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Heritage Science
Characterization ofanunusual coating
onfunerary portraits fromRoman Egypt
circa100-300AD
Daniel P. Kirby1*, Marie Svoboda2, Joy Mazurek3, Lin Rosa Spaabæk4 and John Southon5
Abstract
This paper details the investigation of a discrete coating observed on a group of Egyptian panel paintings, six mummy
portraits and one funerary panel, dating from first-third century CE. Six mummy portraits in this group are encaus-
tic, and the funerary panel is tempera using an animal glue binder. An accretion or coating has been observed on
the surface and recesses of the paint layers on these panels. Examination of the portraits using ultraviolet radiation
revealed an irregular visible fluorescence on the surface. On the mummy portraits, the fluorescence often extends
only as far as where the linen wrappings would have secured the portrait to its mummy. Under magnification, the
coating appears as a crizzled encrustation. Material exhibiting these characteristics was sampled from the surface
of all seven panels. Initial analysis of samples from four panels by gas chromatography mass spectrometry (GC/MS)
and enzyme-linked immunoassay (ELISA) revealed the presence of egg. Subsequent analysis of the coating from all
seven portraits by peptide mass fingerprinting (PMF) and liquid chromatography with tandem mass spectrometry
(LCMSMS) confirmed egg and further characterized the coating as highly deamidated, whole hen egg, or hen egg
white in one instance. Importantly, the 14C date of the coating from two portraits indicates the time of application as
approximately 2000 years ago, implying that the coating, at least in those cases, is not a modern addition. This report
summarizes the examination and analytical characterization of this unusual coating. Possibly applied as an aesthetic
or protective layer, or a symbolic and ritual unguent, the principal function of this coating remains unknown.
Keywords Funerary portrait, Egg coating, Peptide mass fingerprinting, LCMSMS, Radiocarbon dating
Introduction
Ancient funerary portraits are individualized images of
the deceased that are painted on wooden panels. ey
may have been placed in tombs in memory of family
members, or secured over the face of a mummified body
(mummy portrait). In this paper we use “portrait” or
“funerary portrait” to include both funerary and mummy
paintings, and “mummy portrait” when it is necessary to
make a distinction. ese personalized funerary images
were created mainly during the Roman occupation of
Egypt, dating from approximately first–third century
CE, and were reserved only for those who could afford
costly funerary expenses. e painting style and tech-
nique evolved from the Greco-Roman tradition of wall
painting, and the quality of the portrait likely reflects the
*Correspondence:
Daniel P. Kirby
dp.kirby@verizon.net
1 Museum of Fine Arts, Boston and Private Practice, 42 Cliff Road, Milton,
MA 02186, USA
2 Antiquities Conservation, J. Paul Getty Museum, 1200 Getty Center
Drive, Suite 1000V, Los Angeles, CA 90049, USA
3 Getty Conservation Institute, 1200 Getty Center Drive, Suite 700, Los
Angeles, CA 90049, USA
4 Spaabæk Konservering, Burmeistersgade 32,5, 1429 Copenhagen,
Denmark
5 Earth System Science Department, University of California, B321 Croul
Hall, Irvine, CA 92697, USA
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Page 2 of 18
Kirbyetal. Heritage Science (2023) 11:73
economic status of individuals represented [1]. e por-
traits were typically painted with a conventional pallet
of pigments bound in encaustic (wax-based) or tempera
(animal glue or plant gum) media, or a combination of
both [2]. Mummy portraits were secured in place by linen
or stucco wrappings covering their edges, framing the
portrait. Portrait imagery reflects the diverse population
that lived in Roman Egypt during this period–male and
female, young and old–and most portraits were painted
following a standardized and conventional layout, Fig.1.
Approximately 1000 mummy portraits1 are housed in
museum collections worldwide, and there are about 100
intact portrait mummies that provide valuable informa-
tion on their context and construction [3].
Fig. 1 The seven portraits analyzed in this study. Each portrait is shown with visible illumination (left) and ultraviolet-induced visible fluorescence
(UVF) (right)
1 is statistic includes both mummy portraits and non-mummy funerary
portraits.
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Page 3 of 18
Kirbyetal. Heritage Science (2023) 11:73
Ongoing research on funerary portrait production has
focused on the pigments, binders and woods that were
used [4]. Recently, however, an unusual coating has been
observed on the surfaces of six encaustic portraits and
one tempera (animal glue) portrait from collections in
five museums. Whereas coatings (varnishes) and liba-
tions are known to have been applied on certain painted
surfaces in ancient Egypt, coatings on funerary portraits
have not previously been described in publications.
Signicance
Numerous materials employed as paint binders, adhe-
sives, and used in mummification procedures in ancient
Egypt have been described [5–7]. Although egg has been
noted as a binding medium in certain paintings around
the ancient Mediterranean [8, 9], there have been no
definitive identifications of egg as a binder or adhesive
in ancient Egypt to date, nor have any uses of eggs other
than dietary ones been suggested. e use an egg-based
surface coating on several Romano-Egyptian funerary
portraits housed in various museum collections repre-
sents a previously undescribed possible use of egg in the
ancient world. rough the combination of highly sen-
sitive and specific analytical techniques, the coating has
been extensively characterized, and its application date
determined. e data obtained provide evidence for the
use of a whole hen egg or egg glair (white) coating dis-
cretely applied to funerary Romano-Egyptian portraits
likely at the time of their manufacture, thus providing
new insight into the production and ritual use of funerary
portraits.
Study background
In 2013, a collaborative study and comparison of ancient
funerary portraits from museum collections worldwide
was initiated with the goal of identifying their material
composition and manufacturing methods (APPEAR)
[10]. e project has since expanded our understanding
of the painted portraits thus enabling unique features and
trends to be recognized [11].
