Journal of Archaeological Science

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1 Journal of Archaeological Science 36 (2009) Contents lists available at ScienceDirect Journal of Archaeological Science journal homepage: Extraction and sequencing of human and Neanderthal mature enamel proteins using MALDI-TOF/TOF MS Christina M. Nielsen-Marsh a, Christin Stegemann b, Ralf Hoffmann b, Tanya Smith a,c, Robin Feeney a, Michel Toussaint d, Katerina Harvati a, Eleni Panagopoulou e, Jean-Jacques Hublin a, Michael P. Richards a,f, * a Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, Leipzig, Germany b Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, Leipzig, Germany c Department of Anthropology, Harvard University, 11 Divinity Avenue, Cambridge, MA 02138, USA d Direction de l Archéologie, Ministère de la région Wallone, 1 Rue des Brigades d Irlande, 5100 Namur, Belgium e Ephoreia of Palaeoanthropology-Speleology of Southern Greece, Ardittou 34b, Athens, Greece f Department of Anthropology, University of British Columbia, Vancouver BC, Canada V6T 1Z1 article info abstract Article history: Received 22 December 2008 Received in revised form 29 March 2009 Accepted 3 April 2009 Keywords: Proteomics Enamel Neanderthal MALDI-TOF-TOF We report here the first results of a method to extract and sequence mature enamel proteins from modern human and Neanderthal tooth enamel. Using MALDI-TOF/TOF mass spectrometry and a combination of direct sequencing and peptide mass mapping we have sequenced a peptide from the tyrosine-rich amelogenin peptide (TRAP) of the X isoform of the amelogenin protein for modern and recent human samples. We also report our results from two Neanderthal enamel samples where we were also able to recover fragments of the TRAP protein, which had a similar sequence to the modern human samples. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Proteins and DNA, when successfully extracted from ancient skeletal remains, can be used to provide information on the climate, genetics, diet and ecology of ancient humans and Neanderthals and associated animals (Lee-Thorp and Sponheimer, 2006; Nielsen- Marsh et al., 2005; Lalueza-Fox et al., 2006). However, despite some notable successes, neither collagen, the most abundant protein in bone, nor DNA, survive well in the burial environment and studies relying on these molecules to provide usable biomolecular information are frequently limited by both time and location. Though the relatively recent successes with osteocalcin offer another informative molecule for archaeological scientists to utilise (Nielsen-Marsh et al., 2005; Ostrom et al., 2006), the survival potential of all these molecules (DNA, collagen and osteocalcin), is limited by the bone substrate in which they are embedded. To date, * Corresponding author. Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, Leipzig, Germany. Tel.: þ ; fax: þ address: (M.P. Richards). only a few early modern human and Neanderthal remains have contained enough surviving biomolecules to provide information on the life histories of these individuals; and these two species are the only two members of our genus for which any biomolecular information has been successfully recovered. If we are to increase our chances at obtaining useable organic material from valuable, but poorly preserved specimens, or perhaps more significantly, investigate deeper into our lineage and study older hominin fossils, from more hostile environments, then more enduring molecules need to be identified and utilised. Fossil tooth enamel represents an, as yet, untapped source of biomolecular information which may allow us to extend palaeodietary and phylogenetic studies back to our earlier ancestors. Enamel is the hardest substance in the body and is, typically, the best-preserved element in ancient skeletal remains. A variety of information have been obtained from fossil teeth, both at the physical (Dean, 2006; Smith et al., 2007) and also chemical levels, where analyses on fossil tooth enamel have been employed to provide palaeoclimatic and palaeodietary information (Nielsen- Marsh et al., 2005; Koch, 1998; Passey and Cerling, 2006). However, although enamel mineral has been used to provide life history data for fossil hominins, as yet, no such valuable information has ever /$ see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi: /j.jas

