Optimization of tick identification via MALDI-TOF MS for implementation in routine diagnostics

Abstract Ticks are important vectors of pathogens with substantial medical and veterinary relevance, a threat that may be further increased with the spread of invasive tick species. Reliable tick identification is relevant for accurate diagnostic elucidation of transmission pathways, and the development of targeted surveillance strategies and risk assessments. A standardized, rapid, and cost-effective identification workflow that can be implemented across laboratories is needed. Previous studies have shown that MALDI-TOF MS is a promising, objective, and economical approach for tick identification. However, the methodology remains insufficiently standardized and is not yet routinely applied for vector identification. The aim of this study was therefore to optimize MALDI-TOF MS-based tick identification for routine use in diagnostic laboratories. A simplified and standardized protocol for protein extraction from whole tick legs via brief incubation in 70% formic acid is presented. This protocol provides a robust basis for the development of a curated MALDI Biotyper® reference library, enabling reliable, expert-independent tick identification and supporting epidemiological surveillance of vectors and vector-borne pathogens.