At the first APPEAR meeting in 2013, observation of
an unusual surface accretion on several. J. Paul Getty
Museum (Getty) portraits was presented as a possible
direction of study. Prior to this meeting, binding media
analyses carried out on three wooden panel paintings
(74.AP.20–22) in the Getty collection indicated the pres-
ence of egg. Initially presumed to be from a restoration
treatment, cross sections later showed that it was a dis-
crete surface layer [12]. Subsequent analysisof a similar
accretion or coating observed on two mummy portraits
in the Ny Carlsberg Glyptotek collection (Glyptotek) also
revealed the presence of egg. During the examination
of Glyptotek portrait AE.I.N 684 under UV radiation,
conservators had noted a “distinct fluorescent border at
the bottom of the portrait and a faint one at its top. e
coating is not present where the mummy wrappings would
have covered the panel.” [13]
At this point, existing and new samples from the four
Getty and Glyptotek portraits were analyzed by PMF
and LCMSMS. ree additional portraits from other
collections likely containing similar coatings were subse-
quently identified through the APPEAR database based
on their visible appearance and fluorescence response,
and samples from these were also analyzed by the same
techniques.
Experimental background
GC/MS, ELISA
In the initial phase of the current study, coatings from
the Getty and Glyptotek paintings were analyzed by GC/
MS and/or ELISA. Quantitative amino acid analysis by
GC/MS is a well-established method for identification
of proteins such as animal or fish glue, egg and casein
in artworks [14, 15]. e relative amounts of the stand-
ard twenty amino acids (or a subset of them) in a sample
provide a fingerprint characteristic of a specific protein
source. Mixtures of proteins and the effects of aging and
pigment interaction may complicate confident identifica-
tions. GC/MS can distinguish between egg yolk and egg
glair based on the presence or absence of yolk lipids (pal-
mitic and stearic acids), but it is not clear how lipid mate-
rial would behave as a coating over thousands of years, as
fatty acids are volatile and evaporate with time. GC/MS
cannot be used to establish the species from which the
glue or egg proteins originated.
ELISA is an antibody-based technique commonly
used in biological research to identify proteins or other
biological macromolecules by means of a colorimetric
assay. Since 2005, numerous ELISA techniques have been
developed and applied specifically to works of art because
they require small samples, are extremely sensitive, and
can identify complex mixtures of proteins [16–21]. e
methodology used in this study has been described pre-
viously [16]. In the case of the funerary portraits, ELISA
was utilized primarily to identify complex mixtures of
proteins such as egg and animal glue that could not be
identified solely by GC/MS [8].
GC/MS analysis of the coating samples from the Getty
and Glyptotek portraits showed that they best matched
egg glair without added oil, wax, or tree resins (Table1).
e egg coating samples did not contain lipid compo-
nents, such as glycerol, palmitic and stearic acids. ELISA
also confirmed the identification of ovalbumin, the main
protein found in egg glair. is was a significant finding
since previous analyses had shown that the paint medium
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Page 4 of 18
Kirbyetal. Heritage Science (2023) 11:73
of three of these four portraits was encaustic, and the
medium of the other was animal glue, leaving the impor-
tance of the finding of egg protein uncertain. ese anal-
yses provided a starting point for in-depth analysis of the
egg coatings by PMF and LCMSMS, which is the focus of
this paper.
PMF, LCMSMS
Heritage science has benefitted immensely from adop-
tion and adaptation of analytical methods from biotech-
nology, particularly in regard to our understanding of the
use of proteinaceous materials [22]. Two of these meth-
ods, PMF [23–25] and LCMSMS with protein database
searching [26–28], have been applied to a variety of art-
works and objects from cultural heritage and were used
in this study to investigate the coating observed on the
portraits.
PMF analysis involves the enzymatic digestion of pro-
teins followed by Matrix Assisted Laser Desorption-
Ionization Time of Flight Mass Spectrometric analysis
(MALDI) of the resultant peptide mixture [29–32]. For
each protein, the amino acid sequence is unique, thus,
the mixture of peptides is unique−a “peptide mass fin-
gerprint.” Marker ions [33] in the MALDI spectra from
known reference materials are compared with those from
unknown samples for identification.
e use of PMF, also termed Zooarchaeology by Mass
Spectrometry (ZooMS) [34] for identification of the
sources of collagen-based materials has been well devel-
oped and applied successfully to a wide range of objects
and artefacts from both cultural heritage and archaeol-
ogy [35–37]. With PMF, some mammalian groups can
be identified to the family level (cervidae (deer) and cani-
dae (dogs), for example), whereas others may be further
identified to species level (many cetaceans, for example)
[38]. Minimal sample requirements plus high sensitivity
and specificity have made this method a powerful tool for
the conservator as well as the archaeologist, and there is
ongoing activity to extend the methodology to keratin-
based materials [39], as well as fish [40] and shells [41].
Proteinaceous materials commonly found in artworks,
such as egg, animal glues and casein, are also amenable
to analysis by PMF [42, 43], as is the case with the pre-
sent work. Markers for common proteinaceous materi-
als found in artworks, as well as keratin, which is often
encountered as a surface contaminant, have been pub-
lished [29, 44], and a list of those relevant to this study is
shown in Table2.
