly

-J. Stenger

-K. Eremin

-L. Lee

-E. Mysak

-N. Khandekar

Harvard Biomolecule Mass Spectrometry & Proteomics

-W. Lane

-R. Robinson

The Andrew W. Mellon Foundation (D.K.)

*correspondence:

Dp.kirby@verizon.net

SummaryMaldi mass fingerprinting has been used to

identify several classes of proteins in a

variety of artworks. In several cases these

results have been verified with ELISA .

A simple, rapid, one-tube protein

solubilization and digestion procedure is

described. The method offers an alternative

to conservation scientists for protein

identification in artworks and historic artifacts.

Mass fingerprinting is sensitive and specific

and offers the potential for determining

species origin of proteins commonly

encountered in artworks.

ELISA is shown to be sensitive but limited by

available antibodies. It can provide a means

of identification as well as validation of other

methods of protein identification used in

conservation science.

Harvard / Straus

Art Museum

Proteins in artworks•What

-Animal glues-rabbit skin glue

hide glue, French glue,

gelatin, parchment size

-Casein-milk paint

-Egg-whole, white, yolk

-Fish-fish glue, Isinglass

-Garlic

•Where

-Grounds

-Binders

-Matrices

-Coatings

-Gilded surfaces

•Current methods

-Hydrolysis AA analysis

-Immunostaining/ELISA

-HPLC-UV or MS

-IR/Raman

-Proteomics (MFP, LCMSMS)

Animal glues

Pulse Voltage: 2100Laser Energy: 17622 July 09 A RPep MX6 L22 97-7-ct-R (PepMix1)

m/z1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950

%0

100

%

0

100

01Sep09_RP_MX7_C16_IV-ct_M1S4 99 (1.649) Sb (99,10.00 ); Sm (SG, 2x3.00); Cm (6:99) TOF LD+ 1.01e31095.54

1079.52

1045.53

1106.58

1501.81

1465.77

1107.57

1453.83

1267.681235.621125.56

1303.63 1451.72

1311.63

1502.80

1532.85

1562.891586.87

1587.89 1707.92

1608.86 1848.941708.86

1760.90

1948.051850.91

22Jul09A_RPep_MX6_L22_97-7-ct(R) 140 (2.332) Sb (99,10.00 ); Sm (SG, 2x3.00); Cm (139:181) TOF LD+ 2.62e31105.65

1095.63

1079.64

1106.64

1586.841562.88

1435.74

1107.661427.80

1267.741111.67

1193.741333.70

1334.71

1401.70

1459.77

1532.85

1587.84

1648.91

1588.831833.89

1707.86

1708.841760.761976.95

1834.87

1947.931850.87

Rabbit skin

glue reference

Mass fingerprint from

a single red flake

from Rothko’s mural

Mark Rothko

1963

Harvard

Mural

consistent collagen markers

Reference standards from Harvard’s Gettens Collection

Description Maldi fingerprinting ELISA

Gettens Plate 53-26

Parchment cast film

12/7/1933.

Collagen Collagen ++ from HRP

Rabbit skin glue (Spain), cast

filmCollagen Collagen ++ from HRP

Lab gelatin, cast film Collagen Collagen + from HRP

French glue, cast film Collagen Collagen + from HRP

Master of the Fogg Pietà

Italian (1300 - 1350)

The Lamentation over the Dead Christ, c. 1330

Tempera and gold on panel

42.23 x 50.01 cm (16 5/8 x 19 11/16 in.)

Creation Place: Florence, Tuscany, Italy

Pulse Voltage: 2200Laser Energy: 20001 Oct 09 RP MX7 A4 VI-ct #2

m/z1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100

%

0

100

01Oct09_RP_MX7_A4_VI-ct_2 362 (6.034) Sm (SG, 2x3.00); Sm (SG, 2x3.00); Sb (99,10.00 ); Sm (SG, 2x3.00); Cm (65:399) TOF LD+ 2.05e31106.5975

1435.7734

1165.6093

1267.6974

1241.7065

1334.7761

1401.7676

1459.7909

1892.11241648.9857

1562.9436

1532.8215

1586.8937

1832.96621688.9823

1774.0006

2132.10161977.0428

1941.9857

2058.0188

2021.0640

200 µm

Egg Tempera

References cited

aWang, et al., J. Proteome Res. 2005 Nov-Dec;

4(6):2397-403.

bSchultz, J.: Immunolgische Methoden zur Analytik

tierischer Bindemittel – Möglichkeiten und Grenzen.

