Potential of MALDI−TOF MS-based proteomic fingerprinting for species identification of Cnidaria across classes, species, regions and developmental stages

Morphological identification of cnidarian species can be difficult throughout all life stages due to the lack of distinct morphological characters. Moreover, in some cnidarian taxa genetic markers are not fully informative, and in these cases combinations of different markers or additional morphological verifications may be required. Proteomic fingerprinting based on MALDI-TOF mass spectra was previously shown to provide reliable species identification in different metazoans including some cnidarian taxa. For the first time, we tested the method across four cnidarian classes (Staurozoa, Scyphozoa, Anthozoa, Hydrozoa) and included different scyphozoan life-history stages (polyp, ephyra, medusa) into our dataset. Our results revealed reliable species identification based on MALDI-TOF mass spectra across all taxa with species-specific clusters for all 23 analyzed species. In addition, proteomic fingerprinting was successful for distinguishing developmental stages, still by retaining a species specific signal. Furthermore, we identified the impact of different salinities in different regions (North Sea and Baltic Sea) on proteomic fingerprints to be negligible. In conclusion, the effects of environmental factors and developmental stages on proteomic fingerprints seem to be low in cnidarians. This would allow using reference libraries built up entirely of adult or cultured cnidarian specimens for the identification of their juvenile stages or specimens from different geographic regions in future biodiversity assessment studies. Methods In total, 278 specimens of Cnidaria belonging to 23 different species from four classes were analyzed. Field specimens were morphologically identified to species level by taxonomic experts immediately after collection, before complete specimens or subsamples were preserved in undenatured ethanol (80 - 96%). From each specimen, a small tissue fragment (max. 1 mm³) was incubated for 5 minutes with 5 µl of alpha-cyano-4-hydroxycinnamic acid (HCCA) matrix. Of this incubated solution, 1 to 1.5 µl were transferred to a target plate on one to nine spots for co-crystallization of matrix and analytes. Each spot was measured one to three times using a Microflex LT/SH System (Bruker Daltonics). Employing the flexControl 3.4. (Bruker Daltonics) software, molecule masses were measured from 2 to 20k Dalton (kDA). A centroid peak detection algorithm was carried out for peak evaluation by analyzing the mass peak range from 2 to 20 kDa. Furthermore, peak evaluation was carried out by a signal-to-noise threshold of two and a minimum intensity threshold of 600 with a peak resolution higher than 400. To validate fuzzy control, the proteins/oligonucleotide method was employed by maximal resolution of ten times above the threshold. To create a sum spectrum, a total of at least 120 laser shots were applied to a spot. Measurements were carried out using the same instrument at different occasions between 2013 and 2019. MALDI-TOF data processing MALDI-TOF raw data were imported to R, Version 4.1.0 (R-Core-Team, 2022) and processed using R packages MALDIquantForeign, Version 0.12 (Gibb, 2015) and MALDIquant, Version 1.20 (Gibb and Strimmer, 2012). Spectra were square-root transformed, smoothed using the Savitzky Golay method (Savitzky and Golay, 1964), baseline corrected using the SNIP method (Ryan et al., 1988) and spectra normalized using the TIC method. Repeated measurements were averaged by using mean intensities. Peak picking was carried out using a signal to noise ratio (SNR) of 12 and a half window size of 13. Mass peaks smaller than a SNR of 12 were however retained, if they occurred in other mass spectra as long as these were larger than a SNR value of 1.75, which is assumed as a lower detection limit. Repeated peak binning was carried out to align homologous mass peaks. Resulting data was Hellinger transformed (Legendre and Gallagher, 2001) and used for further analyses.