2 C.M. Nielsen-Marsh et al. / Journal of Archaeological Science 36 (2009) been obtained from biomolecules extracted from fossil enamel extracts. It is the heavily mineralised structure of enamel that gives its hardness and durability, leading to survival over very long time periods, but it is also this characteristic that may potentially hinder our ability to extract useable quantities of biomolecules for molecular palaeontological research. Developing enamel contains around 30% proteinaceous material, but approximately 90% of all protein is lost during maturation (Glimcher et al., 1977). Mature enamel contains only traces of structural proteins (around 0.1 w 0.03%), including enamelin and the sheath protein ameloblastin (Glimcher et al., 1990; Fincham et al., 1999). Also present, in low but isolatable quantities, is a proteolytic fragment of amelogenin, known as the tyrosine-rich amelogenin peptide or TRAP (Brookes et al., 1995). Amelogenin makes up 90% of the protein content of immature enamel; however as enamel mineralization progresses, amelogenin is subjected to proteolytic activity and eliminated from the enamel environment, leaving only low molecular weight peptides. TRAP is one hydrolytic product of amelogenin, cleaved during maturation between Trp45 and Leu46 by the enzyme EMSP1; also formed is the leucine-rich amelogenin peptide, or LRAP. The amelogenin gene is expressed from both the X (AMELX) and Y (AMELY) chromosomes resulting in a difference between the amelogenin protein sequences (Salido et al., 1992). The first 28 amino acids at the N-terminus are identical. The methionine at residue 29 of the Y form is conserved, but absent from the X form, due to a 3-bp deletion in the AMELX gene. As there are only very small quantities of protein present in mature enamel, and there is likely to be even less in degraded fossil specimens, we used matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) in this study to identify and sequence peptides and proteins recovered from the enamel extracts. MALDI-TOF/TOF MS has been successfully used in previous studies for sequencing protein extracts from ancient bones (Nielsen-Marsh et al., 2005, 2002; Ostrom et al., 2006). A combination of high sensitivity enabling femtomolar quantities of proteins and peptides (which do not need to be intact) to be directly sequenced, and a tolerance for low quantities of salts make MALDI-TOF/TOF MS an ideal tool when working with fossil extracts, which often require intensive, multiple purification steps. 2. Results Following tryptic digestions, peaks were observed in all samples and in some cases were sufficiently high for sequencing after highenergy collision of selected ions. Peptide mass fingerprint data were submitted to FindPept (SwissProt; and fragment ion data were submitted to the Mascot MS/MS Ion Search tool (www.matrixscience.com) to obtain identification of peak masses. The FindPept search tool enables the identification of peptides that result from unspecific cleavage of proteins from their experimental masses in addition to identifying masses that correspond to autolysis and keratin digestion. Keratin is a common contaminant in MALDI spectra and can be a serious issue when dealing with protein identification. Although rigorous use of lab coats and gloves can reduce keratin introduction into the sample, even very small quantities become a significant problem when dealing with samples of limited availability, containing very low amounts of protein, as for fossil samples. All MS/MS data obtained from fragment ions in this study (modern, medieval and fossil) were checked against keratin sequences where masses matched those of keratin tryptic peptides Modern and medieval samples The MS data from the digested medieval and clinical enamel extracts possessed masses corresponding to peptides formed from both specific and unspecific cleavage of enamel proteins (Fig. 1). Ion intensities from one recurrent peptide, observed in all the samples, which matched the mass of a peptide produced from the partial tryptic cleavage of the X isoform of TRAP (i.e. one end of this Fig. 1. MALDI mass spectra of tryptic digests of the enamel protein extracts from a) a clinical and b) a medieval sample. Only the ion with m/z 1306 from both extracts was intense enough to perform CID MS, but peaks corresponding to tryptic fragments and unspecific cleavage of enamel proteins were observed in both spectra.

3 1760 C.M. Nielsen-Marsh et al. / Journal of Archaeological Science 36 (2009) Fig. 2. CID product ion spectra of peptide Trp-25-Pro-34 from a) clinical and b) medieval human samples as well as c) a synthetic peptide. The precursor ion [M þ H] þ is represented by the peak at the highest m/z (1306.6). Peptide sequence ions produced on cleavage of peptide bonds are labeled using Roepstorff nomenclature (Roepstorff, 2000). peptide has been formed from tryptic cleavage, the other from unspecific cleavage), were sufficiently high enough to attempt sequencing using the CID (Collision-Induced Dissociation) product ion MS to confirm its identity (Fig. 2A and B). The sequence was further confirmed by a synthetic peptide (Fig. 2C), which displayed a very similar fragmentation pattern with respect to m/z values and relative intensities above m/z 150. Most importantly, the y 4 and b 6 ions dominated all the spectra, being indicative for this sequence even at very low peptide amounts Fossil samples Both the Scladina and the Lakonis