Table 1 Summary of analyses of seven funerary portraits
Oval ovalbumin; Vit2 vitellogenin-2; Lysc lysozyme; Apov1 apovitellenin-1; Trfe ovotransferrin; Vit1 vitellogenin-1; Ovaly ovalbumin-related protein Y; ApoB
apolipoprotein B; Iovo ovomucoid; Ovalx ovalbumin-related protein X; Apob apolipoprotein B; Apoai apolipoprotein A-I
* All show keratin contamination
Institution Accession No Medium ELISA GCMS Sample
Method PMF Result* LCMSMS
Result* Hen egg
proteins found
by Mascot
14C Date
Getty 74.AP.11 Encaustic Egg Egg Sample Stick Whole hen egg Whole hen egg,
mammalian
collagen
Oval, Vit2, Lysc,
Apov1, Trfe,
Vit1, Iovo, Apob,
Ovaly
25–128 CE
Getty 74.AP.20 Tempera Egg, glue Glue Sample Stick Whole hen
egg, trace col-
lagen
Whole hen egg,
mammalian
collagen
Oval, Vit2, Lysc,
Apov1, Trfe, Vit1
Glyptotek AE.I.N 681 Encaustic N/A Egg Remnant from
GCMS analysis Whole hen egg Whole hen egg,
mammalian
collagen
Oval, Vit2, Lysc,
Apov1, Vit1, Trfe,
Ovaly, Apob
Glyptotek AE.I.N 684 Encaustic N/A Egg Remnant from
GCMS analysis Whole hen egg Whole hen egg,
mammalian
collagen
Oval, Lysc, Trfe,
Ovaly, Apov1,
Iovo
Norton Simon F.1978.19.P Encaustic N/A N/A Sample Stick Whole hen egg Whole hen egg,
mammalian
collagen
Oval, Vit2,
Vit1, Trfe, Lysc,
Apov1, Ovaly,
Iovo, Ovalx,
Apob, Apoa1
Vienna X 297 Encaustic N/A N/A Sample Stick Whole hen egg Whole hen egg,
mammalian
collagen
Oval, Lysc, Trfe,
Apov1, Vit2,
Ovaly
42 BCE-108 CE
Cleveland 1971.137 Encaustic N/A N/A Sample Stick Hen egg glair Hen egg glair,
mammalian
collagen
Oval, Lysc, Trfe,
Iovo, Ovaly
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Kirbyetal. Heritage Science (2023) 11:73
LCMSMS with database searching is a mainstay of
biotechnology. Its sensitivity is ideally matched to the
minimal samples generally available with cultural objects,
and its specificity allows more precise identification of
proteinaceous materials than may be possible with other
techniques [45]. In the present work it is used either to
identify proteinaceous materials and/or to confirm those
found with PMF.
Methods andmaterials
Samples
Samples of the portrait coatings were taken by multiple
conservators, and not all sample sites were documented
with micrographs. All portraits had a clearly visible sur-
face layer and characteristic visual appearance, and the
UVF response guided the conservators to specific sam-
ple sites. Although the coating was sampled carefully
under magnification, the possibility of unintended inclu-
sion of underlying paint and/or ground layers cannot be
excluded, and those layers could be potential sources of
egg protein. However, a recent study of binding media in
61 Romano-Egyptian paintings, of which 51 were funer-
ary mummy portraits, identified egg in only two paint
layers by ELISA [8]. us, the paint binding medium
seems unlikely to be the source of egg discussed in this
paper.
Several recently introduced sampling methods using,
for example, eraser crumbs, [46, 47] and fine abrasive
films [42, 47, 48], can abrade and entrap small amounts
of material from an object/surface for subsequent pro-
tein analysis. Eraser crumbs are more appropriate for fri-
able surfaces or loosely adhered layers whereas abrasive
films are more appropriate for solid objects or hard sur-
face coatings. Even when surface alteration is not of criti-
cal importance, sampling with an abrasive film may be
easier and more controllable than, for instance, excision
with a scalpel. For the present work, samples from the
Glyptotek portraits were remnants from previous analy-
ses [13] received in Eppendorf tubes as a fine powder. All
other samples were obtained by museum conservators
using “sample sticks,” which are small pieces of 30 µm
alumina grit polishing film attached to a polystyrene sup-
port (Fig.2). After sampling, the tip of the sample stick
containing the abrasive film and entrapped sample is cut
off, placed in an Eppendorf tube, and forwarded for anal-
ysis. Only a single sample was taken from each portrait.
Portions of the sample prepared for PMF were also uti-
lized for LCMSMS.2
Table 2 Markers used to identify materials found in artworks
and objects of cultural heritage
Markers are collected and validated from several sources including published
research data and protein sequences, LCMSMS data from known samples, and
analysis of known reference samples. If a majority of the expected markers is
observed in the MALDI spectrum, the indicated protein or material is considered
to be positively identied. Egg yolk and white markers were derived from
database searching of PMF spectra of authentic samples with Mascot, (www.
Matri xscie nce. com) as well as LCMSMS data from known references. Keratin
markers were also derived from database searching of PMF spectra with Mascot.
The keratin markers noted here are a truncated list of the most intense and
persistent ions (attributed to the Uniprot IDs: P35527, P13645 and P04264)
(* usually more intense)
Egg Yolk Egg White Animal Glue Keratin
1048.6* 1345.7* 1095.6* 1179.7*
1077.6* 1428.6 1105.6* 1235.6
1085.7* 1555.7 1241.8 1300.6
1164.5* 1687.8* 1267.8* 1475.8*
1324.7 1773.9 1427.7* 1493.8
1401.7* 1859.9* 1435.8* 1707.8
1406.6* 1913.0 1459.7* 1765.8
1445.7 2009.0* 1473.7 1791.8
1560.7 1586.9 2384.0*
1561.7* 1648.8* 2705.2
1591.7 1655.9
1891.0 1923.0
1892.0 1963.0
1893.0* 1976.0
2236.1 2705.3*
Fig. 2 Sample stick comprised of a polystyrene support with
attached micro grit polishing film
2 We estimate that this method removes a few 10’s of micrograms of material
from a hard surface. A quantitative study is forthcoming.
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Page 6 of 18
Kirbyetal. Heritage Science (2023) 11:73
PMF
Digestion
60μL 50mM ammonium bicarbonate (AMBI) was added
to each sample and heated to 75°C for 60min. After
cooling, 8μL Promega Sequence Grade trypsin (0.02μg/
μL in 50mM AMBI) was added and digestion proceeded
overnight at 37°C.
