A multidisciplinary study of archaeological grape seeds.

Abstract

We report here the first integrated investigation of both ancient DNA and proteins in archaeobotanical samples: medieval grape (Vitis vinifera L.) seeds, preserved by anoxic waterlogging, from an early medieval (seventh-eighth century A.D.) Byzantine rural settlement in the Salento area (Lecce, Italy) and a late (fourteenth-fifteenth century A.D.) medieval site in York (England). Pyrolysis gas chromatography mass spectrometry documented good carbohydrate preservation, whilst amino acid analysis revealed approximately 90% loss of the original protein content. In the York sample, mass spectrometry-based sequencing identified several degraded ancient peptides. Nuclear microsatellite locus (VVS2, VVMD5, VVMD7, ZAG62 and ZAG79) analysis permitted a tentative comparison of the genetic profiles of both the ancient samples with the modern varieties. The ability to recover microsatellite DNA has potential to improve biomolecular analysis on ancient grape seeds from archaeological contexts. Although the investigation of five microsatellite loci cannot assign the ancient samples to any geographic region or modern cultivar, the results allow speculation that the material from York was not grown locally, whilst the remains from Supersano could represent a trace of contacts with the eastern Mediterranean.

Full text

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ORIGINAL PAPER
A multidisciplinary study of archaeological grape seeds
Enrico Cappellini & M. Thomas P. Gilbert & Filippo Geuna & Girolamo Fiorentino &
Allan Hall & Jane Thomas-Oates & Peter D. Ashton & David A. Ashford & Paul Arthur &
Paula F. Campos & Johan Kool & Eske Willerslev & Matthew J. Collins
Received: 29 May 2009 /Revised: 6 August 2009 /Accepted: 18 November 2009 /Published online: 23 December 2009
# The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract We report here the first integrated investigation
of both ancient DNA and proteins in archaeobotanical
samples: medieval grape (Vitis vinifera L.) seeds, preserved
by anoxic waterlogging, from an early medieval (seventh–
eighth century A.D.) Byzantine rural settlement in the
Salento area (Lecce, Italy) and a late (fourteenth–fifteenth
century A.D.) medieval site in York (England). Pyrolysis
gas chromatography mass spectrometry documented good
carbohydrate preservation, whilst amino acid analysis
revealed approximately 90% loss of the original protein
content. In the York sample, mass spectrometry-based
sequencing identified several degraded ancient peptides.
Nuclear microsatellite locus (VVS2, VVMD5, VVMD7,
ZAG62 and ZAG79) analysis permitted a tentative com-
parison of the genetic profiles of both the ancient samples
with the modern varieties. The ability to recover micro-
satellite DNA has potential to improve biomolecular
analysis on ancient grape seeds from archaeological
contexts. Although the investigation of five microsatellite
loci cannot assign the ancient samples to any geographic
region or modern cultivar, the results allow speculation that
the material from York was not grown locally, whilst the
remains from Supersano could represent a trace of contacts
with the eastern Mediterranean.
Data deposition note All the mass spectrometric raw data associated
with this manuscript may be downloaded from ProteomeCommons.
org Tranche, http://tranche.proteomecommons.org, using the
following hash: wY9mO2A9E/dcKFY7ZzWvX4Aa0Bi
MqU9G3BPyhvsMEUI9x3o7fls8thTmEts0CtbOrQrenfWw3vUPJwfz
k0DtPJZHFxgAAAAAAA+RnA== or this link: http://www.
proteomecommons.org/data-downloader.jsp?fileName=
wY9mO2A9E/dcKFY7ZzWvX4Aa0BiMqU9G3BPyhvsMEUI9x3o
7fls8thTmEts0CtbOrQrenfWw3vUPJwfzk0DtPJZHFxgAAAAAAA%
2BRnA==. The hash may be used to prove exactly what files were
published as part of this manuscript's dataset, and the hash may also
be used to check that the data have not changed since publication.
Electronic supplementary material The online version of this article
(doi:10.1007/s00114-009-0629-3) contains supplementary material,
which is available to authorized users.
E. Cappellini (*):M. J. Collins
Departments of Biology, Archaeology and Chemistry, BioArCh,
University of York,
P.O. Box 373, YO10 5YW York, UK
e-mail: ecappellini@googlemail.com
M. T. P. Gilbert:P. F. Campos:E. Willerslev
Natural History Museum of Denmark, University of Copenhagen,
Øster Voldgade 5-7,
DK1350 Copenhagen, Denmark
F. Geuna
Dipartimento di Produzione Vegetale, University of Milan,
Via Celoria 2,
20133 Milan, Italy
G. Fiorentino:P. Arthur
Dipartimento di Beni Culturali, University of Salento,
Via D. Birago 64,
73100 Lecce, Italy
Naturwissenschaften (2010) 97:205–217
DOI 10.1007/s00114-009-0629-3

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Keywords Archaeobotany.Grape.AncientDNA.Ancient
proteins.Massspectrometry.Proteomics.Vitis vinifera
Introduction
Wine represents one of the most important products of plant
domestication during the development of human civilisa-
tion in western Eurasia. It is intimately bound up with the
expansion of agriculture, trade and commerce and is also
important in socioreligious, cultural and political aspects of
many societies, particularly around the Mediterranean.
Winemaking enabled humans to produce a beverage which
was the most widespread drug and medicine of antiquity, a
drug upon which economies and communities were built
(Bassermann-Jordan 1975; Olmo 1976).