Neanderthal MALDI mass spectra contained peptides following tryptic digest of the extracts (Fig. 3). As can be seen in Fig. 3, the Scladina extract contained a great deal more protein than that of the enamel extract from the Lakonis Neanderthal. A search against collagen digest peptides was also performed for this extract using the FindPept tool, as the large number of peaks in the spectra suggests the presence of collagen contamination. As with the clinical and medieval samples, CID MS was attempted on some of the masses to confirm identity. The TRAP sequenced in the medieval and clinical extracts at m/z was not present in either of the Neanderthal extracts; however the Scladina extract did contain a peak at m/z (Fig. 3). This peptide had the greatest ion intensity of any other in the Scladina extract and was intense enough for sequencing using CID MS (Fig. 4A). The peak at m/z could correspond to a tryptic fragment of human keratin (SwissProt P35527), however the TRAP better matched the product ion data, assuming that the mass increase of 1 u was present in the N-terminal part of the sequence

4 C.M. Nielsen-Marsh et al. / Journal of Archaeological Science 36 (2009) Fig. 3. MALDI mass spectra of tryptic digests of Neanderthal enamel protein extracts from a) Scladina and b) Lakonis. The enamel extract from Scladina, the much better preserved fossil, contains peaks corresponding to human collagen tryptic fragments. Both fossil extracts contain peaks corresponding to tryptic fragments and unspecific cleavage of human enamel proteins, but none of these were able to be conclusively identified using CID MS. A peak at m/z 842.5, matching trypsin autolysis was also observed in both spectra, indicating that protein levels in both extracts were very low. when the presumed b 6 -ion was also shifted by 1 u. Thus we hypothesized that the ion at m/z might have resulted from desamidation of the glutamine in position three of the TRAP. Following analysis using Data Explorer, both the desamidated TRAP WYESIRPYP and the keratin peptide IKFEMEQNLR were synthesized on solid phase. As peptide fragmentation in tandem mass spectrometry depends on both the ionization/fragmentation conditions, i.e. parameters selected on a specific instrument, and the peptide sequence, it is possible to confirm or disprove a given sequence by comparing the tandem mass spectra of the native peptide versus synthetic peptides. In other words, the tandem mass spectra of the synthetic peptide should display not only the same b- and y-ions but their relative intensities should also match. Thus the fragment ions detected at m/z and confirmed the TRAP sequence (Fig. 4). At the same time it clearly disproves the possibility that this is a keratin sequence, although two small signals at m/z 175 and 530 may indicate a minor contamination with the human keratin sequence. The proposed desamidation of the glutamine could be due either to the mutation of this position in the Neanderthal genome, or chemical desamidation occurring in the fossils in the burial environment. Peptides with m/z corresponding to unspecific cleavage of enamel proteins and human collagen (alpha-1(i) chain) were also detected in the Neanderthal extracts, but were too weak for successful CID MS. 3. Discussion That enamel proteins can be successfully extracted from modern mature enamel has already been confirmed by earlier studies (Porto et al., 2006), but these studies also confirm that the recoverable amount of protein from enamel in the later maturation stages is very low. The MS and MS/MS data obtained from the medieval and clinical samples in this study suggest that extraction and sequencing of proteins from mature tooth enamel using MALDI-TOF/TOF MS is possible. The quantities recovered were very limited, and only one peptide was able to be confirmed using CID MS, but this result verified the presence of a fragment of the TRAP X isoform using both the Data Explorer software (Applied Biosystems) and CID MS on three synthetic peptides. When submitted to a MASCOT MS/MS ion search of the NCBInr database, however, no significant hits were found for the ion at m/z The sequenced peptide does not appear to have been produced wholly from tryptic digestion, but may have been partially formed from enamel maturation processes, or during the extraction procedure. Though a single peptide may be searched using MASCOT MS/MS ion search, this search tool is much more powerful when analysing MS/MS runs containing data from multiple peptides, and therefore a combination of unspecific cleavage and limited MS/MS data may be hindering identification. Other masses in the spectra, not able to be sequenced, did match up with peptides formed from unspecific cleavage of enamel proteins (enamelin, ameloblastin, LRAP), and in some cases these masses corresponded to tryptic fragments from these proteins; however as no CID MS could be performed we cannot yet be certain that these proteins were present in the enamel extracts and further work is necessary to confirm their presence. The results from the Neanderthal samples are encouraging. Despite the likelihood that collagen contamination from dentine is present in the Scladina extract, the CID MS data strongly suggest that an enamel protein, namely a peptide from the TRAP X isoform, is present in sequencable amounts. The Lakonis sample is in a much worse preservation state, yet peaks, which do correspond to fragments of human enamel proteins, are present in the MALDI mass spectra. Although more research is necessary, these preliminary

