Project PXD000412

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Human dental calculus LC-MS/MS


The purpose of this study was to investigate oral microbiome and host proteins in archaeological human dental tissues using a shotgun proteomics approach. The research focuses on dental calculus (mineralized plaque), dentine, a carious lesion, and an alveolar bone abscess from the medieval site of Dalheim, Germany (ca. AD 950-1200). For comparison, proteins were also analyzed from archaeological faunal dental tissues and human dental calculus samples from modern Swiss dental patient controls. Protein extraction and generation of tryptic peptides from tooth and dental calculus specimens was performed using a filter-aided sample preparation (FASP) protocol, modified for mineralized and degraded samples. Total protein extraction was performed on a total of fourteen samples: four ancient human calculus samples (indicated as: G12, B71, B61, and B78), four ancient human tooth root samples (indicated as: G12, B17, B61, and B78), one carious lesion (indicated as: B17), one alveolar bone abscess (indicated as: B17), two ancient fauna crown cementum/calculus samples (indicated as: F1 [sheep] and F5 [cattle]), and two modern dental calculus samples from clinical patients (indicated as: P1 and P2). All samples were extracted at the Centre for Evolutionary Medicine (ZEM) at the University of Zürich with the exception of dental calculus from G12, P1, and P2, which were extracted at the Center for GeoGenetics (CGG) at the University of Copenhagen. Two samples (G12 and B61 calculus) were extracted a second time in an independent laboratory at the University of York (YORK) for comparison. Sample extracts were then sequenced (LC-MS/MS) at the Functional Genomics Center Zürich (FGCZ) using an LTQ-Orbitrap Velos, at the Novo Nordisk Foundation Center for Protein Research (CPR) using a Q-Exactive Hybrid Quadrupole Orbitrap, and at the University of York’s Proteomics and Analytical Biochemistry Laboratories (PABL) using a MaXis UHR-Qq-TOF.

Sample Processing Protocol

Tryptic peptides were generated using a modified FASP protocol. LTQ-Orbitrap Velos (FGCZ): Samples were analyzed on an LTQ-Orbitrap VELOS mass spectrometer (Thermo Fischer Scientific, Bremen, Germany) coupled to an Eksigent-NanoLC-Ultra 1D plus HPLC system (Eksigent Technologies, Dublin (CA), USA). Solvent composition at the two channels was 0.2% formic acid, 1% acetonitrile for channel A and 0.2% formic acid, 100% acetonitrile for channel B. Peptides were loaded on a self-made tip column (75 µm × 80 mm) packed with reverse phase C18 material (AQ, 3 μm 200 Å, Bischoff GmbH, Leonberg, Germany) and eluted with a flow rate of 250 nl per min by a gradient from 0.8% to 4.8% of B in 2 min, 35% B at 57 min, 48% B at 60 min, 97% at 65 min. Full-scan MS spectra (300−1700 m/z) were acquired in the Orbitrap with a resolution of 30 000 at 400 m/z after accumulation to a target value of 1,000, 000. Higher energy collision induced dissociation (HCD) MS/MS spectra were recorded in data dependent manner in the Orbitrap with a resolution of 7500 at 400 m/z after accumulation to a target value of 100, 000. Precursors were isolated from the ten most intense signals above a threshold of 500 arbitrary units with an isolation window of 2 Da. Three collision energy steps were applied with a step width of 15.0% around a normalized collision energy of 40% and an activation time of 0.1 ms. Charge state screening was enabled excluding non-charge state assigned and singly charged ions from MS/MS experiments. Precursor masses already selected for MS/MS were excluded for further selection for 45 s with an exclusion window of 20 ppm. The size of the exclusion list was set to a maximum of 500 entries. Q-Exactive Hybrid Quadrupole Orbitrap (CPR): The LC-MS system consisted of an EASY-nLC™system (Thermo Scientific, Odense, Denmark) connected to the Q-Exactive (Thermo Scientific, Bremen, Germany) through a nano electrospray ion source. 5 uL of each peptide sample was auto-sampled onto and directly separated in a 15cm analytical column (75 μm inner diameter) in-house packed with 3μm C18 beads (Reprosil-AQ Pur, Dr. Maisch) with a 130 minute linear gradient from 5% to 26% acetonitrile followed by a steeper linear 20 minute gradient from 26% to 48% acetonitrile. Throughout the gradients a fixed concentration of 0.5% acetic acid and a flow rate of 250 nL/min were set. A final washout and column re-equilibration added an additional 15 minutes to each acquisition. The effluent from the HPLC was directly electrosprayed into the mass spectrometer by applying 2.0 kV through a platinum-based liquid-junction. The Q-Exactive was operated in data-dependent mode to automatically switch between full scan MS and MS/MS acquisition. Software control was Tune version 2.0-1428 and Excalibur version 2.2.42, and the settings were adjusted for ‘sensitive’ acquisition. Briefly, each full scan MS was followed by up to 10 MS/MS events. The isolation window was set at 1.6 Th and a dynamic exclusion of 90 seconds was used to avoid repeated sequencing. Only precursor charge states above 1 and below 6 were considered for fragmentation. A minimum intensity threshold for triggering fragment MS/MS was set at 1e5. Full scan MS were recorded at resolution of 70,000 at m/z 200 in a mass range of 300-1700 m/z with a target value of 1e6 and a maximum injection time of 20 ms. Fragment MS/MS were recorded with a fixed ion injection time set to 120 ms through a target value set to 1e6 and recorded at a resolution of 35,000 with a fixed first mass set to 100 m/z. MaXis UHR-Qq-TOF (PABL): The nanoLC system was interfaced with a maXis LC-MS/MS System (Bruker Daltonics) with a Bruker nano-electrospray source fitted with a steel emitter needle (180 µm O.D. x 30 µm I.D., Thermo [Proxeon]). Positive ESI- MS & MS/MS spectra were acquired using AutoMSMS mode. Instrument control, data acquisition and processing were performed using Compass 1.3 SR3 software (microTOF control, Hystar and DataAnalysis, Bruker Daltonics). Instrument settings were: ion spray voltage: 1,400 V, dry gas: 4 L/min, dry gas temperature 160°C, ion acquisition range: m/z 50-2,200. AutoMSMS settings were: MS: 0.5 s (acquisition of survey spectrum), MS/MS (CID with N2 as collision gas): ion acquisition range: m/z 300-1,500, 0.1 s acquisition for precursor intensities above 100,000 counts, for signals of lower intensities down to 1,000 counts acquisition time increased linear to 1s, the collision energy and isolation width settings were automatically calculated using the AutoMSMS fragmentation table: eight precursor ions, absolute threshold 1,000 counts, preferred charge states: 2–4, singly charged ions excluded. One MS/MS spectrum was acquired for each precursor, and former target ions were excluded for 30 s.

