DOI: 10.5061/dryad.3r2280gkc
title: Data from: Proteomic fingerprinting enables quantitative biodiversity assessments of species and ontogenetic stages in Calanus congeners (Copepoda, Crustacea) from the Arctic Ocean

description: Species identification is pivotal in biodiversity assessments, and
 proteomic fingerprinting by MALDI-TOF mass spectrometry has already been
 shown to reliably identify calanoid copepods to species level. However,
 MALDI-TOF data may contain more information beyond mere species
 identification. In this study, we investigated different ontogenetic
 stages (copepodids C1-C6 females) of three co-occurring Calanus species
 from the Arctic Fram Strait, which cannot be identified to species level
 based on morphological characters alone. Differentiation of the three
 species based on mass spectrometry data was without any error. In
 addition, a clear stage-specific signal was detected in all species,
 supported by clustering approaches as well as machine learning using
 Random Forest. More complex mass spectra in later ontogenetic stages as
 well as relative intensities of certain mass peaks were found as the main
 drivers of stage distinction in these species. Through a dilution series,
 we were able to show that this did not result from the higher amount of
 biomass that was used in tissue processing of the larger stages. Finally,
 the data were tested in a simulation for application in a real
 biodiversity assessment by using Random Forest for stage classification of
 specimens absent from the training data. This resulted in a successful
 stage-identification rate of almost 90%, making proteomic fingerprinting a
 promising tool to investigate polewards shifts of Atlantic Calanus species
 and, in general, to assess stage compositions in biodiversity assessments
 of Calanoida, which can be notoriously difficult using conventional
 identification methods.
description: The anterior prosome body parts
 of 179 specimens were used for MALDI-TOF MS measurements. Depending on
 size, these were incubated in 3 – 30 µl (in adults: 5 µl for <em>C.
 finmarchicus</em>, <em>C. glacialis</em> in 10 µl and C.
 hyperboreus in 30 µl) of a matrix solution covering the entire specimen
 with some supernatant. The matrix contained α-Cyano-4-hydroxycinnamic acid
 (HCCA) as a saturated solution in 50% acetonitrile, 47.5% molecular grade
 water, and 2.5% trifluoroacetic acid. After 5 min of incubation, 1.5 µl
 was transferred to a target plate for co-crystallization of matrix and
 molecules. Subsequently, measurements were carried out 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 160 laser shots were applied to a spot. Each
 spot was measured three times. 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 & Strimmer, 2012). Spectra were square-root transformed,
 smoothed using the Savitzky Golay method (Savitzky & 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 & Gallagher, 2001) and used for further analyses.
description: Data was processed in R. Mass spectrometry data can
 be imported to R using the Package MaldiQuantForeign and further be
 processed using the package MaldiQuant. Gibb, S. (2015). MALDIquantForeign:
 Import/Export routines for MALDIquant. A package for R. https://CRAN.R-project.org/package=MALDIquantForeign Gibb, S., and Strimmer, Korbinian (2012). MALDIquant: Quantitative Analysis of Mass Spectrometry Data. Bioinformatics 28, 2270--2271. DOI: 10.1093/bioinformatics/bts447.