Master thesis. HAWK University of Applied Sciences

and Arts of Hildesheim, Holzminden, Göttingen,

Germany. 2006.

cBaumeister, Mechthild et al., “A Splendid Welcome

to the „House of Praises, Glorious Deeds and

Magnanimity,‟ ‖ Preprints of the IIC Congress,

Istanbul 20-24 September, 2010, in press.

dRizzo, A, et al., “A rediscovered opulence: the

surface decoration of an early 18th century

Damascene reception room at The Metropolitan

Museum of Art,” Postprints of the 4th International

Architectural Paint Research Conference, 3-6

August 2010, in preparation.

eSchultz, J., et al., ―The Use of ELISA for the

Identification of Proteins and Gums Using the

Example of a Syrian Reception Room,‖ IIC

Congress, Istanbul 20-24 September, 2010, in

press.

fRichard Mulholland, ―From Dreams and Visions

and Things Not Known‖: Technique and Process in

David Smith’s Drawings, a thesis submitted in

partial fulfillment of the requirements of the Royal

College of Art for the degree of Doctor of Philosophy

gChambery, A. et al., Anal. Bioanal. Chem (2009)

395:2281-2291..

A number of paints from the Nur al-Din room have been studied by GCMS and

ELISA . Some were also studied by Maldi mass fingerprinting. An example from

the smalt paint, used extensively as blue background for the inscriptions and

obtained from the reverse of a panel, is shown here. A more expansive

description of the analytical study is the subject of forthcoming publications. c, d, e

Milk Paint

Nur al-Din Room, dated 1119 A.H. / 1707 A.D.;

Ottoman: Attributed to Damascus, Syria.

Wood, marble, stucco, glass, mother-of-pearl,

ceramics, tile, stone, iron, colors, gold.

The Metropolitan Museum of Art

Gift of The Hagop Kevorkian Fund, 1970

Inscription panel: smalt paint used as background for

calligraphy. Darkened due to multiple layers of varnishing.

APOV1 VIT1 VIT2 OVALTR OVAL LYSC

922.44 X X

1048.64 X

1077.56 X X

1164.54 X

1324.69 X

1345.69 X

1374.78 X

1401.67 X

1406.60 X

1418.70 X X

1445.70 X

1555.68 X

1560.69 X

1561.67 X

1591.71 X

1687.80 X X

1773.88 X X X

1777.88 X X

1859.90 X X

1890.99 X

1891.98 X

1892.97 X

1912.98 X X

1914.05 X

1972.91 2X

2144.05 X X

2236.07 2X

2278.12 X X

Pulse Voltage: 2200Laser Energy: 210VII-ct (1:2 aC) A. Rizzo #2

m/z900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950

%

0

100

09Feb10_RP_MX8_F2_Rizzo2 130 (4.333) Sm (SG, 7x3.00); Sb (15,10.00 ); Sm (SG, 2x3.00); Cm (107:179) TOF LD+ 2.03e31078.6206

1048.6964

922.5126

947.5422

981.5439

1401.7571

1164.6006

1317.7205

1271.6721

1221.6708

1372.7628

1343.7421

1892.10901446.7921

1688.9362

1562.7909

1608.82861830.0570

consistent egg yolk markers

Maldi mass fingerprinting indicated the

presence of yolk only. ELISA was

positive for ovalbumin, an egg white

protein, indicating possible cross-

reactivity of the commercial antibody

used in the analysis with egg yolk

proteins or a greater sensitivity of

ELISA for ovalbumin. These ELISA

issues are the subject of ongoing

investigations.

Egg tempera marker ions

David Smith, Untitled; 1952 (73-52-39)

300 µmDavid Smith (1906-1965), an American abstract

expressionist sculptor of large, steel abstract geometric

sculptures, also produced a body of abstract sketches

such as Untitled; 1952 (73-52-39) shown here. He was

thought to use a casein-based white paint for many of

his sketchesf, but there were no technical studies to

confirm this. Maldi mass fingerprinting was able to

confirm the artist’s use of casein (milk paint) and, based

on a recent publication by Chambery, et al.g, determine

that bovine casein was used.

Multiple alignment of alpha S1 caseins (after Chambery, et al.)g

Pulse Voltage: 2100Laser Energy: 18622 July 09 A RPep MX6 N8 97-11-ct (PepMix1)

m/z1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750

%

0

100

%

0

100

22Jul09A_RPep_MX6_N8_97-11-ct 136 (2.266) Sm (SG, 2x3.00); Sb (99,10.00 ); Cm (97:137) TOF LD+ 8.63e31267.76

1252.67

1251.76

1268.75

1383.85

1269.76

1270.751329.67

1314.64

1384.83

1760.95

1385.83

1619.981386.87

1446.74 1624.89

1761.96

1762.95

01Oct09_RP_MX7_A8_VI-ct_4 298 (4.968) Sb (99,10.00 ); Sm (SG, 5x3.00); Cm (202:331) TOF LD+ 3.34e31267.86

1251.85

1268.85

1269.86

1270.85

1271.891383.97

1272.88 1382.941324.92

1385.961421.03

1450.01 1512.03 1592.121531.11

1626.11 1644.13 1679.18

FVVAPFPEVFR = 1306.707 Da (sheep, goat)

FFVAPFPEVFGK = 1383.723 Da (bovine)

Gettens plate 53-24

Casein with lime

ELISA confirmed casein in

this reference sample.

David Smith

Untitled: 1952

Mass fingerprint from a

small sample from

Master of the Fogg Pietà

Yolk White