Sequential trypsin and ProAlanase digestions unearth immunological protein biomarkers shrouded by skeletal collagen

Shevan Wilkin; Liam T Lanigan; Nuria Montes; Mukul Sharma; Charlotte Avanzi; Donikë Sejdiu; Kerttu Majander; Saskia Pfrengle; Yun Chiang; Laura Kunz; Antje Dittmann; Frank Rühli; Pushpendra Singh; Maria Fontanals Coll; Matthew J Collins; Alberto J Taurozzi; Verena J Schuenemann

doi:10.1016/j.isci.2024.109663

Sequential trypsin and ProAlanase digestions unearth immunological protein biomarkers shrouded by skeletal collagen

iScience. 2024 Apr 3;27(5):109663. doi: 10.1016/j.isci.2024.109663. eCollection 2024 May 17.

Authors

Shevan Wilkin^{1

2

3}, Liam T Lanigan⁴, Nuria Montes⁵, Mukul Sharma⁶, Charlotte Avanzi⁷, Donikë Sejdiu¹, Kerttu Majander¹, Saskia Pfrengle^{1

8}, Yun Chiang⁴, Laura Kunz⁹, Antje Dittmann⁹, Frank Rühli¹, Pushpendra Singh^{6

10}, Maria Fontanals Coll¹¹, Matthew J Collins^{4

12}, Alberto J Taurozzi⁴, Verena J Schuenemann¹

Affiliations

¹ Institute of Evolutionary Medicine, University of Zurich, Zurich, Switzerland.
² Max Planck Institute of Geoanthropology, Jena, Germany.
³ Australian Research Centre for Human Evolution, Griffith University, Brisbane, QLD, Australia.
⁴ Globe Institute, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
⁵ Unitat d'Antropologia Biològica, Departament de Biologia Animal, Biologia Vegetal i Ecologia, Universitat Autònoma de Barcelona, Barcelona, Spain.
⁶ Microbial Pathogenesis and Genomics, National Institute of Research in Tribal Health, Jabalpur, MP, India.
⁷ Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO, USA.
⁸ Institute for Archaeological Sciences, University of Tübingen, Tübingen, Germany.
⁹ Functional Genomics Center Zurich, University of Zurich, Zurich, Switzerland.
¹⁰ Model Rural Health Research Unit, Badoni, Datia (MP), India.
¹¹ BioArCh - Department of Archaeology, University of York, York, UK.
¹² McDonald Institute for Archaeological Research, University of Cambridge, West Tower, Downing St, Cambridge CB2 3ER, UK.

Abstract

This study investigates the efficacy of proteomic analysis of human remains to identify active infections in the past through the detection of pathogens and the host response to infection. We advance leprosy as a case study due to the sequestering of sufferers in leprosaria and the suggestive skeletal lesions that can result from the disease. Here we present a sequential enzyme extraction protocol, using trypsin followed by ProAlanase, to reduce the abundance of collagen peptides and in so doing increase the detection of non-collagenous proteins. Through our study of five individuals from an 11th to 18th century leprosarium, as well as four from a contemporaneous non-leprosy associated cemetery in Barcelona, we show that samples from 2 out of 5 leprosarium individuals extracted with the sequential digestion methodology contain numerous host immune proteins associated with modern leprosy. In contrast, individuals from the non-leprosy associated cemetery and all samples extracted with a trypsin-only protocol did not. Through this study, we advance a palaeoproteomic methodology to gain insights into the health of archaeological individuals and take a step toward a proteomics-based method to study immune responses in past populations.

Keywords: Biochemistry; Biochemistry applications; Biochemistry methods; Medical biochemistry; Proteomics.