MALDI analysis
2μL of the digest were added to 20μL 40% acetoni-
trile (ACN), 0.1% trifluoroacetic acid (TFA) with satu-
ratedα-Cyano-4-hydroxycinnamic acid (CHCA) matrix.
0.65μL of the mixture was spotted onto the MALDI plate.
Spectra were obtained with an ABI/Sciex 5800 MALDI-
TOF-TOF instrument operated in reflector mode. Cali-
bration was done with a standard mixture of peptides:
757.3992Da, 1046.5418Da, 1296.6848Da, 1347.7354Da,
1619.8223 Da, 2093.0867 Da, 2465.1983 Da, and
3147.4710 Da. Spectra were coadditions of 1200–2000
laser shots. Acquired spectra were exported from ABI
(Applied Biosystems) Data Explorer software as.txt files
and imported into mMass [49] for analysis. Markers used
to identify egg glair and yolk and animal glue as well as
contaminant keratin are listed in Table2.
LCMSMS
LCMSMS analyses were done on a ermo Q Exactive-
Orbitrap mass spectrometer interfaced with an Ulti-
Mate™3000 chromatographic system. e mass
spectrometer was operated in data dependent mode (full
scan 350-1800Da at a resolution of 70,000, top 10 ions
were selected for fragmentation at a resolution of 17,
500, detected peptides were placed on an exclusion list
for 10s.). e LC buffers were A: 0.1% formic acid and
B: 0.1% formic acid in ACN, and a gradient from 2–50%
B over 60min. was used. One micro-liter of each of the
digests that was analyzed by PMF was injected onto a
nanocolumn that was 75μm ID × 15 cm long, packed
with Reprosil AQ C18 with 1.9 um beads (Dr. Maisch,
Germany). e column had an integrated tip (pulled in-
house at Northeastern University by laser) with a tapered
internal ID of ~ 5–10μm. e column was connected to
the HPLC autosampler using a Pico View nESI source
(New Objective, Woburn, MA) and high voltage was sup-
plied to the back end of the column from the Pico View
source using a conductive tee.
LCMSMS data files were converted to mgf format
(mascot generic format, Matrix Science [50]) with
msConvert software from ProteoWizard Tools [51] and
searched on-line through Mascot. Mascot searches were
against the SwisProt and contaminants databases, tax-
onomy: Chordata, enzyme: trypsin, one missed cleavage,
and variable modifications: N, Q deamidation and M, K,
P oxidation. Peptide tolerance was 0.3Da and MSMS tol-
erance was 0.5Da.
Radiocarbon dating
None of the analytical techniques that detected the pres-
ence of egg coating could indicate whether the coating
was ancient or contemporary. As a result, samples from
Getty 74.AP.11 and Norton Simon F.1978.19.P were
submitted for 14C dating [52]. e two samples submit-
ted were small scrapings of the egg coating (~ 1mg total
each) obtained using a scalpel under a binocular micro-
scope. e samples were combusted under vacuum with
CuO at 900 °C in sealed quartz tubes, graphitized by
Fe-catalyzed hydrogen reduction [53], and measured by
Accelerator Mass Spectrometry (AMS) at the Keck AMS
facility at University of California, Irvine [54]. Given the
small sample sizes and the difficulty in separating egg
protein from possible proteinaceous contamination by
human keratin due to recent handling, no attempt was
made to chemically clean the samples or to isolate pro-
tein. Instead, the samples were simply combusted as
received and compared to results on 14C-free wood to
evaluate process backgrounds from the combustion and
graphitization. e rationale for this decision was that
since the mummies were not disturbed prior to excava-
tion in the 19th and early twentieth centuries [55], any
contamination would bias the samples to younger ages;
hence an old date would be convincing evidence that
the coating was applied prior to the mummy burial, i.e.,
during or shortly after the portrait painting, rather than
post-excavation.
UVF
Getty andNorton Simon
Images were captured using a Canon 80D 24.2 megapixel
modified camera with the UV-IR blocking filter removed,
hence providing UV–VIS-IR functionality. e camera
was outfitted with a Zeiss Milvus 50mm macro lens. As
control targets, a Labsphere Spectralon 99% reflectance
target and Passport Color Checker were used.
Two Wildfire Long throw series (365 nm peak) UV
radiation sources. UVF filters used were X-nite CC1,
Peca 916 or 918 and Kodak Wratten 2e filters.
Glyptotek
Images were captured using aCanon EOS 5D Mark II,
digital camera with a Tiffen 2A equivalent to Kodak
Wratten 2A-filter, 400nm UV-filter. Phillips UV bulbs.
A Gretag Macbeth Munsell Color was used as control
target.
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Page 7 of 18
Kirbyetal. Heritage Science (2023) 11:73
Vienna
Nikon D80 (10 Mpixel, APS-C sensor); Nikon lens AF D
Nikkor 28 – 105mm f/3,5–4,5
UV lamps: UVLS- 28 EL 365nm and Original Hanan,
Fluorotest 366nm.
Cleveland
1971.137 UVA visible fluorescence Camera: Canon
5DSR, 50mm lens with X-Nite CC1, PECA 918 and 2E
filters. Lighting: Two Wildfire long wave radiation lamps.
Materials
Ammonium bicarbonate (AMBI), trifluoro acetic acid
(TFA) and formic acid (FA) were reagent grade from
various suppliers and used without further purification;
HPLC-grade acetonitrile and water were from re-
moFisher Scientific; Sequencing Grade Modified Trypsin
was from Promega; α-Cyano-4-hydroxycinnamic acid
(CHCA): Sigma-Aldrich #476870-10g; Polystyrene strips
were from Walthers, www. walth ers. com; and aluminum
oxide polishing film with pressure sensitive adhesive was
from Precision Fiber Products, Inc., www. preci sionfi berp
roduc ts. com.