Archaeobotanical evidence of Vitis sp. consists principally
of waterlogged, mineralised and charred seeds. Morpholog-
ical differences can only tentatively distinguish wild and
cultivated subspecies (Mangafa and Kotsakis 1996). Further-
more,inmanycases,thisdiscriminationreliesmoreonindirect
evidence, such as the absence of wild Vitis in the geographic
area, or on the archaeological context and the time period.
Vitis seeds and in particular those that are waterlogged store
biomolecular information, which can provide greater detail on
viticulture diffusion and the wine trade.
Genetics have advanced our understanding of crop
domestication, for example, studies of modern grapes have
revealed changes brought about by human selection
(Meredith 2001; This et al. 2006; Arroyo-García et al.
2006; Vouillamoz and Grando 2006). Ancient DNA
(aDNA) recovered from archaeological plant residues has
also begun to play a role by shaping our understanding of
past grape diffusion (Manen et al. 2003). With their
morphology and structure specifically designed to store
genetic information, seeds are a promising target for studies
on ancient biomolecules, in some cases germinating after
centuries (Sallon et al. 2008). In grape seeds in particular,
protection is further enhanced by the hard coat and the high
concentrations of antioxidant molecules (Yilmaz and
Toledo 2004). Remarkable preservation of lipids and
nucleic acids has been reported in 1,400-year-old radish
(Raphanus) seeds by O'Donoghue et al. (1996). Previous
work on blackberry (Rubus fruticosus) seeds and crab apple
(Malus silvestris) pips from York revealed that the
preservation is good for lignin and cellulose but only
limited for proteins (McCobb et al. 2001).
In this study, we have undertaken a combined DNA/
carbohydrate/protein analysis of several ancient grape
seeds. We explored the survival of polymerase chain
reaction (PCR) amplifiable nuclear DNA (nuDNA) using
five microsatellite loci in order to tentatively compare
ancient samples with modern grape varieties. Such infor-
mation can help to clarify ambiguities about indigenous
cultivation and commercial trade of grapes and wine in two
different archaeological contexts: a seventh–eighth century
A.D., Byzantine rural settlement in the ‘heel’ of the Italian
peninsula, near the town of Supersano (Salento area, Lecce,
Italy; Arthur 1999, 2004; Arthur et al. 2008), and a late
medieval, fourteenth–fifteenth century A.D., site on Low
Petergate, in York city centre (northern England; Akeret et
al. 2006; Reeves 2006). The present-day climate in York is
unsuitable for outdoor grape cultivation, but the chronology
of the site, between the Medieval Warm Period and the
Little Ice Age (Lamb 1965; Bradley et al. 2003), does not
completely exclude the possibility that viticulture was
practised in the area at that time.
Considering that in ancient samples DNA survival may be
relatedtogeneral biomolecularpreservation,wealsoanalysed
carbohydrates and proteins from the same samples of
archaeological grape seeds, adopting multiple approaches.
Pyrolysis gas chromatography mass spectrometry (py-GC/
MS) was used to investigate carbohydrate preservation.
Protein degradation was estimated observing amino acid
composition and racemisation and by direct sequencing of
preserved peptides.
The protein component represents an important indicator
of seed and grain quality. Pioneering studies on archaeo-
logical cereal seeds (Derbyshire et al. 1977; Shewry et al.
1982) suggested that the investigation of proteins from
ancient grains could aid in understanding of plant domes-
tication, but this work was hampered by the prevailing
technology of that time. However, the application of mass
spectrometry to fossil bone samples has enabled the identifi-
A. Hall
Department of Archaeology, University of York,
P.O. Box 373, YO10 5YW York, UK
J. Thomas-Oates
Department of Chemistry and Centre of Excellence in Mass
Spectrometry, University of York,
P.O. Box 373, YO10 5YW York, UK
P. D. Ashton
Bioinformatics Laboratory, Technology Facility,
Departments of Biology,
University of York,
P.O. Box 373, YO10 5YW York, UK
D. A. Ashford
Proteomics and Analytical Biochemistry Laboratory,
Technology Facility, Departments of Biology and Centre of
Excellence in Mass Spectrometry, University of York,
P.O. Box 373, YO10 5YW York, UK
J. Kool
Institute for Geo- and Bioarchaeology, Faculty of Earth and Life
Sciences, VU University Amsterdam,
De Boelelaan 1085,
1081 HV Amsterdam, The Netherlands
206Naturwissenschaften (2010) 97:205–217

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cation and sequencing of single ancient proteins, namely
osteocalcin (Nielsen-Marsh et al. 2002, 2005; Humpula et al.
2007) and collagen (Asara et al. 2007; Schweitzer et al.
2009; but see also Buckley et al. 2008). Recently Solazzo et
al. (2008) were able to identify protein remains in archae-
ological potsherds, whilst Hollemeyer et al. (2008) used
matrix-assisted laser desorption/ionisation time-of-flight
(MALDI-TOF) mass spectrometry to analyse some frag-
ments of the 5,300-year-old Tyrolean mummy's clothing.
The integration of a range of technical improvements has
enabled rapid and sensitive identification of proteins present
in small and complex biological samples. To test the potential
of this ensemble of technologies for the analysis of archae-
obotanicalsamplesandtocharacterisetheeffects oflong-term
protein degradation processes, we have extracted and ana-
lysed the proteins from ancient grape seeds, using both
electrospray ionisation (ESI) and MALDI.
