Renata Soares

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.

Differential proteomics of dehydration and rehydration in bryophytes: evidence towards a common desiccation tolerance mechanism

All bryophytes evolved desiccation tolerance (DT) mechanisms during the invasion of terrestrial habitats by early land plants. Are these DT mechanisms still present in bryophytes that colonize aquatic habitats? The aquatic bryophyte Fontinalis antipyretica Hedw. was subjected to two drying regimes and alterations in protein profiles and sucrose accumulation during dehydration and rehydration were investigated. Results show that during fast dehydration, there is very little variation in protein profiles, and upon rehydration proteins are leaked. On the other hand, slow dehydration induces changes in both dehydration and rehydration protein profiles, being similar to the protein profiles displayed by the terrestrial bryophytes Physcomitrella patens (Hedw.) Bruch and Schimp. and, to what is comparable with Syntrichia ruralis (Hedw.) F. Weber and D. Mohr. During dehydration there was a reduction in proteins associated with photosynthesis and the cytoskeleton, and an associated accumulation of proteins involved in sugar metabolism and plant defence mechanisms. Upon rehydration, protein accumulation patterns return to control values for both photosynthesis and cytoskeleton whereas proteins associated with sugar metabolism and defence proteins remain high. The current results suggest that bryophytes from different ecological adaptations may share common DT mechanisms.

Chronic sustained hypoxia-induced redox remodeling causes contractile dysfunction in mouse sternohyoid muscle

Chronic sustained hypoxia (CH) induces structural and functional adaptations in respiratory muscles of animal models, however the underlying molecular mechanisms are unclear. This study explores the putative role of CH-induced redox remodeling in a translational mouse model, with a focus on the sternohyoid—a representative upper airway dilator muscle involved in the control of pharyngeal airway caliber. We hypothesized that exposure to CH induces redox disturbance in mouse sternohyoid muscle in a time-dependent manner affecting metabolic capacity and contractile performance. C57Bl6/J mice were exposed to normoxia or normobaric CH (FiO2 = 0.1) for 1, 3, or 6 weeks. A second cohort of animals was exposed to CH for 6 weeks with and without antioxidant supplementation (tempol or N-acetyl cysteine in the drinking water). Following CH exposure, we performed 2D redox proteomics with mass spectrometry, metabolic enzyme activity assays, and cell-signaling assays. Additionally, we assessed isotonic contractile and endurance properties ex vivo. Temporal changes in protein oxidation and glycolytic enzyme activities were observed. Redox modulation of sternohyoid muscle proteins key to contraction, metabolism and cellular homeostasis was identified. There was no change in redox-sensitive proteasome activity or HIF-1α content, but CH decreased phospho-JNK content independent of antioxidant supplementation. CH was detrimental to sternohyoid force- and power-generating capacity and this was prevented by chronic antioxidant supplementation. We conclude that CH causes upper airway dilator muscle dysfunction due to redox modulation of proteins key to function and homeostasis. Such changes could serve to further disrupt respiratory homeostasis in diseases characterized by CH such as chronic obstructive pulmonary disease. Antioxidants may have potential use as an adjunctive therapy in hypoxic respiratory disease.

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.

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

Mitochondrial proteomics of the acetic acid – induced programmed cell death response in a highly tolerant Zygosaccharomyces bailii – derived hybrid strain

Very high concentrations of acetic acid at low pH induce programmed cell death (PCD) in both the experimental model Saccharomyces cerevisiae and in Zygosaccharomyces bailii, the latter being considered the most problematic acidic food spoilage yeast due to its remarkable intrinsic resistance to this food preservative. However, while the mechanisms underlying S. cerevisiae PCD induced by acetic acid have been previously examined, the corresponding molecular players remain largely unknown in Z. bailii. Also, the reason why acetic acid concentrations known to be necrotic for S. cerevisiae induce PCD with an apoptotic phenotype in Z. bailii remains to be elucidated. In this study, a 2-DE-based expression mitochondrial proteomic analysis was explored to obtain new insights into the mechanisms involved in PCD in the Z. bailii derived hybrid strain ISA1307. This allowed the quantitative assessment of expression of protein species derived from each of the parental strains, with special emphasis on the processes taking place in the mitochondria known to play a key role in acetic acid - induced PCD. A marked decrease in the content of proteins involved in mitochondrial metabolism, in particular, in respiratory metabolism (Cor1, Rip1, Lpd1, Lat1 and Pdb1), with a concomitant increase in the abundance of proteins involved in fermentation (Pdc1, Ald4, Dld3) was registered. Other differentially expressed identified proteins also suggest the involvement of the oxidative stress response, protein translation, amino acid and nucleotide metabolism, among other processes, in the PCD response. Overall, the results strengthen the emerging concept of the importance of metabolic regulation of yeast PCD.

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.