Experimental results
UVF andvisual examination
e paintings from which samples were analyzed—
a single sample from each—are shown in Fig.1, along
with UVF images of each overall painting. All of the
painted panels are mummy portraits except Getty
74.AP.20, which is a funerary portrait. Localized areas
on all showed generally the same surface appearances:
the coating layer looked visually like an irregular, dis-
tinct surface accretion that is often absent where the
wrappings would have secured the panel to its mummy.
Under magnification, the material can be described as
“dried islands of a yellowed, glassy accretion,” and it is
usually pooled in the recesses of both encaustic and
Fig. 3 Magnified surfaces from four of the portraits illustrating the characteristic surface appearance of the coating
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Page 8 of 18
Kirbyetal. Heritage Science (2023) 11:73
tempera paint, Fig. 3. A blue or bluish/yellow visible
fluorescence of the coating observed with ultraviolet
radiation further connected these portraits and assisted
with the identification of other potentially coated por-
traits. Often the coating had incrustations and surface
deposits (sand/dirt) suggesting that it was not restora-
tion material.
Cross sections from two of the paintings (Getty
74.AP.20 [12] and Glyptotek AE.I.N 684, Fig. 4) con-
firmed that the fluorescent material was definitely con-
centrated in a discrete surface layer.
Following the initial examination and analysis of the
four painted panels from Getty and Glyptotek, three
additional portraits displaying similar visual surface
characteristics were identified in the APPEAR database.
Based on the criteria described above, portraits from
the Cleveland Museum of Art (1971.137), Norton Simon
Museum (F.1978.19.P) and Kunsthistorisches Museum,
Vienna (X 297) were sampled and analyzed, Fig.1.
Initial analysis of four portraits from Getty and Glyp-
totek by GC/MS and/or ELISA [12, 13] indicated the
presence of egg, even though the media had been deter-
mined to be encaustic and should therefore exclude egg,
Table1. A cross section from paint on Getty 74.AP.20
indicated that egg was present as a coating and not part
of the paint [12], as did a cross section from Glyptotek
AE.I.N 684, Fig. 4. us, it was assumed that the egg
originated from a coating separate from the paint bind-
ing medium, and analyses by PMF and LCMSMS were
undertaken to further characterize the coating on the
Getty and Glyptotek portraits, as well as coatings on
Norton Simon, Vienna and Cleveland portraits that were
identified in the APPEAR database.
PMF analysis
Figure 5 (blue trace) is the PMF spectrum from coat-
ing sampled from Getty portrait 74.AP.11 compared
with unaged, whole egg reference (red trace). Many of
the ions in the Getty sample can be attributed to surface
contamination (unlabeled ions are mainly contaminat-
ing human epithelial keratin), but a number of ions are
similar to but not identical with those in egg glair and
yolk, which are indicated in the red trace by green circles.
Closer examination of the “near” matching ions led to
the realization that the Getty sample was whole egg but
likely highly deamidated. Several of the more intense egg
ions observed in both the coating and reference spectra,
Fig. 4 UVF photomicrograph of a cross section from Glyptotek AE.I.N
684 showing characteristic fluorescence of egg on the top layer
Fig. 5 PMF spectra from the coating on Getty 74.AP.11 (top) and whole egg reference (bottom). Egg yolk and glair markers (Table 2) are highlighted
with green circles in the lower spectrum
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Page 9 of 18
Kirbyetal. Heritage Science (2023) 11:73
along with their sequences as determined by LCMSMS,
are shown in Fig.6A–F. Sequences 6A–E, all of which
contain Q and/or N residues, are shifted to higher mass,
likely due to deamidation. Sequence 6F, which contains
neither Q nor N residues, is not shifted, supporting
deamidation as the source of the mass shifts in the other
spectra.
Deamidation is a chemical reaction of the amino acids
with an amide side group (asparagine, N, and glutamine,
Q) resulting in a 1Da increase in mass from the loss of
the amine side group and replacement with a carboxyl
side group [34]. Deamidation can originate from sev-
eral sources including age, chemical exposure, heat,
and humidity and does not necessarily proceed to 100%
completion, so there may be a mixture of amidated and
deamidated species present resulting in a complex mix-
ture and isotopic pattern. An example is shown in Fig.7
for GGLEPINFQTAADQAR (Fig. 6C) to illustrate the
interpretation of the isotopic pattern resulting from the
overlap of amidated and deamidated forms of the same
peptide.
Figure8 compares spectra from all seven portraits that
were analyzed by PMF. All exhibit both egg glair and
yolk markers with varying degrees of intensity, with the
exception of the Cleveland portrait, which has only egg
glair markers. All spectra show highly deamidated pep-
tides, as indicated by their isotopic patterns. Four ions
common to all seven samples, and which were usually the
most intense, are labeled.3 Because of their relatively low
intensity and the high keratin background, only the more
intense egg-related ions were usually observed, and glair
ions were the most abundant. PMF spectra are included
in Additional file3 as mMass (.msd) files. It should also
be noted that the egg here is specifically from chicken,
Fig. 6 Expanded spectra from selected ions in Fig. 5 (blue traces) and their sequences as determined by LCMSMS. Masses of the non-deamidated
peptides from whole egg reference are shown in the red traces. Isotopic envelopes from the sample for sequences with N or Q residues are
shifted to higher mass (A–E) whereas the sequence without N or Q residues is not (F). The sequence of the 1892.1 Da ion (E) observed in egg glair
reference (not shown) is unknown
3 e 1892Da ion cluster in Cleveland is from glair and is usually obscured by
the 1893Da cluster from yolk. See Fig.6E.
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Page 10 of 18
Kirbyetal. Heritage Science (2023) 11:73
Gallus gallus domesticus, a domesticated fowl used
mostly for egg and meat production and which is widely
distributed over the globe.
