Results
The data set contained 1,246 specimens from 198 taxa including
echinoderms (Asteroidea, Echinoidea and Ophiuroidea), molluscs
(Bivalvia, Gastropoda, Polyplacophora and Cephalopoda), arthropods
(Crustacea, Pantopoda) and chordates (Tunicata, Vertebrata: Teleostei,
Elasmobranchii). For 1,139 specimens a published COI barcode or another
molecular identifier is available (supplementary table S1). The
remaining specimens were identified morphologically. For 226 specimens
attempts to obtain mass spectra either failed or were of minor quality
and discarded.
Sample preparation
To determine the concentration range for successful measurements,
weighted tissue samples were mixed with varying amounts of
α-cyano-4-hydroxycinnamic acid (HCCA). In total, 15 different
tissue/matrix concentrations were tested ranging from 0.01 µg
µl-1 to 200 µg µl-1 (Fig. 1A).
Despite variations between samples, high quality mass spectra were
generally assessed in a concentration range from to 3.1 µg
µl-1 to 12.5 µg µl-1. The largest
concentration range for successful measurements was recorded for the
echinoderm Stichastrella rosea (sample MT03612), with successful
measurements across almost the entire concentration range. No
measurements were obtained for concentrations of 200 µg
µl-1. It was only when concentrations reached 12.5
µg/µl or lower that results were obtained for all specimens.
Good measurements were obtained from small tissue samples, when these
were completely submersed in the HCCA solution within a 1.5 ml
microcentrifuge tube (Fig. 1B). Before samples/matrix ratio was too low
to detect a signal, an increase in baseline height in the lower masses
was recorded (Fig. 1C). When sample to matrix ratio was increased, an
increase in noise was observed (Fig. 1D). Quality improvement of spectra
from high tissue-to-HCCA matrix ratios was achieved by dilution. This
was tested using tissue from the crustacean Cancer pagurus(sample MT01453). Concentrations were diluted from an initial
concentration of 200 µg µl-1 that resulted in no mass
spectra at all. The measurements from diluted preparations then showed
similar results as measurements made with the respective concentrations
from undiluted sample preparations (compare Fig. 1A brown and red
results).