Introduction
Correct and cost-effective species identification is crucial in various research areas, including biodiversity assessments, where obtaining reliable information on species’ occurrences and distributions is pivotal. If species cannot be morphologically identified to the species level, they are often assigned to higher taxonomic levels, leading to less detailed analyses and consequently imprecise conclusions (Bailey et al., 2001; Timms et al., 2013). However, identification of samples using COI-barcoding is expensive, time consuming (Rossel et al., 2019) and therefore not feasible in large biodiversity assessments including large numbers of specimens.
Matrix Assisted Laser Desorption/Ionization Time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid species identification method that measures a proteome fingerprint to identify specimens using a reference library. With few preparation steps, peptides and proteins are extracted from tissue and embedded in a matrix absorbing laser radiation while measuring ionized, intact compounds in a mass spectrometer (Singhal et al., 2015). This method is routinely applied for the identification of microorganisms such as bacteria, viruses and fungi (Fenselau and Demirev, 2001; Sandrin et al., 2013; Calderaro et al., 2014). It was also used for food fraud detection (Mazzeo et al., 2008; Flaudrops et al., 2015) or to check food adulteration (Sassi et al., 2015). In pilot studies, it was successfully applied for identification of metazoans such as copepods (Laakmann et al., 2013; Kaiser et al., 2018; Rossel and Martínez Arbizu, 2019; Renz et al., 2021; Yeom et al., 2021; Rossel et al., 2022; Peters et al., 2023), isopods (Kürzel et al., 2022; Paulus et al., 2022), different groups of Cnidaria (Holst et al., 2019; Park et al., 2021; Korfhage et al., 2022), molluscs (Wilke et al., 2020), fish (Mazzeo et al., 2008; Volta et al., 2012) and especially disease vectors such as ticks, sandflies or mosquitoes (Yssouf et al., 2013; Chavy et al., 2019; Rakotonirina et al., 2020, 2022; Nabet et al., 2021). Most studies only analyzed a few species or were limited to a certain taxonomic group while studies across different classes and phyla are still missing. Also, no gold standard protocol for metazoan analytics has been established yet. Systematic tests, how data processing will affect the identification success and whether and how pipelines need to be adapted to higher-taxonomic-level identification are also missing.
For the first time, we present a generalized workflow for species identification of metazoans as well as the subsequent bioinformatics using a wide spectrum of marine taxa. We emphasize the importance of adjusting bioinformatics to the data set and finally prove the power of proteomic fingerprinting for differentiation of morphologically cryptic, closely related marine species and beyond mere species identification on sex level, making it a promising tool for ecological research.