LCMSMS analysis
Digest aliquots from all seven portraits analyzed by
MALDI were subsequently analyzed by LCMSMS, and
the MSMS spectra were searched through Mascot to
corroborate the PMF identifications and their degree of
deamidation. Data from all portraits provided similar,
consistent results confirming the interpretation of the
PMF spectra as being indicative of highly deamidated,
whole chicken egg.
e presence of egg glair was indicated primarily by
identification of ovalbumin, lysozyme, and ovotransfer-
rin; egg yolk was indicated primarily by identification of
vitellogenin-1, vitellogenin-2, and apovitellenin-1. ese
were usually the main egg proteins found in the samples,
and, as noted with the PMF spectra, abundances varied
from sample to sample. Consistent with the PMF results,
both amidated and deamidated peptides were identified,
and high levels of contaminating human epithelial kera-
tin were also present.
e PMF spectrum from Getty 74.AP.20 was the only
result that indicated the presence of a trace of mamma-
lian collagen, consistent with ELISA and GC/MS results,
Table1. e LCMSMS results from all seven portraits,
however, indicated the presence of low levels of mam-
malian collagen, although the data were insufficient to
conclusively determine the species. e presence of col-
lagen is surprising in that, except for Getty 74.AP.20, the
portraits are encaustic. Collagen was observed in only
one PMF spectrum likely because of its low level and the
high levels of keratin contamination in all samples. Many
of the proteins identified by Mascot were contaminating
human epithelial keratin types, also consistent with the
high background observed in the PMF spectra.
LCMSMS data (.mgf format) and Mascot Summary
Reports for all samples are included in the Additional
file1, 2.
Mascot search results, examples
Table 3 is the Mascot summary of the ions used to
identify ovalbumin (egg glair, Gallus gallus) in Getty
74.AP.20, and it includes several of the ions shown in
Fig.6. As expected, based on the PMF data, the 1345Da
peptide (HIATNAVLFFGR), the 1688 Da peptide
(GGLEPINFQTAADQAR) and the 1859 Da peptide
(ELINSWVESQTNGIIR) show deamidation, whereas
the 2009Da peptide (EVVGSAEAGVDAASVSEEFR) ion
does not. Peptides are represented multiple times in the
table because of the short exclude time between MSMS
spectral acquisitions.
Figures9 and 10 are examples of MSMS spectra that
were used to identify ovalbumin in the Mascot analysis,
Table3. Figure9 is the ovalbumin 1687Da ion identified
Fig. 7 Isotopic pattern formed by the overlap of amidated and
deamidated species of the same peptide. From bottom to top, the
peptide GGLEPINFQTAADQAR with 0, 1, 2 and 3 deamidations and
the resulting overlapped spectra. Compare the top spectrum here
with Fig. 6C
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Page 11 of 18
Kirbyetal. Heritage Science (2023) 11:73
as non-deamidated GGLEPINFQTAADQAR, and Fig.10
is the ovalbumin1690Da ion identified as triply deami-
dated GGLEPINFQTAADQAR at N7, Q9 and Q14.
Table4 is the Mascot summary of ions used to identify
apovitellenin (egg yolk, Gallus gallus) in Getty 74.AP.11.
Consistent with the PMF data, both are deamidated.
Figures11 and 12 are examples of MSMS spectra used
to identify apovitellenin in the Mascot analysis, Table4.
Figure11 is the apovitellenin 1078Da ion identified as
NFLINETAR deamidated at N5. Figure12 is the apovi-
tellenin1893Da ion sequence identified as AGQFLLDVS-
QTTVVSGIR deamidated at Q3 and Q10.
14C analysis
Although visual examination of the portraits with egg-
containing coatings suggested that the coatings were not
from recent restorations, analyses of samples from them
provided no direct support for this conclusion. In order
to establish their age, samples of the coatings from two
paintings were analyzed by 14C dating.
e radiocarbon age for the whole hen egg coat-
ing on Getty portrait (74.AP.11) was reported as
UCIAMS-232793: 1935 ± 15 BP (14C years before AD
1950). e calibrated age range, determined using
the Calib 8.2 software with the IntCal20 dataset [56],
Fig. 8 PMF spectra from coatings on the seven funerary portraits. Labeled ion clusters were usually the most intense and were common to each
sample. Ions indicative of both egg glair and yolk were detected in all of these samples except for Cleveland, where only glair was observed
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 12 of 18
Kirbyetal. Heritage Science (2023) 11:73
Table 3 The Mascot summary of peptides used to identify ovalbumin (Gallus gallus) in the LCMSMS analysis of the sample from Getty
74.AP.20 including HIATNAVLFFGR, GGLEPINFQTAADQAR, ELINSWVESQTNGIIR and EVVGSAEAGVDAASVSEEFR shown in Fig. 6
As observed in the PMF spectra, HIATNAVLFFGR, ELINSWVESQTNGIIR and GGLEPINFQTAADQAR are deamidated whereas EVVGSAEAGVDAASVSEEFR is not, consistent
with the presence or absence of N and Q residues
Fig. 9 MSMS spectrum from the Getty portrait 74.AP.20 sample that identifies the non-deamidated ovalbumin 1688 Da ion sequence as
GGLEPINFQTAADQAR
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Page 13 of 18
Kirbyetal. Heritage Science (2023) 11:73
indicates a date of around 92 CE (median probabil-
ity), with a full 2 sigma (95% probability) age range of
25–128 CE. is date is consistent with the age range of
the portrait’s linden wood panel, 14C dated in the same
facility as UCIAMS-128007: 1925 ± 20 BP, correspond-
ing to a median probability calibrated age of 103 CE, 2
sigma calibrated age range 28–204 CE.
