Typic: A Practical and Robust Tool to Rank Proteotypic Peptides for Targeted Proteomics.

The selection of a suitable proteotypic peptide remains a challenge for designing a targeted quantitative proteomics assay. Although the criteria are well-established in the literature, the selection of these peptides is often performed in a subjective and time-consuming manner. Here, we have developed a practical and semiautomated workflow implemented in an open-source program named Typic. Typic is designed to run in a command line and a graphical interface to help selecting a list of proteotypic peptides for targeted quantitation. The tool combines the input data and downloads additional data from public repositories to produce a file per protein as output. Each output file includes relevant information to the selection of proteotypic peptides organized in a table, a colored ranking of peptides according to their potential value as targets for quantitation and auxiliary plots to assist users in the task of proteotypic peptides selection. Taken together, Typic leads to a practical and straightforward data extraction from multiple data sets, allowing the identification of most suitable proteotypic peptides based on established criteria, in an unbiased and standardized manner, ultimately leading to a more robust targeted proteomics assay.

Bothrops jararaca venom proteome rearrangement upon neonate to adult transition

The pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Similarly, the diet of this species changes from ectothermic prey in early life to endothermic prey in adulthood. In this study we used large and representative newborn and adult venom samples consisting of pools from 694 and 110 specimens, respectively, and demonstrate a significant ontogenetic shift in the venom proteome complexity of B. jararaca. 2-DE coupled to MS protein identification showed a clear rearrangement of the toxin arsenalboth in terms of the total proteome, as of the glycoproteome. N-glycosylation seems to play a key role in venom protein variability between newborn and adult specimens. Upon the snakedevelopment, the subproteome of metalloproteinases undergoes a shift from a P-III-rich to a P-I-rich profile while the serine proteinase profile does not vary significantly. We also usedisobaric tag labeling (iTRAQ) of venom tryptic peptides for the first time to examine the quantitative changes in the venom toxins of B. jararaca upon neonate to adult transition. TheiTRAQ analysis showed changes in various toxin classes, especially the proteinases. Our study expands the in-depth understanding of venom complexity variation particularly withregard to toxin families that have been associated with envenomation pathogenesis.

Disintegrin-like/cysteine-rich domains of the reprolysin HF3 Site-directed mutagenesis reveals essential role of specific residues

Little is known about the biochemical properties of the non-catalytic domains of snake venom metalloproteinases (SVMPs). The ECD sequence of the disintegrin-like domain (D-domain) has been assigned as the disintegrin motif and, recently, the hyper-variable region (HVR) of the cysteine-rich domain (C-domain) was suggested to constitute a potential protein-protein adhesive interface. Here we show that the recombinant C-domain of HF3, a hemorrhagic SVMP from Bothrops jararaca, as well as three peptides resembling its HVR, inhibit collagen-induced platelet aggregation, which indicates a role for the C-domain and its HVR in targeting HF3 to platelets. Site-directed mutagenesis was used for the first time to identify charged residues essential for the functionality of the disintegrin-like/cysteine-rich domains (DC-domains). Residues of the disintegrin loop (E467 and D469), and of the HVR (K568, K569 and K575) of HF3 were individually mutated to Ala. Interestingly, only the mutant D469A was obtained in soluble form in Escherichia coli and this single mutation caused loss of two functional activities of the DC-domains: inhibition of platelet aggregation and increase of leukocyte rolling in the microcirculation. In summary we demonstrate that the C-domain and its HVR are critical for HF3 to affect platelets and leukocytes, however, the disintegrin loop may be important for the functionality of the D-domain in the context of the C-domain.

A Comparative Study of the Outer Membrane Proteome from an Atypicaland a Typical Enteropathogenic Escherichia coli

This study compared the proteomic profile of outer membrane proteins (OMPs) from one strain of atypicalenteropathogenic Escherichia coli (aEPEC) and one of typical EPEC (tEPEC). The OMPs fractions were obtained using sarcosine extraction, and analyzed by one- and two-dimensional gel electrophoresis (1DE and 2DE, respectively). The 1DE OMPs analysis of typical and atypical EPEC evidenced similar patterns; however, the 2DE OMP profile from the aEPEC revealed more protein spots in the 40- to 70-kDa region. 2DE image analysis identified 159 protein spots in bothstrains whereas 53 protein spots were observed only in tEPEC and 128 were observed only in aEPEC. Remarkably, 41.5% of aEPEC spots showed higher levels of expression compared to tEPEC, some of which with two, others four or even five times more. Twenty-four selected spots were identified using MALDI-TOF mass spectrometry and they corresponded to proteins involved in cell structure and metabolism, as well as in gene regulation. Some of these proteins showed similarity with proteins identified in other E. coli pathotypes. Besides, the differential expression of some proteins in aEPEC may suggest that it could be related to their features that ascertain the adaptation to distinct environments and the worldwidespread distribution of these pathogens.

High resolution analysis of snake venom metalloproteinase (SVMP) peptide bond cleavage specificity using proteome based peptide libraries and mass spectrometry. (AU)

Both serine and metalloproteinases have been shown to play the role of toxins in the venoms of many snakes. Determination of the natural protein substrates of these toxins is an important feature in the toxinological characterization of these proteinases. Furthermore, characterization of their peptide bond specificity is of value for understanding active site preference of the proteinase associated with effective proteolysis as well as of use in the design of peptide substrates and inhibitor lead compounds. Typically the determination of peptide bond cleavage specificity of snake venom serine proteinases (SVSPs) and snake venom metalloproteinases (SVMPs) has been performed using limited sets of peptides or small oligopeptides as experimental substrates. Although this approach has yielded valuable data it is generally limited in scope due to the relatively small sets of substrates used to generate the consensus specificity sequences for these proteinases. In this study we use a large, plasma based, proteome-derived peptide library as substrates along with mass spectrometry to explore the peptide bond specificity of three PI SVMPs and one PIII SVMP to determine their individual peptide cleavage consensus sequences. All of the proteinases assayed displayed a clear preference for a leucine residue in the P1Œ site. Careful analysis of the specificity profiles of the SVMPs examined showed interesting differences in the preferences at the other P and PŒ sites suggesting functional differences between these proteinases.

Autolysis at the disintegrin domain of patagonfibrase, a metalloproteinase fromPhilodryas patagoniensis (Patagonia Green Racer; Dipsadidae) venom

Patagonfibrase is a 57.5-kDa hemorrhagic metalloproteinase isolated from the venom of Philodryas patagoniensis (Patagonia Green Racer), a South American rear-fanged snake. Herein we demonstrate that patagonfibrase undergoes autolysis at its pH optimum (7.5) and at 37 ¡ÆC, primarily producing a ¡­ 32.6 kDa fragment composed of disintegrin-like and cysteine-rich domains, as identified by mass spectrometry and N-terminal sequencing. The autolysis site for production of this fragment is similar to that observed for metalloproteinases from front-fanged Viperidae snake venoms. In the presence of Ca2+, patagonfibrase was only partially autolysed, giving rise mainly to one fragment of ¡­ 52.2 kDa. In addition, calcium markedly enhanced the azocaseinolytic activity of patagonfibrase. Our findings contribute to the understanding of the structural and mechanistic bases of this family of metalloenzymes that are widely distributed among snake venoms, demonstrating that important post-translational modifications such as proteolysis can also contribute to the diversity and complexity of proteins found in rear-fanged snake venoms.