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

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), high-performance 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 nano-electrospray 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 (MS³)and four-stage (MS⁴)tandem experiments, whereas classical tandem mass spectrometry (MS²)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 MS³ and MS⁴ compared with the MS² 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 .