Materials and methods1
Modern and ancient sample description
Vitis vinifera L. modern seeds from well-characterised
grape cultivars ‘Cabernet Sauvignon’, ‘Cabernet Franc’,
‘Pinot Noir’, ‘Syrah’, ‘Cot’, ‘Chardonnay’ and ‘Croatina’
were collected in the Regional Agency for Services to
Agriculture and Forests (ERSAF) experiment collection in
Torrazza Coste, Pavia, Italy. Ancient grape seeds from the
Byzantine rural settlement in ‘Località Scorpo’ to the north
of the small town of Supersano (Lecce, Italy) were found on
July 2007 at the bottom of a 6 m-deep well. Associated
artefacts and radiocarbon dating (Arthur et al. 2008) suggest
that the well was backfilled between the seventh and eighth
centuries A.D. The lower part of the well was found to be
waterlogged and contained large amounts of preserved
organic remains, including hundreds of grape seeds (see
S2a). Ancient grape seeds from York, S2b, were preserved
by anoxic ‘waterlogging’ in the basal fill of a cobble-lined
pit, dated by artefacts to the fourteenth/fifteenth century,
identified during excavation of a series of medieval tene-
ments at 62-8 Low Petergate in York city centre.
Ancient DNA analysis
DNA was extracted from, and PCR was attempted on, nine
ancient grape seeds (three from York, six from Supersano).
The aDNA extractions and PCR setup were performed in a
dedicated ancient DNA laboratory at the University of
Copenhagen, where stringent measures are taken to prevent
contamination with modern sources of DNA. In addition to
the ancient samples, modern grape seeds were extracted in
a separate laboratory. To remove external contaminant
sources of DNA, the seeds were briefly washed in dilute
bleach solution (10% commercial strength) then rinsed in
analytical grade H2O. Following this, the seeds were
allowed to dry naturally, before being manually crushed.
The crushed grape seeds were then digested overnight at
55°C with agitation in 400 μL of a proteinase K-containing
digestion buffer (Gilbert et al. 2007). Post digestion, DNA
was purified from the mixture using two phenol and one
chloroform extractions. Subsequently, the final aqueous
layer was purified further and concentrated using a Qiagen
Qiaquick PCR cleanup column (Qiagen, Valencia, CA,
USA) following the manufacturer's guidelines. In the final
stage, the DNA was eluted from the silica filter in 50 μL
elution buffer after a 10-min incubation at room tempera-
ture. In addition to the nine ancient samples, four extraction
controls were performed to screen for contamination
derived from the laboratory, reagents or between extracts.
The grape extracts were subjected to PCR amplification
using five microsatellite markers (see S3). Initially, all
extracts were screened with marker VVS2 (Thomas and
Scott 1993) to assess for DNA survival. The results
indicated that good quality DNA was present in three of
the samples (two from York, one from Supersano). These
were subsequently PCR-amplified for four additional
microsatellites: VVMD5, VVMD7 (Bowers et al. 1996),
ZAG62 and ZAG79 (Sefc et al. 1999). PCR amplification
was performed in 25 μL reactions, using Amplitaq Gold
(Applied Biosystems). Each reaction contained 1 μL DNA
template, 2.5 mM MgCl2, 1 μL bovine serum albumin
(20 mg/mL), 0.2 mM deoxyribonucleotide triphosphates
(dNTPs), 0.2 μL Amplitaq Gold, 400 nM of each primer
and 1× PCR buffer. PCR parameters were as follows:
enzyme activation 95°C for 4 min, 40 cycles of 95°C for
15 s, 60°C for 30 s, 72°C for 30 s and final extension 72°C
for 7 min. PCR reactions incorporated one PCR blank
reaction for every three ancient samples. Following PCR,
5 μL of the products was visualised on 2% Tris–acetate–
ethylenediaminetetraacetic acid agarose gels stained with
ethidium bromide. The results gave no indication of any
contamination, whether in the extraction or PCR controls.
Successful amplicons were subsequently purified using
Qiaquick kit (Qiagen) then cloned using the Topo TA
cloning system (Invitrogen, Carlsbad, CA, USA), and up to
16 clones were sequenced by a commercial facility (Macro-
gen, Korea). In addition to the cloned amplicons, an
additional amplicon was generated by PCR for each
marker, from each of the three extracts. Following gel
confirmation and Qiagen purification, using Qiaquick kit
(Qiagen), these PCR amplicons were sent to Milan for
capillary short tandem repeat (STR) analysis.
1Further details of the methods used in this work are provided in S1
as electronic supplemental material (ESM).
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Capillary STR analysis was performed on a 1,000-fold
dilution of PCR products received from Copenhagen. All
PCR reactions were carried out in a 20-μL volume using a
PTC-100 (MJ Research, Waltham, MA, USA) thermocy-
cler. Each reaction contained 1 μL DNA template, 2.5 mM
MgCl2, 0.2 mM dNTPs, 1 U Platinum Taq Polymerase
(Invitrogen), 200 nM each primer and 1× PCR buffer
(Invitrogen). PCR parameters were as follows: initial
denaturation 95°C for 2 min, 35 cycles of 95°C for 30 s,
60°C for 30 s, 72°C for 30 s and final extension 72°C for
30 min. Capillary electrophoresis was performed on an
ABI 310 Genetic Analyser (Applied Biosystems). Samples
(1 μL in 12 μL highly deionised formamide) were injected
at 15 kV for 5 s, and separation was performed at 15 kV
and 60°C during 28 min. Data were collected with data
collection software and analysed by GeneMapper 3.7
software (Applied Biosystems, Foster City, CA, USA).
GeneScan-500 (250) standard fragments in the size range
of 75–350 bp were used for the calculation of relative
sizes of microsatellite alleles. It has been well documented
that the adoption of different protocols in different
laboratories can result in perceived size differences of as
much as 5 bp for identical microsatellite alleles (This et al.
