De Novo Sequencing by Nano-Electrospray Multiple-Stage Tandem Mass Spectrometry of An Immune-Induced Peptide of Drosophila Melanogaster

Abstract

To combat infection, the fruit fly Drosophila melanogaster responds by rapid synthesis of a series of immune-induced molecules reported as Drosophila immune-induced molecules (DIMs). Characterization of the primary structure of the DIMs is required to establish their exact function. In order to get such information, previous studies on the elucidation of primary structures of the DIMs were developed using a methodology combining matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), highperformance liquid chromatography (HPLC), enzymatic digestion and Edman degradation. Nevertheless, some of the DIMs were resistant to classical Edman sequencing. Therefore, mass spectrometry was used to characterize the primary structure of one of the DIMs, namely the N-blocked DIM13 peptide. The complete sequence of DIM13 was established by means of a strategy of nanoelectrospray ionisation (ESI) combined with multiple-stage tandem mass spectrometry (MS n ) and then was partially confirmed with a combination of enzymatic digestions and MALDI-MS analyses. Interestingly, most of the amino acid sequences have been determined using three-stage (MS 3 ) and four-stage (MS 4 ) tandem experiments, whereas classical tandem mass spectrometry (MS 2 ) yielded only incomplete sequence information. Finally, DIM13 is a 23 amino acid peptide with a pyroglutamic modification at the N terminal position. This work illustrates the remarkable advantages of MS 3 and MS 4 compared with the MS 2 experiment for de novo peptide sequencing. The use of nano-ESI also makes these experiments compatible with the low amount (picomolar level) of DIM13 peptide available for sequencing by ESI-MS n .

Pherokine‐2 and ‐3

“…Indeed, one hallmark of the response to bacterial or fungal infections is the inducible secretion into the hemolymph of a cocktail of antimicrobial peptides [4,5]. In addition, a large number of Drosophila immune‐induced molecules are also induced in the hemolymph following septic injury [13,14,29]. By contrast, none of these molecules are induced upon DCV infection at the time points analyzed, and we only identified a single induced molecule, Phk‐2, in the hemolymph of DCV‐infected flies.…”

Capillary liquid chromatography with MS3 for the determination of enkephalins in microdialysis samples from the striatum of anesthetized and freely–moving rats

“…While this principle is well known, it has rarely been applied to the determination of trace species. MS 3 has been primarily used in structural characterization or to investigate fragmentation pathways;20–22 however, more recently, MS 3 data were utilized to quantitate structural isomeric23 and isobaric mixtures24 and various toxins25, 26 with good reproducibility and improved detection limits. Gas chromatography coupled to MS 3 detection is becoming important in quantitative environmental27 and drug screening 28.…”

Naturally and externally pulsed electrospray

“…In most bioapplications, the requirements of less sample consumption, high sensitivity, and minimum matrix effects demand developments of micro-ESI and nano-ESI technology (Valaskovic, Kelleher, & Mclafferty, 1996;Wilm et al, 1996). Many efforts have been devoted to make the nano-ESI a routine tool for bio-molecule ionization (Carte et al, 2001;Pope et al, 2001). Currently, the studies on ESI are still a focus for further improvement in sample consumption and ion throughput in terms of its application and mechanism.…”