Differential display of peptides induced during the immune response of Drosophila : A matrix-assisted laser desorption ionization time-of-flight mass spectrometry study

Abstract

We have developed an approach based on a differential mass spectrometric analysis to detect molecules induced during the immune response of Drosophila, regardless of their biological activities. For this, we have applied directly matrix-assisted laser desorption/ionization MS to hemolymph samples from individual f lies before and after an immune challenge. This method provided precise information on the molecular masses of immune-induced molecules and allowed the detection, in the molecular range of 1.5-11 kDa, of 24 Drosophila immune-induced molecules (DIMs). These molecules are all peptides, and four correspond to already characterized antimicrobial peptides. We have further analyzed the induction of the various peptides by immune challenge in wild-type f lies and in mutants with a compromised antimicrobial response. We also describe a methodology combining matrix-assisted laser desorption ionization time-of-f light MS, HPLC, and Edman degradation, which yielded the peptide sequence of three of the DIMs. Finally, molecular cloning and Northern blot analyses revealed that one of the DIMs is produced as a prepropeptide and is inducible on a bacterial challenge.

Primary Structure and in Vitro Antibacterial Properties of the Drosophila melanogaster Attacin C Pro-domain

“…Insect Immunization and Hemolymph Collection-Two-to three-dayold Drosophila melanogaster Oregon R adults were experimentally infected with a mixture of two bacteria (Micrococcus luteus as Grampositive strain and Escherichia coli as Gram-negative strain), and the hemolymph (20 batches of 60 flies) of 24-h challenged flies was collected according to a procedure reported previously (10). The acidified hemolymph was centrifuged at 12,000 ϫ g for 20 min at 4°C and frozen until HPLC analysis.…”

“…A differential display study by matrix-assisted laser desorption/ionization (MALDI) 1 mass spectrometry (MS) and high-performance liquid chromatography (HPLC) on hemolymph collected from experimentally infected and from noninfected flies led to the detection of at least 24 immune-induced molecules referred to as DIMs for Drosophila immune-induced molecules (10,11). The 24 DIMs have molecular masses ranging from 1.5 to 11 kDa.…”

Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays

“…Only one gene in this group, IM-2, was previously identified although its precise function is unknown (30). Drosophila also possesses two genes encoding homologues of IM-2, which display a similar pattern of expression and, like IM-2, are induced by both bacterial and fungal infection.…”

Gambicin: A novel immune responsive antimicrobial peptide from the malaria vector Anopheles gambiae

“…Four-day-old A. gambiae 4a r͞r strain females were infected with bacteria as described (19) and allowed to recover 20 h at 25°C. Hemolymph was collected and purified by RP-HPLC as reported (33). Fractions at the retention time of gambicin were concentrated by ZipTip (Millipore) and analyzed by MALDI-TOF MS.…”