Rapid proteomic amelogenin sex estimation of human and cattle remains using untargeted Evosep-timsTOF mass spectrometry

TY - JOUR

T1 - Rapid proteomic amelogenin sex estimation of human and cattle remains using untargeted Evosep-timsTOF mass spectrometry

AU - Blacka, Charllotte

AU - Dowle, Adam

AU - Lisowski, Mik

AU - Alexander, Michelle Marie

AU - Hendy, Jessica Ruth

AU - Penkman, Kirsty Elizabeth Helena

AU - Mosely, Jackie

N1 - © 2025 John Wiley & Sons Ltd. This is an author-produced version of the published paper. Uploaded in accordance with the University’s Research Publications and Open Access policy.

PY - 2025/4/15

Y1 - 2025/4/15

N2 - Rationale: Sex estimation by analysis of amelogenin peptides in archaeological and fossil material has recently been gaining great traction within the fields of archaeology and palaeontology. Current widely used proteomic amelogenin sex estimation methods are hindered by relatively long mass spectrometric run times, or targeting peptides specific to human amelogenin proteins. Untargeted, high-throughput amelogenin sexing would be invaluable for a range of applications, from sex estimation of remains at mass grave sites to broadening the application of rapid amelogenin sexing to non-hominin species for husbandry and evolutionary studies. Methods: A new acid etch protocol followed by Evosep-LC-TIMS-TOF mass spectrometry is presented for amelogenin analysis, providing global peptide data through rapid mass spectrometric methods in under 20 min per sample (including sample preparation, mass spectrometric acquisition and data processing). This sampling protocol was developed on modern cattle (Bos taurus) teeth, before Evosep-timsTOF partial validation with archaeological cattle and human (Homo sapiens) teeth, demonstrating the potential of straightforward application of this rapid amelogenin sexing method to a range of taxa. Results: The rapid Evosep-LC-TIMS-TOF mass spectrometry methods gave comparable peptide counts to conventional long untargeted methods, while maintaining similar (or faster) acquisition times to those reported in methods targeting specific human amelogenin peptides. Implementation of the novel acid etch sampling approach also streamlined sample preparation without compromising peptide counts. Conclusions: Rapid, untargeted Evosep-LC-TIMS-TOF mass spectrometry was successfully implemented in sex estimation of modern and archaeological material from Bos taurus and Homo sapiens teeth. This demonstrates an advancement in low-cost, high-throughput amelogenin sex estimation, for both human and zooarchaeological applications.

AB - Rationale: Sex estimation by analysis of amelogenin peptides in archaeological and fossil material has recently been gaining great traction within the fields of archaeology and palaeontology. Current widely used proteomic amelogenin sex estimation methods are hindered by relatively long mass spectrometric run times, or targeting peptides specific to human amelogenin proteins. Untargeted, high-throughput amelogenin sexing would be invaluable for a range of applications, from sex estimation of remains at mass grave sites to broadening the application of rapid amelogenin sexing to non-hominin species for husbandry and evolutionary studies. Methods: A new acid etch protocol followed by Evosep-LC-TIMS-TOF mass spectrometry is presented for amelogenin analysis, providing global peptide data through rapid mass spectrometric methods in under 20 min per sample (including sample preparation, mass spectrometric acquisition and data processing). This sampling protocol was developed on modern cattle (Bos taurus) teeth, before Evosep-timsTOF partial validation with archaeological cattle and human (Homo sapiens) teeth, demonstrating the potential of straightforward application of this rapid amelogenin sexing method to a range of taxa. Results: The rapid Evosep-LC-TIMS-TOF mass spectrometry methods gave comparable peptide counts to conventional long untargeted methods, while maintaining similar (or faster) acquisition times to those reported in methods targeting specific human amelogenin peptides. Implementation of the novel acid etch sampling approach also streamlined sample preparation without compromising peptide counts. Conclusions: Rapid, untargeted Evosep-LC-TIMS-TOF mass spectrometry was successfully implemented in sex estimation of modern and archaeological material from Bos taurus and Homo sapiens teeth. This demonstrates an advancement in low-cost, high-throughput amelogenin sex estimation, for both human and zooarchaeological applications.

KW - amelogenin

KW - LC–MS/MS

KW - palaeoproteomics

KW - sex estimation

KW - TIMS-TOF

UR - http://www.scopus.com/inward/record.url?scp=105000140789&partnerID=8YFLogxK

U2 - 10.1002/rcm.10022

DO - 10.1002/rcm.10022

M3 - Article

C2 - 40067017

AN - SCOPUS:105000140789

SN - 0951-4198

VL - 39

JO - Rapid communications in mass spectrometry

JF - Rapid communications in mass spectrometry

IS - 11

M1 - e10022

ER -