Elisabete Pires

Iminoboronates: a new strategy for reversible protein modification.

Protein modification has entered the limelight of chemical and biological sciences, since, by appending small molecules into proteins surfaces, fundamental biological and biophysical processes may be studied and even modulated in a physiological context. Herein we present a new strategy to modify the lysines ε-amino group and the proteins N-terminal, based on the formation of stable iminoboronates in aqueous media. This functionality enables the stable and complete modification of these amine groups, which can be reversible upon the addition of fructose, dopamine, or glutathione. A detailed DFT study is also presented to rationalize the observed stability toward hydrolysis of the iminoboronate constructs.

Mass spectrometry and animal science: Protein identification strategies and particularities of farm animal species ☆

Proteomic approaches are gaining increasing importance in the context of all fields of animal and veterinary sciences, including physiology, productive characterization, and disease/parasite tolerance, among others. Proteomic studies mainly aim the proteome characterization of a certain organ, tissue, cell type or organism, either in a specific condition or comparing protein differential expression within two or more selected situations. Due to the high complexity of samples, usually total protein extracts, proteomics relies heavily on separation procedures, being 2D-electrophoresis and HPLC the most common, as well as on protein identification using mass spectrometry (MS) based methodologies. Despite the increasing importance of MS in the context of animal and veterinary science studies, the usefulness of such tools is still poorly perceived by the animal science community. This is primarily due to the limited knowledge on mass spectrometry by animal scientists. Additionally, confidence and success in protein identification is hindered by the lack of information in public databases for most of farm animal species and their pathogens, with the exception of cattle (Bos taurus), pig (Sus scrofa) and chicken (Gallus gallus). In this article, we will briefly summarize the main methodologies available for protein identification using mass spectrometry providing a case study of specific applications in the field of animal science. We will also address the difficulties inherent to protein identification using MS, with particular reference to experiments using animal species poorly described in public databases. Additionally, we will suggest strategies to increase the rate of successful identifications when working with farm animal species.

Understanding regeneration through proteomics

Regeneration is a complex cellular process that, rather than simply forming a scar following injury, the animal forms a new functional tissue. Regeneration is a widespread process among metazoa, although not uniformly. Planaria, starfish, and some worms can regenerate most of their body, whereas many other species can only regenerate parts of specific tissues or fail to accomplish a functional regrowth, as is the case of mammals CNS. Research in regenerative medicine will possibly culminate in the regeneration of organs/tissues originally not prone to this process. Despite the complexity of the interactions and regulatory systems involved, the variety of tissues and organs these cells differentiate into has so far impaired the success of direct transplantation to restore damaged tissues. For this reason, a study, at the molecular level of the regeneration mechanisms developed by different animal models is likely to provide answers to why these processes are not readily activated in mammals. Proteomic‐based approaches are being recognized as extremely useful to study of regeneration events, also because there is a relevant contribution of posttranscriptional processes that involve frequently the occurrence of a broad range of PTMs. The present review focuses on the significant knowledge brought up by proteomics in diverse aspects of regeneration research on different animal models, tissues, and organs.

Comparative Proteomic Profiling of Ehrlichia ruminantium Pathogenic Strain and Its High-Passaged Attenuated Strain Reveals Virulence and Attenuation-Associated Proteins.

The obligate intracellular bacterium Ehrlichia ruminantium (ER) causes heartwater, a fatal tick-borne disease in livestock. In the field, ER strains present different levels of virulence, limiting vaccine efficacy, for which the molecular basis remains unknown. Moreover, there are no genetic tools currently available for ER manipulation, thus limiting the knowledge of the genes/proteins that are essential for ER pathogenesis and biology. As such, to identify proteins and/or mechanisms involved in ER virulence, we performed the first exhaustive comparative proteomic analysis between a virulent strain (ERGvir) and its high-passaged attenuated strain (ERGatt). Despite their different behaviors in vivo and in vitro, our results from 1DE-nanoLC-MS/MS showed that ERGvir and ERGatt share 80% of their proteins; this core proteome includes chaperones, proteins involved in metabolism, protein-DNA-RNA biosynthesis and processing, and bacterial effectors. Conventional 2DE revealed that 85% of the identified proteins are proteoforms, suggesting that post-translational modifications (namely glycosylation) are important in ER biology. Strain-specific proteins were also identified: while ERGatt has an increased number and overexpression of proteins involved in cell division, metabolism, transport and protein processing, ERGvir shows an overexpression of proteins and proteoforms (DIGE experiments) involved in pathogenesis such as Lpd, AnkA, VirB9 and B10, providing molecular evidence for its increased virulence in vivo and in vitro. Overall, our work reveals that ERGvir and ERGatt proteomes are streamlined to fulfill their biological function (maximum virulence for ERGvir and replicative capacity for ERGatt), and we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium.

Adhesive proteins of stalked and acorn barnacles display homology with low sequence similarities.