5 1762 C.M. Nielsen-Marsh et al. / Journal of Archaeological Science 36 (2009) Fig. 4. CID product ion spectra of the precursor ion m/z from a) Scladina, b) the synthesized peptide Trp-25-Pro-34 (Gln27Glu) from the human TRAP X isoform and c) the synthesized peptide Ile-241-Arg-254 from human keratin (P35527 SwissProt) to see which produced the most complete ion series. The ion series consistent with b) Trp-Tyr-Glu- Ser-Ile-Arg-Pro-Pro-Tyr-Pro, from the synthesized TRAP produced the best fit and only two minor m/z signals at 175 and 530 might indicate very minor impurities from the keratin sequence. Peptide sequence ions produced on cleavage of peptide bonds are labeled by using Roepstorff nomenclature (Roepstorff, 2000). data suggest that proteins recovered from fossil enamel of significant age (i.e. z100,000 years) can be extracted, purified and sequenced. It is not yet clear to us why the TRAP (Trp-25-Pro-34) is the most dominant ion in the MALDI mass spectra of the protein extracts analysed. Amelogenin is the most abundant protein in enamel and during maturation proteolysis causes the formation of polypeptide fragments namely TRAP and LRAP, so it may simply be that TRAP is the most abundant peptide present in mature enamel, and therefore it dominates the MALDI mass spectra. However, we have not yet seen any evidence that TRAP is present in its whole form in the enamel extracts. No other tryptic peptides of TRAP were

6 C.M. Nielsen-Marsh et al. / Journal of Archaeological Science 36 (2009) observed in the MALDI mass spectra and the peptide sequenced is only, apparently, partially formed from tryptic digestion. That it is identifiable as the X isoform is not surprising. The X isoform is z10 more abundant than the Y isoform (Salido et al., 1992), and is therefore much more likely to be ionized and sequenced. We have never observed the Y isoform in our extracts, despite having analysed male samples. However, if the Y isoform could be recovered and sequenced, it suggests the intriguing possibility of using TRAP from fossil enamel extracts to sex fossil specimens. 4. Methods Three modern, clinical human teeth (molars, all male), one medieval human tooth from the UK (sex unknown) and two Neanderthal teeth; one from Lakonis, Greece, dating to approximately 40,000 years old (Harvati et al., 2003) and the other from Scladina Cave, Belgium dating to z100,000 years old (Toussaint and Pirson, 2006), were analysed. For the clinical and medieval samples, tooth fragments were cleaned and powdered (Dremel micro-drill) and the Manly Hodge separation method was used to separate dentine from enamel (Manly and Hodge, 1939). In this centrifugal method a mixture containing 91 volume % bromoform and 9 volume % acetone provides the optimum density (2.7 g ml 1 ) for separation of enamel and dentine. Careful separation of the enamel and dentine is crucial to the successful extraction and sequencing of enamel proteins, as any collagen remaining in the dentine could swamp the ionisation of the much less abundant enamel peptides present, hindering identification and sequencing using MALDI-TOF/TOF MS. For the Neanderthal samples, enamel was removed from the tooth by drilling (Dremel micro-drill) and care was taken to avoid the enamel dentine junction where possible, particularly in the case of the Scladina specimen, a well preserved fossil which yielded both collagen and DNA in earlier studies (Bocherens et al., 1999). We used minimum sample sizes (z2 mg of enamel); therefore the results reported for the Neanderthals are for one extraction per sample. Following drilling and enamel dentine separation, a modified version of the method described in Porto et al. (2006) was used on all samples to extract the enamel proteins. z1 2 mg of enamel powder was placed in a 0.5 ml eppendorf and 10% trichloroacetic acid (TCA) (1 mg enamel powder 200 ml 1 TCA) containing a set of protease inhibitors (Complete Mini; Roche Applied Sciences) was added. After 1 h, 2 ml of sodium deoxycholate (20 mg ml 1 )was added and the samples were stored at 4 C for 2 3 days and then centrifuged (4 C, 3,000 g, 45 min). Following centrifugation the protein pellet was washed with acetone (200 ml, 20 C) and centrifuged (4 C, 13,000 g, 5 min), this wash step was repeated and the sample was dried (CentriVap, 10 min). An in-solution digest was then performed; the pellet was re-suspended in 100 ml of 25mM ammonium bicarbonate (ABC, ph 8.0), 5 ml of 200 mm dithiothreitol (DTT) in 25 mm ABC was then added and the sample was vortexed and incubated (95 C, 10 min). 