High-throughput targeted paleoproteomics sex identification on medieval Great Moravia individuals using MALDI-CASI-FTICR mass spectrometry

The site of Mikulčice, located in south Moravia (Czech Republic), contains the cemetery associated with the 3rd church, the largest Great Moravian church excavated to date. The burial ground was used during the 9th–10th century AD (Ungerman – Kavánová 2010; Klanica et al. 2019) and is characterized by a high intensity of interments. The individuals buried here represent a medieval Central European population belonging to the Great Moravian Empire. The identification of the biological sex of archaeological remains is crucial information in archaeology, anthropology, and forensic sciences. To date, three techniques are used in the field for identify the biological sex: osteomorphology, genomics and proteomics [1]. Of these, the last two techniques mentioned allow for reliable and accurate sex estimation in subadult individuals [2], [3]. Here we introduce a new method MALDI-CASI-FTICR MS analysis for the identification of biological sex. Materials and methods Samples and osteological analysis In total, 28 modern human teeth (14 males, 14 females) form adult were collected at the Forensic Taphonomy and Anatomy Unit, the School of Dentistry, University of Lille, France, for the development of the assay. 30 archaeological adult teeth originating from the excavation of the 3rd church at medieval Mikulčice (9th-10th centuries, Czech Republic). The sex of adult individuals was previously estimated/assessed by Stloukal [17], [18] and revised by Zazvonilová et al. [19] using various osteomorphological methods (Table 2). 100 archaeological juvenile teeth originating from the excavations of medieval Rajhard (9th-10th centuries, Czech Republic). The sex of 13 of them was previously identified by Pospíšilová [20] using tooth morphological metric method (Table 2). Reagents and chemicals All aqueous solutions were prepared from ultrapure grade water obtained by water filtration with a Direct-Q3 sytem (Merck Millipore, Burlington, Massachusetts, United States). All chemicals, biochemicals and solvents were purchased from Merck (Merck KGaA, Darmstadt, Germany) and used without additional purification. All solvents were MS analytical grade (ThermoFisher, Kandel, Germany). Peptide extraction by acid etching and purification Each tooth was individually cleaned with LC-MS-grade water. The tooth was then incubated in 200 μL of 0.6 N HCl at room temperature for 1 h. The crown of the tooth was incubated in the cap of 1.5-mL microcentrifuge tube. Enamel peptide solutions were then purified using 96-well plates C18 (Affinisep, Petit-Couronne, France). Peptides were resuspended in 10 µL of 0.1% formic acid in H2O. MALDI-FTICR MS experiments were carried out on a Bruker 9.4 Tesla SolariX XR FTICR mass spectrometer controlled by FTMSControl software and equipped with a CombiSource and a ParaCell (Bruker Daltonics, Bremen, Germany). A Bruker Smartbeam-II Laser System was used for irradiation at a frequency of 1,000 Hz and using the “Minimum” predefined shot pattern. MALDI-CASI-FTICR MS spectra were generated from 500 laser shots in the m/z range from 600 to 3,500 with 1 M data points (i.e., transient length of 2.564 s). Fifteen spectra were averaged. The transfer time of the ICR cell was set to 1.2 ms and the quadrupole mass filter operating in RF-only mode was set at m/z 600. Five ions were selected: m/z 829.4566 (SIRPPYP, protein AMELX, name AMELX1), 879.4393 (SM(ox)IRPPY, protein AMELY, name AMELYL), 889.4491 (SM(ox)IR*PPY, protein AMELY, name AMELYH), 1079.5520 (SIRPPYPSY, protein AMEL X, name AMELX2L), 1089.5621 (SIR*PPYPSY, protein AMEL X, name AMELX2H), with isolation windows at m/z 10. The acquisition time for one sample is less than 1 min. MALDI-FTICR analysis Desalted peptides were diluted 2 times in 0.1% formic acid in H2O. Diluted peptides (0.6 µL) were deposited on 384 Ground steel MALDI plates (Bruker Daltonics, Bremen, Germany) with 0.6 µL of isotope-labeled peptides (AQUA™ Peptides, Merck KGaA) at 6 fmol for AMELX and AMELY (table 1) and 1 µL of HCCA matrix at 10 mg/mL in ACN/H2O (80/20, v/v). TFA (0.1%) was added for each sample spot and dried at ambient temperature. Table 1. List of isotope-labeled peptides for AMELX and AMELY (AQUA™ Peptides). R* = isotope-labeled arginine which induces a shift of +10 Da, and (ox) = oxidation of methionine which induces a shift of +15.99 Da. Peptide sequence Protein name Uniprot code-isoform m/z heavy m/z light SM(ox)IR*PPY AMELY Q99218-1 889.4491 879.4393 SIR*PPYPSY AMELX Q99217-1 1089.5621 1079.5520 Peptides identification MS raw data from MALDI-FTICR were processed using DataAnalysis 5.0. FTMS algorithm was employed with the following parameters of S/N > 4, Relative intensity threshold 0.01% and Absolute intensity threshold 100. The list of peptides markers was searched with home-made Python program (version 3.12.7). The list of [M+H]+ masses is indicated in the program, and the search is carried out with an error of 1 ppm. Log2 for AMELX, Y intensities and heavy/light peptides ratio were calculated. Samples with a Log2 AMELX < 20, Log2 AMELY < 16, ratio 889/879 > 10 are considered indeterminate. The graphical representations are created using R studio (version 2025.09.2+418).