2004). To counter against this, for each capillary run,
possible microsatellite size shift due to the experimental
procedure adopted was corrected comparing the size of
the microsatellites of the ancient samples with standards
from the seven modern reference grape cultivars,
reported above. Runs were repeated at least in triplicate
for each sample until the best peak height and resolution
were attained. The cloning procedure followed by
amplicon sequencing, commonly adopted as a good
practice procedure in ancient DNA investigation, also
allowed confirmation of the size of the alleles. When
allele sizes at one or more loci diverged from the odd-
even profile shown by the reference accessions, both the
original and the corrected values (allele size −1 base)
were used for both the GeneClass2 and the genetic
distance calculations.
Area-specific allele frequencies were used in a likelihood-
based assignment test (Paetkau et al. 1995) that calculates
the probability of each individual's assignment to a
particular area as the cumulative products of each allele's
frequency of occurrence in any of several European areas
under examination. We generated significance levels for
individuals that cross-assigned to a neighbouring area
using the simulation routine within GeneClass 2.0 soft-
ware (Paetkau et al. 2004; Piry et al. 2004). Significance
levels were determined by comparing the individual
genotypes of cross-assigned individuals with a simulated
set of 10,000 genotypes that were generated using area-
specific allele frequencies using a type I (alpha) error
threshold set at 0.01.
In addition, genetic distance and cluster analyses were
performed to compare the three ancient samples with a set
of modern accessions representative of grapevines cultivat-
ed in Europe. The reference dataset consisted of 167
accessions described in the database of Sefc et al. (2000),
also available at http://www.dagz.boku.ac.at/15271.html.
Loci VVS2, VVMD5, VVMD7, ZAG62 and ZAG79 were
included in the analysis. Distance matrices were calculated
by the programme GenAlEx 6.1 (Peakall and Smouse
2006) using the ‘codominant’ and the ‘total distance only’
options. Cluster analysis was performed by the Neighbour
programme of the PHYLIP package (Felsenstein 2005)
using the neighbour-joining (Saitou and Nei 1987) algo-
rithm. Dendrograms were plotted by the NJplot 2.2
programme (Perrière and Gouy 1996).
Amino acid analysis and Py-GC/MS
A 10.41 mg of powdered modern sample, 4.93 mg of
ancient material from York and 1.18 mg from Supersano
were analysed to evaluate amino acid composition and
racemisation rate following the procedure previously
reported (Penkman et al. 2008). The York sample was
analysed using Curie point Py-GC/MS. The sample was
ground and compressed onto a ferromagnetic wire using a
hydraulic press. The temperature of the pyrolysis unit was
set at 250°C. After heating to a temperature of 600°C for
5 s, the pyrolysis products were separated on a Fison
Instruments GC8060 gas chromatograph fitted with a
fused silica capillary column (25 m×0.25 mm ID) with a
CP-SIL 5CB-MS coating, film thickness 0.40 μm. The GC
oven was set at 40°C and was heated to 320°C at a rate of
7°C/min; finally, it was kept isothermal for 15 min. The
GC was connected to a Fison Instruments MD800
quadrupole mass spectrometer, operating at 70 eV and
scanning the m/z range 45–650 at a cycle time of 1.5 s.
Helium was used as the carrier gas with a constant flow
rate of 0.7 mL/min.
Mass spectrometric protein analysis
Three pools, made of six modern grape seeds, six ancient
seeds from Supersano and four ancient seeds from York
were powdered in liquid nitrogen; 136.81 mg of the modern
sample, 98.16 mg of the ancient one from York and
141.24 mg from Supersano were extracted adopting the
phenol-based protocol number four reported by Vincent et
al. (2006), with minor changes. Since it was not possible to
recover a pellet from the Supersano sample, this pool was
not processed further. The other pellets were resuspended in
50 mM ammonium bicarbonate pH 8.00 and were digested
overnight at 37°C using Sequencing Grade Modified
Trypsin (Promega Corporation, Madison, WI, USA).
208 Naturwissenschaften (2010) 97:205–217

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Three microlitres of the digest solution was loaded onto
an Ultimate micro-capillary high-performance liquid chro-
matography (HPLC) system; the eluate was on-line mixed
with a matrix solution containing α-cyano-4-hydroxy
cinnamic and then collected onto a MALDI sample plate.
Plates were loaded into an AB 4700 Proteomics Analyser
TOF/TOF (Applied Biosystems, Foster City, CA, USA),
mass spectrometer operated in positive ion reflectron mode.
The same amount of the digest solution was also loaded
onto a micro-capillary HPLC system interfaced with a
QSTAR API Pulsar LC-MS/MS System (Applied Biosys-
tems) with a Micro Ion Spray source. Positive ESI-MS and
MS/MS spectra were acquired using information dependent
acquisition. Peak lists generated by the software associated
with the mass spectrometers were searched using the
Mascot 2.2 algorithm (Matrix Science Ltd., London, UK),
in December 2007, after the grape genome was made
publicly available (Jaillon et al. 2007; Velasco et al. 2007).
Post-translational modifications, semi-tryptic peptides and
single amino acid substitutions were searched for by
selecting the Mascot automatic error tolerant database
search option. Each tryptic digest solution was labelled
using iTRAQ reagents (Applied Biosystems), reporter mass
114 for the modern and 116 for the ancient sample, and
analysed on the MALDI mass spectrometer following the
manufacturer’s instructions. The quantitative proteomic
approach has been used very recently on modern grape
samples (Lucker et al. 2009).