5 ml of 200 mm iodoacetamide (IAA) in 25 mm ABC was added, the sample vortexed and incubated in the dark for 45 min. A further 20 ml of 200 mm DTT in 25 mm ABC was then added and the samples were left to stand (45 min, RT). The samples were digested with trypsin (5 ml) (Promega) overnight at 37 C, after which 1% trifluoroacetic acid (TFA) was added to stop the reaction. The samples were concentrated down to 10 ml (CentriVap) and ZipTips (Millipore) were then used to further concentrate the protein extracts and remove any salts. Following extraction, digestion and purification the samples were plated onto a MALDI target and sequencing was performed via a combination of peptide mass mapping and Collision-Induced Dissociation (CID) product ion MS of selected peptides using an Applied Biosystems 4700 Proteomics Analyzer (Applied Biosystems GmbH, Darmstadt, Germany). The spectra were analysed with the Data Explorer software package (Version 4.6, Applied Biosystems GmbH). Acknowledgements We would like to thank the Max Planck Gesellschaft for funding, Corinna Sykora for advice and technical assistance with the MALDI- TOF/TOF MS, Raquel Gerlach with assistance with tooth preparation, and David Singer for the synthesis of three peptides. References Bocherens, H., Billiou, D., Mariotti, A., Palaeoenvironmental and palaeodietary implications of isotopic biogeochemistry of last interglacial Neanderthal and mammal bones in Scladina Cave (Belgium). J. Archaeol. Sci. 26, Brookes, S.J., Robinson, C., Kirkham, J., Bonass, W.A., Biochemistry and molecular biology of amelogenin proteins of developing dental enamel. Arch. Oral Biol. 40, Dean, M.C., Tooth microstructure tracks the pace of human life-history evolution. Proc. Royal Soc. B. 273, Fincham, A., Moradian-Oldak, J., Simmer, J., The structural biology of the developing dental enamel. J. Struct. Biol. 126, Glimcher, M., Brickley-Parsons, D., Levine, P., Studies of enamel proteins during maturation. Calcif. Tissue Res. 24, Glimcher, M., Cohensolal, L., Dericqles, A., Biochemical analyses of fossil enamel and dentin. Paleobiology 16, Harvati, K., Panagopoulou, E., Karkanas, P., First Neanderthal remains from Greece: the evidence from Lakonis. J. Hum. Evol. 45, Koch, P., Isotopic reconstruction of past continental environments. Annu. Rev. Earth Planet. Sci. 26, Lalueza-Fox, C., Krause, J., Caramelli, D., Catalano, G., Milani, L., Sampietro, M., Calafell, F., Martinez-Maza, C., Bastir, M., Garcia-Tabernero, A., de la Rasilla, M., Fortea, J., Paabo, S., Bertranpetit, J., Rosas, A., Mitochondrial DNA of an Iberian Neandertal suggests a population affinity with other European Neandertals. Curr. Biol. 16, R629 R630. Lee-Thorp, J., Sponheimer, M., Contributions of biogeochemistry to understanding hominin dietary ecology. Yearb. Phys. Anthropol. 4, Manly, R., Hodge, H., Density and refractive index studies of dental hard tissues: I Methods for separation and determination of purity. J. Dent. Res. 18, Nielsen-Marsh, C., Ostrom, P., Gandhi, H., Shapiro, B., Cooper, A., Collins, M., Sequence preservation of osteocalcin protein and mitochondrial DNA in bison bones older that 55 ka. Geology 30, Nielsen-Marsh, C., Richards, M., Hauschka, P., Thomas-Oates, J., Trinkaus, E., Pettitt, P., Karavanic, I., Poinar, H., Collins, M., Osteocalcin protein sequences of Neanderthals and modern primates. Proc. Natl. Acad. Sci. U.S.A 102, Ostrom, P., Gandhi, H., Strahler, J., Walker, A., Andrews, P., Leykam, J., Stafford Jr., T., Kelly, R., Walker, D., Buckley, M., Humpula, J., Unravelling the sequence and structure of the protein osteocalcin from a 42 ka fossil horse. Geochim. Cosmochim. Acta 70, Passey, B., Cerling, T., In situ stable isotope analysis (d 13 C, d 18 O) of very small teeth using laser ablation GC/IRMS. Chem. Geol. 235, Porto, I., Line, S., Laurre, H., Gerlach, R., Comparison of three methods for enamel protein extraction in different developmental phases of rat lower incisors. Eur. J. Oral Sci. 114, Roepstorff, P., MALDI-TOF mass spectrometry in protein chemistry. In: Jolles, P., Jornvall, H. (Eds.), Proteomics in Functional Genomics. Birkhauser- Verlag, Basel, pp Salido, E., Yen, P., Koprivnikar, K., Yu, L.-C., Shapiro, L., The human enamel protein gene amelogenin is expressed from both the X and Y chromosomes. Am. J. Hum. Genet. 50, Smith, T., Tafforeau, P., Reid, D., Grun, R., Eggins, S., Boutakiout, M., Hublin, J.-J., Earliest evidence of modern human life history in North African early Homo sapiens. Proc. Natl. Acad. Sci. U.S.A 104, Toussaint, M., Pirson, S., Neandertal studies in Belgium: Period Biol. 108,

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