Fig. 10 MSMS spectrum from the Getty portrait 74.AP.20 sample that identifies the ovalbumin 1690 Da ion sequence as GGLEPINFQTAADQAR
deamidated at N7, Q9 and Q14
Table 4 The Mascot summary of peptides used to identify apovitellenin (Gallus gallus) in the LCMSMS analysis of the sample from
Getty 74.AP.11 including NFLINETAR and AGQFLLDVSQTTVVSGIR shown in Fig. 6
As observed in the PMF spectra, both are deamidated
Fig. 11 MSMS spectrum from the Getty portrait 74.AP.11 sample that identifies the apovitellenin 1079 Da ion sequence as NFLINETAR deamidated
at N1 and N5
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Page 14 of 18
Kirbyetal. Heritage Science (2023) 11:73
e radiocarbon age for the egg coating on the Nor-
ton Simon portrait (F.1978.19.P) was UCIAMS-237981:
1990 ± 20 BP, with a median probability calibrated age
of 24 CE and a full 2 sigma age range of 42 BCE-108
CE.
e dates established for both portraits support that
the application of the egg coatings is ancient and not a
recent intervention. Although the coatings on only two of
the portraits have been 14C dated, it is likely that the coat-
ings on the others were also applied in antiquity based
on their similar visual and UVF characteristics and the
detection of highly deamidated hen egg on all of them.
Discussion
Egg, binding media andcoatings inantiquity
e chicken was not native to Egypt and is believed to
have arrived from Asia via Europe around the seventh
century BCE, possibly earlier, in sporadic appearances
and disappearances [57, 58]. Artistic representation sup-
ports the presence of the chicken between 1425–1123
BCE [59], primarily for sport, such as cock fighting, or as
an auspicious symbol and sign of victory going into bat-
tle [60]. Due to a lack of archeological evidence (bones
and shells), it is thought that the chicken as a source of
food was only exploited much later. Pliny the Elder, AD
23/24—79, discusses both the use of chickens for cock
fighting, food and possible artificial hatching [61]. An
increase in both written and archaeological evidence
during the Roman period further supports the domesti-
cation of the chicken and egg not only as a source of food
but also as an industry in Egypt through the invention of
egg incubators [62].
e identification of ancient binding media is com-
plex, and the absence of published information or the
presence of dubious results to date supports this. ere
are a few comprehensive studies on the chemistry of egg
and numerous overviews of the use of egg, yoke or glair,
added to binders and used historically as a varnish [63–
65]. A 2016 compilation of paint media analysis from a
group of ancient paintings reported the identification of
egg, yoke or glair, typically mixed with glue on 12 out of
14 panels analyzed [66]. e use of egg glair as a binder
on medieval manuscript illuminations is well known [67].
Medieval treatises also mention that egg glair varnishes
were applied to prevent materials from sticking to freshly
painted surfaces providing protection from external
forces [65, 68]. is benefit might have been useful to the
ancient portrait painters before burying mummies in the
hot Egyptian dessert sand. Egg glair has been thoroughly
documented from the Renaissance to the nineteenth cen-
tury, primarily as a coating to provide luster and gloss
to painted surfaces. Various types of coatings have been
identified on late period funerary painted artifacts, such
as sarcophagi, including oils, pistachia spp.resins, pine
resins, and bitumen, alone or modified [6, 8, 69, 70].
While these unguents had a practical and aesthetic pur-
pose, their primary function was part of the funerary cer-
emony. e use of egg associated with this ritual has not,
to the authors’ knowledge, been documented to date.
e egg as a symbol of life and rebirth was of cos-
mic significance to the ancient Egyptians. Representing
renewal and everlasting light, the egg was believed to be
the center of the creation myth spanning into the Chris-
tian Era [71, 72]. e profound symbolism of the egg in
combination with esteemed ancient Egyptian funerary
practices may support the application of an egg coating
as a ritual function, thus providing compelling evidence
for its use beyond practical or aesthetic reasons. Our
Fig. 12 MSMS spectrum from the Getty portrait 74.AP.11 sample that identifies the apovitellenin 1893 Da ion sequence as AGQFLLDVSQTT VVSGIR
deamidated at Q3 and Q10
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Page 15 of 18
Kirbyetal. Heritage Science (2023) 11:73
confirmation of the existence of this intentional coat-
ing may also provide clues to how ancient artists worked
and define workshop and/or regional methods. As we
continue to characterize materials with sensitive analyti-
cal techniques, we will gain more insights into when and
how such coatings were applied and a better understand-
ing of the ancient artistic and ceremonial customs of
which they were a part.
Unexplained observations
LCMSMS data consistently point to the presence of
low levels of animal glue in the coating. Whether this is
admixed material from a ground, although unlikely based
on the thickness of the paint layers, or it was intentionally
added to the egg mixture, cannot be determined from
the present data. Its consistent appearance in all sam-
ples included in this study, however, may argue against it
being due to a later conservation intervention.
e egg coating on all seven portraits examined here
was highly deamidated. While these coatings are all
believed to be ancient, and age can be a factor in deami-
dation, the extent of deamidation still seems out of place,
especially when the underlying paint layers are visually
unaffected. Further study will be needed to determine the
exact circumstances behind the coatings’ physical and
chemical characteristics.
Finally, the addition of the coating after the portrait has
been affixed to its mummy is significant, but unexplained.
If the coating were applied to the entire panel as part of
its manufacture, it might suggest a protective “varnish.”
However, application after insertion into the mummy
broadens the possibilities of its function or intent, and at
this point we can only speculate as to what the original
purpose might have been.
Conclusions
is study has focused on the characterization of a
unique coating observed on a group of Romano-Egyptian
funerary portraits. e possible presence of such a coat-
ing can be strongly suggested by close visual observation,
or more importantly, by observation of UVF. A unique
feature of the coatings on the mummy portraits is that
they seem to terminate where the mummy wrappings
secured the panels in place, suggesting that the coating
was applied after the portrait had been placed on the
mummy. e coating on six funerary portraits confirmed
the presence of deamidated whole hen egg and on one
portrait, egg glair alone. As noted above, only a single
sample was analyzed from each painting so, whether the
glair-only result for the Cleveland portrait is representa-
tive of its overall coating cannot be determined without
additional sampling. Significantly, since the LCMSMS
data fully confirmed the interpretation of the PMF data,
future analyses will be possible with PMF alone.
e identification of whole hen egg is quite unusual as
egg has not to date been confirmed as the sole binder for
funerary portraits, and literature on historic egg coatings,
from well after ancient times, discusses only the use of
egg glair alone.