Results
Protein and carbohydrate preservation
Py-GC/MS analysis demonstrated that lignin- and cellulose-
derived compounds are present in the coat of the York seeds
(Fig. 1). The catechyl and guaiacyl compounds detected are
pyrolytic breakdown products of the lignin structure of the
seed coat. The data confirm that V. vinifera L. has no
syringyl compounds in its lignin structure. Within the early-
eluting compounds, there are some indicators of the presence
of sugars, e.g. 3-hydroxy-2-methyl-2-cyclopenten-1-one, in
the sample, as well as some compounds linked to the
presence of proteins, e.g. toluene and phenol (Ralph and
Hatfield 1991).
Amino acid analysis documents a high level of biomo-
lecular degradation for the Supersano samples. Compared to
modern grape seeds, the samples from Supersano have lost
87% of their amino acids, slightly less than the York seeds
(89%).Thelevelsofasparticacidandasparagine(Asx=Asp+
Asn) racemisation, however, are higher in the Supersano
sample, D/L Asx 0.17 (n=3, SD=0.01), than in the York
sample, D/L Asx=0.13 (n=3, SD=0.01).
A more detailed characterisation of the proteinaceous
residues was achieved using a phenol-based protocol to
extract proteins from three sample pools generated on
powdering (a) six modern seeds, (b) six ancient seeds
from Supersano and (c) four ancient seeds from York. It
was possible to extract approximately 62.5 mg of
proteins per gram of modern grape seed (6.25% by
wt), as estimated by Bradford assay and confirmed by
amino acid analysis. The medieval York sample
contained an order of magnitude less protein; no protein
pellet was recovered from the Supersano sample. The
sodium dodecyl sulfate polyacrylamide gel electropho-
retic (SDS-PAGE) profile of the protein pellet from the
York sample revealed a 23-kDa band with other more
diffuse bands between 6 and 14 kDa (Fig. 2), all
emerging from a continuous background smear.
In the absence of a sedimented protein pellet from the
Supersano sample, nano-LC MS/MS was only performed
on the late medieval sample from York. Protein extracts of
the modern and the York samples were digested using
trypsin, isobarically labelled (iTRAQ), and the resulting
peptide mixtures were resolved using a micro-capillary
HPLC system for analysis on both MALDI TOF/TOF and
ESI quadrupole TOF mass spectrometers. The MS/MS peak
lists from the MALDI and ESI experiments were searched
using the Mascot search engine against the National Center
for Biotechnology Information protein database. Despite
the age of the sample, the quality of the mass spectra from
the ancient sample is, in some cases, remarkable. For the
storage protein peptide, for example, DSGFEYVAIK (see
Fig. 3b, c), it was possible to identify almost complete ‘y’
and ‘b’ ion series. However, due to the low protein
recovery, as documented by amino acid analysis, a total
of only six protein accessions, based on 30 unique peptide
attributions, were identified in the ancient sample (Table 1;
S5), representing a few percent of the total number of
proteins typically identified from modern dry seeds (Rajjou
1
5
7
8
10
9
15
14
18
23
24
29
35
23
6
11
12
13
16
17
1922
20
21
2830
27
31
3233
34
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
Time (min)
42
38 40
44
0
2.105
4.105
6.105
8.105
1.106
1.2.106
1.4.106
1.6.106
1.8.106
2.106
2.2.106
2.4.106
4
Intensity
26
25
Fig. 1 Total ion current of on-line flash pyrolysate of the seed coat of
the York samples (for compound identification, see S4 in ESM)
Naturwissenschaften (2010) 97:205–217 209

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et al. 2006; Chibani et al. 2006). 2S albumin was directly
identified (Li and Gray 2005), whilst a Basic Local
Alignment Search Tool (BLAST) search (see S1 on ESM)
allowed confident identification of different seed storage
and metabolic proteins: 7S and 11S globulins, peptidase A1
and a non-specific lipid transfer protein.
Quantitative evaluation shows that the seed storage
protein content in the ancient sample is 30–57% of the
values observable from modern samples, significantly higher
than for other proteins (6–20%). Error-tolerant searching of
the data obtained from the medieval sample allowed the
identification of a series of semi-tryptic peptides generated
by hydrolytic processes. A clear example is represented by
the detection of the fragments generated after cleavage of the
peptidic backbone next to five of the eight consecutive
glutamine (Q) residues in the tryptic peptide FYLAGNPQ
NEFQQQQQQQQGSEGQQQQQEGGGSEGR. The full-
length peptide was detected unaltered in the modern sample
and, in a very limited amount, in the ancient one (see S5 and
S6 on ESM). It also appears that, for the ancient sample, the
number of peptides matched is lower using ESI rather than
MALDI.
Ancient DNA
DNAwas successfully PCR amplified from two out of three
York seeds (here referred to as ‘York 1’ and ‘York 2’) and
one out of six Supersano seeds (here referred to as
‘Supersano’). The size of the microsatellites of the ancient
samples, reported in Table 2, are consistent with the size of
the amplicon sequences obtained after cloning the PCR
products (see S7).
The Supersano sample yielded a 228-bp allele at locus
VVMD5 that is not represented in the reference set, whilst
the two York samples showed the allele 201 bp at locus
ZAG62 which is shared by reference samples ‘Agiorgitiko’,
‘Dermatas’, ‘Akominato’ (Greek) and ‘Cortese’ (Italian).
For those loci with homozygous profiles, such as ZAG79
for both the York samples, it is not possible to exclude
undetected heterozygosis due to allele drop after degrada-
tion of the ancient genetic material. However, a certain
amount of homozygosis is likely considering grape her-
maphroditism and its consequent propensity for selfing.
