Venoms, venomics, antivenomics

Venoms comprise mixtures of peptides and proteins tailored by Natural Selection to act on vital systems of the prey or victim. Here we review our proteomic protocols for uncoiling the composition, immunological profile, and evolution of snake venoms. Our long-term goal is to gain a deep insight of all viperid venom proteomes. Knowledge of the inter- and intraspecies ontogenetic, individual, and geographic venom variability has applied importance for the design of immunization protocols aimed at producing more effective polyspecific antivenoms. A practical consequence of assessing the cross-reactivity of heterologous antivenoms is the possibility of circumventing the restricted availability of species-specific antivenoms in some regions. Further, the high degree of target specificity makes toxins valuable scaffolds for drug development.     

Biomarker discovery in asthma‐related inflammation and remodeling

Asthma is a complex inflammatory disease of airways. A network of reciprocal interactions between inflammatory cells, peptidic mediators, extracellular matrix components, and proteases is thought to be involved in the installation and maintenance of asthma‐related airway inflammation and remodeling. To date, new proteic mediators displaying significant activity in the pathophysiology of asthma are still to be unveiled. The main objective of this study was to uncover potential target proteins by using surface‐enhanced laser desorption/ionization‐time of flight‐mass spectrometry (SELDI‐TOF‐MS) on lung samples from mouse models of allergen‐induced airway inflammation and remodeling. In this model, we pointed out several protein or peptide peaks that were preferentially expressed in diseased mice as compared to controls. We report the identification of different five proteins: found inflammatory zone 1 or RELMα (FIZZ‐1), calcyclin (S100A6), clara cell secretory protein 10 (CC10), Ubiquitin, and Histone H4.     

LuMPIS – A modified luminescence‐based mammalian interactome mapping pull‐down assay for the investigation of protein–protein interactions encoded by GC‐low ORFs

The GC content is highly variable among the genomes of different organisms. It has been shown that recombinant gene expression in mammalian cells is much more efficient when GC‐rich coding sequences of a certain protein are used. In order to study protein–protein interactions in Varicella zoster virus, a GC‐low herpesvirus, we have developed a novel luminescence‐based maltose‐binding protein pull‐down interaction screening system (LuMPIS) that is able to overcome the impaired protein expression levels of GC‐low ORFs in mammalian expression systems.     

Proteomic analysis of hepatic ischemia/reperfusion injury and ischemic preconditioning in mice revealed the protective role of ATP5β

Hepatic ischemia/reperfusion (I/R) injury is an inevitable consequence during liver surgery. Ischemic preconditioning (IPC) has been shown to protect the livers from I/R injury, partially mediated by preservation of hepatic ATP contents. However, the precise molecular mechanisms of these events remain poorly elucidated. In this study, liver proteomes of the mice subjected to I/R injury pretreated with or without IPC were analyzed using 2‐DE combined with MALDI‐TOF/TOF mass analysis. Twenty proteins showing more than 1.5‐fold difference were identified in the livers upon I/R injury. Among these proteins, four proteins were further regulated by IPC when compared with nonpretreated controls. One of these proteins, ATP synthase β subunit (ATP5β) catalyzes the rate‐limiting step of ATP formation. The expression level of ATP5β, which was further validated by Western blot analysis, was significantly decreased upon I/R injury while turned over by IPC pretreatment. Change pattern of hepatic ATP corresponded with that of ATP5β expression, indicating that increasing hepatic ATP5β expression might be a reason for ATP‐preserving effect of IPC. In summary, this study provided new clues for understanding the mechanisms of IPC against I/R injury. The protective role of ATP5β might give evidences for developing new therapeutic approaches against hepatic I/R injury.     

HUPO World Congress Publication Committee Meeting August 2008, Amsterdam,The Netherlands

The plenary session of the Publications Committee of the Human Proteome Organisation at the 7^th annual HUPO world congress examined the relationship between journals, proteomics standardization initiatives, such as the work of the HUPO‐PSI, and the public domain data repositories. Delegates from industry, academia and the publishing houses discussed how best to bring these bodies closer to together and facilitate the publication process for the bench scientist.     

Proteome profiling of aging in mouse models: Differential expression of proteins involved in metabolism,transport, and stress response in kidney

Aging is a time‐dependent complex biological phenomenon observed in various organs and organelles of all living organisms. To understand the molecular mechanism of age‐associated functional loss in aging kidneys, we have analyzed the expression of proteins in the kidneys of young (19–22 wk) and old (24 months) C57/BL6 male mice using 2‐DE followed by LC‐MS/MS. We found that expression levels of 49 proteins were upregulated (p ≤ 0.05), while that of only ten proteins were downregulated (p ≤ 0.05) due to aging. The proteins identified belong to three broad functional categories: (i) metabolism (e.g., aldehyde dehydrogenase family, ATP synthase β‐subunit, malate dehydrogenase, NADH dehydrogenase (ubiquinone), hydroxy acid oxidase 2), (ii) transport (e.g., transferrin), and (iii) chaperone/stress response (e.g., Ig‐binding protein, low density lipoprotein receptor‐related protein associated protein 1, selenium‐binding proteins (SBPs)). Some proteins with unknown functions were also identified as being differentially expressed. ATP synthase β subunit, transferrin, fumarate hydratase, SBPs, and albumin are present in multiple forms, possibly arising due to proteolysis or PTMs. The above functional categories suggest specific mechanisms and pathways for age‐related kidney degeneration.     

Mixed-model of ANOVA for measurement reproducibility in proteomics

This work is a statistical analysis of reproducibility of a MALDI-TOF mass spectrometry experiment. Its aim is to evaluate measurement variability and compare peak intensities from two types of MALDI-TOF platforms. We compared and commented on the abilities of Principal Component Analysis and mixed-model analysis of variance to evaluate the biological variability and the technical variability of peak intensities in different patients. The properties and hypotheses of both methods are summarized and applied to spectra from plasma of patients with Hodgkin lymphoma. Principal Component Analysis checks rapidly the balance between the two variabilities; however, a mixed-model analysis of variance is necessary to quantify the biological and technical components of the experimental variance as well as their interactions and to split the total variance into between-subjects and within-subject components. The latter method helped to assess the reproducibility of measurements from two MALDI-TOF platforms and to decompose the technical variability according to the experimental design.