14C dating results from samples taken from two por-
traits confirm that their coating was applied approxi-
mately 2000years ago, most likely at the final stage of the
mummification ritual. While the 14 C date for only two
portraits does not confirm when the others in this group
were coated, the presence of deamidated hen egg on the
group studied may suggest that the coating was applied
in antiquity for those exhibiting similar characteristics.
Understanding the purpose of the coating remains
enigmatic. It is evident that hen egg was selected as a
material distinct from the common materials used for
painting the portraits. Whether this was a part of the
funerary ritual, or a protective measure to preserve the
painted portrait is uncertain, but, as more examples are
identified and dated, a better understanding of ancient
Egyptian funerary and artistic practices will be possible.
e work presented here required the collaboration
of a multidisciplinary team employing complementary
analytical techniques to recognize, identify, date and bet-
ter understand a discrete surface coating observed on a
group of funerary portraits from Egypt during the Roman
period. is study was made possible by our ability to vis-
ually compare portraits from various museum collections
through the APPEAR project as well as the opportunity
to pursue in-depth technical studies to confirm unique,
parallel features. is project provided the very rare
opportunity to discover and characterize the application
of a hen egg coating in antiquity. Although the compara-
tive data is not extensive—seven confirmed egg coatings,
six as whole hen egg and one as hen egg glair and two
with confirmed ancient dates—this study presents new
information about the manufacture of funerary portraits
and underscores the need for further investigations and
an expanded exploration of ancient coating materials and
practices. anks to analytical investigations, these stud-
ies are possible and “the history of ancient media is only
starting to be written.”
Photo credits
Ny Carlsberg Glyptotek
Portrait of a bearded young man AE.I.N 681 and Por-
trait of a bearded man. AE.I.N 684: Images: Maria Louise
Sargent.
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Page 16 of 18
Kirbyetal. Heritage Science (2023) 11:73
J. Paul Getty
Mummy Portrait of a Bearded Man, Romano-Egyptian,
AD 150–170. Encaustic on linden panel, 37 × 21 cm (14
9/16 × 8 1/4 in.). Los Angeles, J. Paul Getty Museum,
74.AP.11.
Portrait of a Bearded Man, Romano-Egyptian, AD 100.
Tempera on panel,
36 × 37.5 × 0.3 cm (14 3/16 × 14 3/4 × 1/8 in.) Los
Angeles, J. Paul Getty Museum, 74.AP.20.
Norton Simon
Portrait of a Man, second century AD. F.1978.19.P.
Encaustic on wood, 12–1/8 × 6–5/8 in. (30.8 × 16.8cm).
e Norton Simon Foundation, Gift of Mr. Norton
Simon.
Cleveland
Funerary Portrait of a Young Girl, c. AD 25–37. Egypt,
Roman Empire, late Tiberian. Encaustic on wood; over-
all: 39.4 × 17.4 cm (15 1/2 × 6 7/8 in.). e Cleveland
Museum of Art, John L. Severance Fund 1971.137.
For the overall and UV images.
© Elena Mars, courtesy of e Cleveland Museum of
Art.
Photomicrograph
© Colleen Snyder and Dean Yoder, courtesy of e Cleve-
land Museum of Art.
Vienna
Portrait of a Lady, Romano-Egyptian, AD 117–138.
er-Rubayat. Encaustic on wood, 40 × 20 cm (15 ¾ × 7
7/8 in.). Vienna, Kunsthistorisches Museum, Antiken-
sammlung, X 297. KHM-Museumsverband.
Abbreviations
AMS Accelerator mass spectrometry
APPEAR Ancient Panel Painting: Examination, Analysis and Research
Project
ELISA Enzyme-linked Immunosorbent assay
GC/MS Gas chromatography coupled with mass spectrometry
LCMSMS Liquid chromatography coupled with tandem mass spectrometry
PMF Peptide mass fingerprinting
ZooMS Zooarchaeology by mass spectrometry
MALDI Matrix assisted laser desorption-ionization mass spectrometry
UVF Ultraviolet-induced visible fluorescence
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s40494- 023- 00908-5.
Additional le1: Mascot Summary Reports.
Additional le2: LCMSMS .mgf files.
Additional le3: mMass .msd files.
Acknowledgements
The authors would like to thank the institutions who generously allowed sam-
pling of their portraits to make this study possible: the Norton Simon Museum;
Cleveland Museum of Art; Kunsthistorische Museum, Vienna; Ny Carlsberg
Glyptotek, J. Paul Getty Museum. DPK thanks Sue Abbatiello, formerly North-
eastern now Waters Corp. and Richard Newman for technical support and
encouragement. MS thanks Richard Newman, Salima Ikram, Kathlyn Cooney,
Alessia Amenta and Tamar Hodos for sharing their valuable knowledge of egg,
and Susanne Gänsicke and Michael Schilling for their support.
Author contributions
Conceptualization, preliminary investigation and imaging, MS, LRS; methodol-
ogy and data analyses, JM, DK, JS; writing—original draft preparation DK, MS;
writing review and editing, DK, MS, JM, LRS, JS. All authors read and approved
the final manuscript.
Funding
Not applicable.
Availability of data and materials
All data generated or analyzed during this study are included in this published
article and its additional files.
Declarations
Competing interests
The authors declare no competing interest.
Received: 21 December 2022 Accepted: 15 March 2023
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