GeneClass2 (Piry et al. 2004) was used to statistically
assign the archaeological specimens to European popula-
tions of V. vinifera cultivars using the database of Sefc et al.
(2000). The input data list contains all possible genotype
combinations per specimen as described in Manen et al.
(2003). The profile of the two York samples is identical
except for one of the two ZAG62 alleles. ‘York 2’ was
placed, by likelihood-based assignment testing (Paetkau et
al. 1995), in the Greek, Italian, Austrian/German and
French groups with probabilities of 0.106, 0.023, 0.019
and 0.013, respectively. Sample ‘York 1’ was placed only
in the Greek group with a probability of 0.020. No
assignment was possible for the ‘Supersano’ sample.
Cluster analysis placed the ancient samples next to each
other, on particularly long branches. The closest group of
modern cultivars includes the Spanish accessions ‘Ondar-
rabi Beltza’ and ‘Blanca Cayetana’, the Italian ‘Cortese’,
the Portuguese ‘Boal Ratinho’ and the Greek ‘Dafnia’,
‘Kristalli’, ‘Syriki’, ‘Dermatas’ and ‘Agiorgitiko’ (see
Fig. 4; S8 on the ESM).
Discussion
Biomolecular preservation
Py-GC/MS reveals the high abundance of both 4-methyl-
catechol and 4-methyl-guaiacol, considered indicators of
good preservation as the loss of methyl-side chains is
commonly associated to lignin degradation (van Bergen et
al. 1994). The abundance of the protein-derived compounds
is lower than is commonly seen in waterlogged archaeo-
logical seeds (McCobb et al. 2001; Johan Kool, unpub-
lished data).
Ancient protein degradation is also confirmed by the
diffuse smear present in the electrophoretic profile (Fig. 2),
as previously reported by Shewry et al. (1982), and by the
‘hump’ in the nano-LC chromatogram (cf. Fig. 3a with
Fig. 2 SDS-PAGE separation of archaeological grape seed proteins.
The proteins were resolved in a 4–12% (w/v) gradient acrylamide gel
and stained with colloidal Coomassie Blue. MW lane, molecular mass
markers. The molecular mass (in kilodaltons) of each protein is shown
on the left. Lane 1, ancient proteins isolated by phenolic extraction
from medieval grape seeds excavated in York. Lane 2, modern grape
seed proteins extracted under the same conditions
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inset of modern sample with flat baseline; Moreno et al.
2005). Most of the proteins identified by mass spectrometry
are storage proteins, which represent a source of carbon,
nitrogen and sulfur for the shoot. They are usually present
in large amounts in discrete deposits called protein bodies,
and their high initial abundance in the seeds is probably the
crucial factor for the persistence of residual fragments
amenable to identification after centuries. Storage proteins
are characterised by a high level of polymorphism, due to
the presence of multigene families and to post-translational
modifications (Shewry 1995; Shewry and Halford 2002).
Consequently, a mass spectrometry-based approach, investi-
gating storage protein isoform distribution, could be possibly
developed for species or sub-species attribution of archaeo-
logical seeds, integrating conventional DNA-based methods.
Preliminary results, documenting distinctive protein profiles
between grape seeds from different modern varieties, have
recently been reported (Pesavento et al. 2007).
The molecular investigation of this protein subset from
archaeological seeds enabled, for the first time, a more
detailed understanding of protein damage mechanisms over
extended time periods. The role of asparagine and gluta-
mine in protein ageing reactions clearly emerges. These
amino acids are subject to deamidation (Ritz-Timme and
Collins 2002), one of the most relevant hydrolytic protein
degradation mechanisms (Groenen et al. 1994). The
importance of deamidation/racemisation control for main-
tenance of seed viability is documented by the detection of
active protein L-isoaspartyl methyltransferase in seeds that
remained viable for centuries. This enzyme repairs L-
isoaspartyl residues accumulating in ageing proteins as a
consequence of deamidation/racemisation (Shen-Miller
et al. 1995; Shen-Miller 2002; Clarke 2003). We found,
in the ancient sample only, deamidation of Q3 in the
peptide LHQGAMVLPYYNVNAH and of Q12 in
SSVTGYDLPVLQK (see S5 and S9 in ESM). The
estimated in vitro deamidation half-time life for glutamine
in these contexts is more than 2 and 16 years, respectively
(Robinson et al. 2004). Although it is possible that some
diagenesis may occur as an artefact of laboratory prepara-
tion, the slow rates of these reactions convince us that they
represent real products of the prolonged action of time.
Fig. 3 Example of nano-LC/
tandem MS analysis results for
the archaeological grape seed
protein extract from York:
a nano-LC chromatogram of the
tryptic digest. The baseline in
the ancient sample is raised,
compared to the modern one
reported in the inset, indicating
the presence of degraded pro-
teinaceous material. b Mass
spectrum at the retention time of
14.3 min (m/z 650–4,400).
c MS/MS spectrum of m/z
1,128.59, which identified the
peptide DSGFEYVAIK, unique
to the unnamed protein product
gi|157350579 from V. vinifera
showing high homology,
BLAST score=379, with a seed
storage protein from Juglans
regia (accession no.
gi|56788031)
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Table 1 List of the ancient proteins extracted from medieval grape seeds excavated in York
Modern sample
York medieval sample
Quantification
BLAST homology attributiond
Accession
number
No. of
peptidesa
Coverage
%b
Mowse
scorec
No. of
peptidesa
Coverage
%b
Mowse
scorec
Ancient/
modern %
Accession
number
Description
Score
(bits)
E value
gi|157350579
12
54
723/627
10
37
442/262
29.8
gi|56788031
Seed storage protein (Juglans regia)
379
3.00 E−103
gi|157350582
11
57
684/488
8
33
299/149
57.0
gi|56788031
Seed storage protein (Juglans regia)
294
5.00 E−78
gi|157347968
16
42
709/357
6
11
585/59
7.6
gi|13183177
7S globulin (Sesamum indicum)
442
3.00 E−122
gi|147821120
7
27
511/282
3
8
178/-
18.9
gi|87240526
Peptidase A1, pepsin
(Medicago truncatula)
414
5.00 E−114
gi|30421130
4
35
369/52
2
15
203/-
0.5
N.A.