Barnacle adhesion underwater is an important phenomenon to understand for the prevention of biofouling and potential biotechnological innovations, yet so far, identifying what makes barnacle glue proteins ‘sticky’ has proved elusive. Examination of a broad range of species within the barnacles may be instructive to identify conserved adhesive domains. We add to extensive information from the acorn barnacles (order Sessilia) by providing the first protein analysis of a stalked barnacle adhesive, Lepas anatifera (order Lepadiformes). It was possible to separate the L. anatifera adhesive into at least 10 protein bands using SDS-PAGE. Intense bands were present at approximately 30, 70, 90 and 110 kilodaltons (kDa). Mass spectrometry for protein identification was followed by de novo sequencing which detected 52 peptides of 7–16 amino acids in length. None of the peptides matched published or unpublished transcriptome sequences, but some amino acid sequence similarity was apparent between L. anatifera and closely-related Dosima fascicularis. Antibodies against two acorn barnacle proteins (ab-cp-52k and ab-cp-68k) showed cross-reactivity in the adhesive glands of L. anatifera. We also analysed the similarity of adhesive proteins across several barnacle taxa, including Pollicipes pollicipes (a stalked barnacle in the order Scalpelliformes). Sequence alignment of published expressed sequence tags clearly indicated that P. pollicipes possesses homologues for the 19 kDa and 100 kDa proteins in acorn barnacles. Homology aside, sequence similarity in amino acid and gene sequences tended to decline as taxonomic distance increased, with minimum similarities of 18–26%, depending on the gene. The results indicate that some adhesive proteins (e.g. 100 kDa) are more conserved within barnacles than others (20 kDa).

Radial nerve cord protein phosphorylation dynamics during starfish arm tip wound healing events

Echinoderms, as invertebrate deuterostomes, have amazing neuronal intrinsic growth aptitude triggered at any time point during the animal lifespan leading to successful functional tissue regrowth. This trait is known to be in opposition to their mammal close phylogenic relatives that have lost the ability to regenerate their central nervous system. Despite the promising nature of this intrinsic echinoderm trait, it was only recently that this complex biological event started to be unveiled. In the present study, a 2DE gel‐based phosphoproteomics approach was used to investigate changes in starfish neuronal protein phosphorylation states at two different wound healing time‐graded events following arm tip amputation, 48 h and 13 days. Among the resolved protein spots in 3.0–5.6 NL pH IEF strips, 190, 142, and 124 had a phosphoprotein signal in the control and the two injury experimental groups, respectively. Gel image analysis, highlighted 129 spots with an injury‐related protein phosphorylation dynamics, several being exclusively phosphorylated in controls (72 spots), injured nerves (8 spots) or, showing significantly different phosphorylation ratios (37 spots). Within these, a total of 43 proteins were identified with MALDI‐TOF/TOF. Altogether, several intervening proteins of important injury‐signaling pathways that seem to be modulated through phosphorylation, were identified for the first time in starfish radial nerve cord early regeneration events. These include cytoskeleton re‐organization toward the formation of the neuronal growth cones; cell membrane rearrangements, actin filaments, and microtubules dynamics; mRNA binding and transport; lipid signaling; Notch pathway; and neuropeptide processing.

Tandem Mass Spectrometry of Peptides

Renata Soares1, Elisabete Pires1, Andre M. Almeida1,2, Romana Santos1,3, Ricardo Gomes1, Kamila Koci1, Catarina Ferraz Franco1 and Ana Varela Coelho1 1ITQB/Universidade Nova de Lisboa 2Instituto de Investigacao Cientifica Tropical& Centro Interdisciplinar de Investigacao em Sanidade Animal 3Unidade de Investigacao em Ciencias Orais e Biomedicas, Faculdade de Medicina Dentaria/Universidade de Lisboa Portugal

Mass spectrometry for the veterinary and farm animal world

Proteomics approaches are gaining increasing importance in the context of all fields of animal and veterinary sciences, including physiology, productive characterization, and disease/parasite tolerance, among others. Proteomic studies mainly aim at the proteome characterization of a certain organ, tissue, cell type or organism, either in a specific condition or comparing protein differential expression within two or more selected situations. Due to the high complexity of samples, usually total protein extracts, proteomic studies rely heavily on protein identification and quantification using mass spectrometry (MS) based methodologies or with coupling to other methodologies, like liquid chromatography and gel electrophoresis. Despite the increasing importance of MS in the context of animal and veterinary sciences studies, the usefulness of such tools is still poorly perceived by the Animal Science community. This is primarily due to limited knowledge on Mass Spectrometry by animal scientists, which use nowadays still requires a high level of specialization. Additionally, confidence and success in protein identification is hindered by the lack of information in public databases for most of farm animal species and their pathogens, with the exception of cattle (Bos taurus), pig (Sus scrofa) and chicken (Gallusgallus). During this lecture, a description of the main MS methodologies available for proteome characterization and differential proteomic studies will be presented.