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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
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