Figure 1.
Workflow for the identification of bioactive peptides.
On the one hand the thermolysin-digested hemolymph sample was analyzed directly by nanoHPLC-FTMS. On the other hand the thermolysin-digested sample was pre-fractionated using a HPLC-system and the collected samples were tested for bioactivity for a more detailed analysis of immuno-relevant peptides.
Table 1.
Peptides identified in the bioactive fractions.
Figure 2.
MALDI-Orbitrap MS/MS measurement with higher-energy collisional dissociation mode (HCD).
The [M+H]+ precursor ion at m/z 631.3872 was detected in the bioactive fraction A1. The presented peptide sequence KAERK was determined by de novo sequencing (CBS).
Figure 3.
Venn diagram comparing identified peptides obtained by using different approaches.
In total, 127 peptides were identified. 25 peptides were identified with the de novo approach, 78 with the standard database search and 40 with the experimental database.
Figure 4.
Validation of the identified peptide VV-9 in hemolymph fraction B.
The MS/MS spectrum of the precursor ion m/z 886.46 was acquired with accurate mass using CID(35) as fragmentation technique. The sequence of this peptide was determined as VDGKSAPNV by database search. The fragmentation pattern of the synthetic peptide is in good accordance with the MS/MS spectrum of the identified peptide in the bioactive fraction.
Figure 5.
Bioactivity test of the identified peptides against (living) Micrococcus bacteria.
The activity is shown in Gentamycin equivalents inµg/mL. Peptides VV-9, IE-8, FN-9, IN-10, LY-11 and AP-14 exhibit significantly higher immune-stimulatory activity than the used solvent control. Peptide KK-5 shows no activity compared to the solvent control, in contrast. EG-4 and SP-8 show slightly elevated immune activity. Statistically significant differences between activities of larvae injected with peptides and control were determined using Students t-test and are indicated by * (p<0.05) and ** (p<0.005), (n = 4-6 for each peptide).