2S albumin precursor (Vitis vinifera)
N.A.
gi|157344576
3
30
134/109
3
30
77/-
9.0
gi|128378
Nonspecific lipid-transfer protein A
(phospholipid transfer protein)
126
3.00 E−28
Ancient proteins, extracted from medieval grape seeds excavated in York, identified by the Mascot search engine from the data produced by nano-LC/tandem MS
N.A. not applicable
aThe number of peptides reported is calculated as the sum of the unique peptides identified by ESI and MALDI together
bThe same approach was used to calculate the sequence coverage
cFor each protein, the Mowse scores from both MALDI, including quantitative data, and ESI experiments are reported
dThe proteins described as ‘unnamed protein product’ were identified by BLAST homology (see S1 on ESM for details). Quantitative evaluation was based on the data produced using stable
isotope isobaric labelling—iTRAQ
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Where multiple peptides from the same protein are
detected, it is also instructive to identify those with
atypically poor preservation. As an example, the quantita-
tive analysis showed, for the peptide VVPVNPALLNR, a
‘survival level’ of approximately 6% in the ancient sample,
two to four times lower than the other peptides from the
same protein. Asn residues form a heterocyclic succinimide
by attack on the amide of the C-terminally adjacent amino
acid, but in the case of the imine Pro, Asn-Pro cyclisation
leads to spontaneous cleavage of the peptide bond (Capasso
et al. 1996), as previously reported in fossil proteins
(McNulty et al. 2002). GNGYEETICSLR is another
peptide characterised by atypically low recovery in the
ancient sample. The Asn2-Gly3peptide bond is particularly
prone to cleavage, due to the limited steric hindrance of the
glycine residue affording little protection (Bada 1985;
Collins et al. 1998). Our results are consistent with the
proposal that specific positions represent ‘hot spots’ for
protein fragmentation (Dehart and Anderson 2007).
Racemisation and peptide bond cleavage are hydrolytic
reactions (Dal Monte et al. 2002), enhanced in conditions,
such as oxidative stress, that increase polypeptide chain
flexibility (Ingrosso et al. 2002). Oxidative damage of
ancient biomolecules is well documented (Höss et al. 1996)
and carbonylation in particular is the predominant alteration
recently detected in the Arabidopsis thaliana seed pro-
teome, after ageing treatments (Rajjou et al. 2008).
Carbonylation principally converts arginine and proline to
glutamic semialdehyde and lysine to aminoadipic semi-
aldehyde (Nyström 2005), removing from arginine and
lysine side chains the basic motif necessary for trypsin
substrate recognition. The limited tryptic peptide recovery
can thus represent an indicator of oxidative damage, despite
waterlogging and high levels of antioxidant compounds in
grape seeds. The investigation of molecular degradation in
relation to seed viability has practical application beyond
the realms of archaeology, for example, in the forestry
industry, and for the management of seed banks created to
protect plant genetic diversity.
Different preservation of proteins and DNA from the
two archaeological sites is to be expected, given the
considerably warmer climate at Supersano compared with
York, as anticipated by thermal age modelling (Smith et
al. 2001). Supersano (40.02 N, 18.24 W) has a thermal age
of 6kyr @10?C, an order of magnitude higher than that of the
York specimens0:6kyr @10?C
acid racemisation, protein degradation and DNA preser-
vation, however, do not appear to be closely linked. The
presence of aDNA—even nuclear microsatellites—in the
Supersano sample seems inconsistent with our inability to
obtain an insoluble protein extract and consequently
peptide sequences. Further method development is prob-
ably required to improve peptide recovery from archaeo-
logical samples.
??. The results from amino
SampleVVS2 (bp) VVMD5 (bp) VVMD7 (bp) ZAG62 (bp)ZAG79 (bp)
York 1
York 2
Supersano
132
132
130
140
140
142
230
230
228
232
232
228
240
240
246
246
246
248
201
187
197
201
201
197
244
244
246
244
244
248
Table 2 Microsatellite sizes as
determined by capillary
electrophoresis
Fig. 4 Detail of dendrogram, reported in full in S8, based on five
microsatellites, showing the position of the three ancient samples:
‘York 1’, ‘York 2’ and ‘Supersano’ relative to the closest accessions
from the database of Sefc et al. (2000). The bar corresponds to 1 U of
genetic distance
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Ancient DNA
The recovery of five microsatellite loci from ancient grape
seed samples demonstrates good nuclear DNA preservation
and is quite encouraging for future work. The variation in
cloned sequences, however, showed a certain degree of
microsatellite stutter, as expected for aDNA (Burger et al.