Data Processing Protocol

Tandem mass spectra were converted to the Mascot generic format (.mgf) using proteowizard version 2.2.3101 with the vendor peak picking option for MS level two. Corresponding .mgf files were further deisotoped and deconvoluted using the H-Scorer script. Mascot generic format MS/MS peak lists were submitted to Mascot (Matrix Science, London, UK; version 2.3.02) for searching. Selecting semi-trypsin as the proteolytic enzyme, Mascot was set up to search against a non-redundant, concatenated target/decoy protein database consisting of 4198561 forward and 4197958 reversed protein sequences that included all proteins available in UniProtKB/Swiss-Prot (v.20121031; total 1076779 sequences), all proteins available in the Human Oral Microbiome Database as of 2012/10/11 (total 4476028 sequences;, and all proteins available for Genbank genome accessions of bacteria and archaea in which the word “soil” appears in the metadata (total 2843972 sequences) as of 2012/02/22. Our concatenated database is available online ( For data generated on the LTQ Obitrap Velos and Q-Exactive Hybrid Quadrupole Obitrap instruments, Mascot was searched with a fragment ion mass tolerance of ± 0.8 Da; for data generated on the Q-TOF, Mascot was searched with a fragment ion mass tolerance of ± 0.1 Da. For Mascot search of all data, precursor mass tolerance was set at ± 10 ppm, and a maximum of 2 missed cleavages were allowed. Carbamidomethylation of cysteine was specified as a fixed modification, and as already mentioned, acetylation (Protein N-term), deamidation (N, Q), Gln→ pyro-Glu (N-term Q), oxidation (M), and oxidation (P) were specified in Mascot as variable modifications.


Christina Warinner, Centre for Evolutionary Medicine (ZEM)
Christina G Warinner, Department of Anthropology, University of Oklahoma, USA ( lab head )

Submission Date


Publication Date



    Warinner C, Rodrigues JF, Vyas R, Trachsel C, Shved N, Grossmann J, Radini A, Hancock Y, Tito RY, Fiddyment S, Speller C, Hendy J, Charlton S, Luder HU, Salazar-García DC, Eppler E, Seiler R, Hansen LH, Castruita JA, Barkow-Oesterreicher S, Teoh KY, Kelstrup CD, Olsen JV, Nanni P, Kawai T, Willerslev E, von Mering C, Lewis CM Jr, Collins MJ, Gilbert MT, Rühli F, Cappellini E. Pathogens and host immunity in the ancient human oral cavity. Nat Genet. 2014 Apr;46(4):336-44 PubMed: 24562188


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# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 34619 B17 calc Z27 827 1483 586 5529 491
2 34606 B17 disalv Z30 diseased alveolar bone 207 2379 503 4913 786
3 34618 B17 disdent Z29 diseased carious dentine 227 2843 573 5319 946
4 34605 B17 root Z24 257 2988 670 4082 1005
5 34628 B61 calc Y46 Maxis 18 43 16 7324 36
6 34615 B61 calc Z46 Maxis 21 107 26 7810 92
7 34627 B61 calc Z46 466 915 468 5589 398
8 34609 B61 root Z25 260 2812 687 4197 1003
9 34614 B78 calc Z28 771 1361 595 5186 395
10 34626 B78 root Z26 236 2563 593 3441 849