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No biomarker has yet been discovered to identify the reproductive status of the endangered leatherback sea turtle (Dermochelys coriacea). Although vitellogenin (VTG) could be used for this, its sequence is not known in D. coriacea and no quantitative assay has been carried out in this species to date. Using de novo sequencing-based proteomics, we unambiguously characterized sequences of two different VTG isoforms that we named Dc-VTG1 and Dc-VTG2. To our knowledge, this is the first clear evidence of different VTG isoforms and the structural characterization of derived yolk proteins in reptiles. This work illustrates how massive de novo sequencing can characterize novel sequences when working on "exotic" nonmodel species in which even nucleotide sequences are not available. We developed assays for absolute quantitation of these two isoforms using selected reaction monitoring (SRM) mass spectrometry, thus providing the first SRM assays developed specifically for a nonsequenced species. Plasma levels of Dc-VTG1 and Dc-VTG2 decreased as the nesting season proceeded, and were closely related to the increased levels of reproductive effort. The SRM assays developed here therefore provide an original and efficient approach for the reliable monitoring of reproduction cycles not only in D. coriacea, but potentially in other turtle species.

Staphylococcus aureus is responsible for severe bacterial infections in hospitals and healthcare facilities. It produces single and bicomponent toxins (leukotoxins and hemolysins) that hinder innate immune function. Leukotoxin subunits bind to leukocyte cell membrane thus inducing transmembrane pores and subsequently, cell lysis. Leukotoxin LukE/D is a member of the bicomponent toxin family, but to date, no study concerning its involvement in host-pathogen interactions has been reported. In the present study, we performed the proteomic analysis of the secretions recovered after activation of human neutrophils by leukotoxin LukE/D. The neutrophil secretions were purified by RP-HPLC and different fractions were analyzed by Edman sequencing, LC-MS/MS, immunoblotted for chromogranin-derived peptides and further analyzed for antimicrobial properties. Proteomic analysis revealed that neutrophil secretions constitute a large number of proteins related with immune boosting mechanisms, proteolytic degradation, inflammatory process and antioxidant reactions.

Transcription factors and their regulators possess "basic amino acid free domains" which modulate transcriptional gene activation. We aimed at optimizing a MALDI mass spectrometry (MS) analytical method for the characterization of such domains after protein enzymatic digestion. A panel of recombinant transcription factors with different basic residue contents was proteolytically digested with the Asp-N endoprotease and resulting peptide mixtures were analyzed by MALDI-MS with alpha-cyano-4-hydroxy-cinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) as matrix. We found that peptides without lysine, arginine, histidine (Lys/Arg/His free peptides) were efficiently detected in the positive ion mode only when using DHB. These findings proved to be very useful for two different targeted proteomic applications. Indeed, the MALDI-MS/MS identification of the CARM1 proteolytic cleavage site, which happens in a Lys/Arg/His free domain, could only be achieved using the DHB matrix. Moreover, in routine proteomic analyses, the detection efficiency of Lys/Arg/His free C-terminal peptides of two-dimensional gel separated proteins was strongly enhanced when DHB was used instead of CHCA.