1999). The data also indicate that it is highly unlikely that
any of the results derived from contamination either
between samples or from external sources. Specifically, all
samples yielded identifiable grape sequences for all micro-
satellite loci, all samples differed at the markers and no
samples or extraction and PCR controls showed any
example of cross contamination. Although it has been
popular to demand independent replication of aDNA results
(Cooper and Poinar 2000), we subscribe to the view of
Gilbert et al. (2005) that aDNA studies should be validated
using a cognitive approach. As the controls showed no
evidence of contamination, the samples yielded clearly
grape DNA sequences, there was no evidence of any cross
contamination in the sequences and the data were generated
from multiple PCR reactions analysed in two different
ways, i.e. cloning plus sequencing and capillary analysis;
the results are unlikely to derive from contamination, thus
do not require independent validation.
The microsatellite data, however, allow just tentative
comparison of the ancient samples with the modern
varieties. Conventionally at least six and possibly 20
microsatellites are required to fully resolve the cultivar for
modern grape samples (Vouillamoz et al. 2003, 2006; This
et al. 2004). We acknowledge that the investigation of five
microsatellite loci is still not enough to clearly assign the
ancient samples to a geographic region or to a cultivar.
The low resolution resulting from so few microsatellites is
clearly observed in the tree, as closely related cultivars
(Bowers and Meredith 1997; Sefc et al. 1998; Vouillamoz
and Grando 2006) do not group. The results of this
analysis cannot be used to draw conclusions with regard to
the attribution of the ancient samples to modern cultivars
since clusters illustrate similarity rather than kinship or
identity (Karatas et al. 2007). Despite both size calibration
and correction for microsatellite shift, the allelic profiles
of the ancient samples were quite distant from those of all
the modern cultivars they were compared to. Ancient
sample assignment to any geographic region is law, and
their genetic distance from the modern varieties is high
(Fig. 4), suggesting that they either represent unsequenced
or extinct cultivars. Future analysis searching larger
databases may lead to greater precision. Because V.
vinifera is a hermaphrodite, seeds are mostly the result
of selfing; nevertheless, crosses cannot be excluded, and
consequently, analyses of ancient wood remains would
also be useful to characterise old cultivars and compare
their DNA profiles with those from modern ones (This et
al. 2006). Although tentative, the data from the Supersano
archaeological sample suggest shorter distance from the
modern Greek cultivars ‘Dermatas’, ‘Syriki’, ‘Kristalli’,
‘Dafnia’ and ‘Agiorgitiko’. The affinity with Greek
cultivars may suggest grape or wine trading between
southern Italy and the eastern Mediterranean or perhaps
cultivation of similar varieties in both the areas during the
Byzantine period, or even earlier.
All the cultivars in the group closer to the York
archaeological seeds are from regions in the south of
Europe, with no particular resistance to cold environments.
Despite the extreme latitude, viticulture in York cannot be
excluded during the ‘Medieval Warm Period’ (about 1100–
1300 A.D.; Lamb 1965; Seward 1979, p. 128; Goosse et al.
2006). However, assuming the York grape sample belongs
to a variety with climatic requirements similar to those of
the modern cultivars it clusters closer to, a more plausible
explanation than local viticulture is that the seeds were
imported from southern growing areas as dried raisins.
Historical records document consistent import of raisins
and wine from southern Europe into England for centuries
starting from the twelfth century A.D. (Salzmann 1931, p.
410; Seward 1979, p. 135). Also, the lack in the York
context of conventional archaeological evidence, such as
tools or dedicated structures associated to winemaking
activity, is consistent with this option. Further integration of
aDNA and classical pollen data from Roman and medieval
sites in southern areas of Britain could help to clarify the
dynamics of viticulture and winemaking in this region
(Unwin 1990; Brown et al. 2001).
Conclusions
In addition to observing good preservation of the lignin-
and cellulose-derived compounds (as detected by Py-GC-
MS), peptide sequencing allowed us to identify several
ancient proteins and to characterise chemical changes. We
determined that the highly abundant seed storage proteins
were affected by hydrolytic damage and that specific motifs
are particularly prone to cleavage. The finding also defines
the basis for the development of a proteomic approach for
species or sub-species attribution of archaeological seeds to
integrate DNA-based methods.
Archaeological grape seeds have several traits that make
them attractive for future studies on the domestication and
diffusion processes of this important crop. The data suggest
that the material from York was not grown locally, whilst
the remains from Supersano evoke relations with the
eastern Mediterranean. Successful genetic analysis of five
microsatellite markers enabled a tentative comparison of the
ancient profiles with the modern varieties, providing
214Naturwissenschaften (2010) 97:205–217

Page 11

indications to corroborate archaeological hypotheses. This
is a significant achievement with promise of much more
with the integration of high-throughput sequencing (Poinar
et al. 2006; Green et al. 2006; Gilbert et al. 2008) for both
ancient proteins and DNA.
Acknowledgements
Natural Environment Research Council ‘Palaeoproteomics’ grant (no.
NE511148) allotted to MJC and JTO. EC and MJC would like to
acknowledge Dr. Kirsty Penkman and Dr. Caroline Solazzo for
technical support. PFC was supported by the ‘Genetime’ European
Marie Curie Training Network. JK would like to acknowledge Eef
Velthorst for help with Py-GC/MS analysis and Wageningen Univer-
sity for access to their equipment. The Centre of Excellence in Mass
Spectrometry at York, chaired by JTO, is supported by Science City
York and Yorkshire Forward, using funds from the Northern Way
Initiative. The authors express their gratitude to Prof. Raffaele Testolin
of the University of Udine-Italy for the helpful discussion about the
genetic data.
EC, JTO and MJC were supported by the
Open Access
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
This article is distributed under the terms of the
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