Putting the Pieces Together: High-performance LC-MS/MS Provides Network-, Pathway-, and Protein-level Perspectives in Populus

High-performance mass spectrometry (MS)-based proteomics enabled the construction of a detailed proteome atlas for Populus, a woody perennial plant model organism. Optimization of experimental procedures and implementation of current state-of-the-art instrumentation afforded the most detailed look into the predicted proteome space of Populus, offering varying proteome perspectives: (1) network-wide, (2) pathway-specific, and (3) protein-level viewpoints. Together, enhanced protein retrieval through a detergent-based lysis approach and maximized peptide sampling via the dual-pressure linear ion trap mass spectrometer (LTQ Velos), have resulted in the identification of 63,056 tryptic peptides. The technological advancements, specifically spectral-acquisition and sequencing speed, afforded the deepest look into the Populus proteome, with peptide abundances spanning 6 orders of magnitude and mapping to ∼25% of the predicted proteome space. In total, tryptic peptides mapped to 11,689 protein assignments across four organ-types: mature (fully expanded, leaf plastichronic index (LPI) 10–12) leaf, young (juvenile, LPI 4–6) leaf, root, and stem. To resolve protein ambiguity, identified proteins were grouped by sequence similarity (≥ 90%), thereby reducing the protein assignments into 7538 protein groups. In addition, this large-scale data set features the first systems-wide survey of protein expression across different Populus organs. As a demonstration of the precision and comprehensiveness of the semiquantitative analysis, we were able to contrast two stages of leaf development, mature versus young leaf. Statistical comparison through ANOVA analysis revealed 1432 protein groups that exhibited statistically significant (p ≤ 0.01) differences in protein abundance. Experimental validation of the metabolic circuitry expected in mature leaf (characterized by photosynthesis and carbon fixation) compared with young leaf (characterized by rapid growth and moderate photosynthetic activities) strongly testifies to the credibility of the approach. Instead of quantitatively comparing a few proteins, a systems view of all the changes associated with a given cellular perturbation could be made.Mass spectrometry (MS)-based proteomics has experienced tremendous growth in recent years, leading to the establishment of numerous protocols, platforms, and workflows for the characterization of protein expression at the genome level (1). Although these advancements have facilitated comprehensive proteomic investigations of simple bacterial isolates and microbial communities, the application of MS-based proteomics for plants and other higher eukaryotes remains underdeveloped. Recently, large-scale proteomic studies have been directed at characterization of Populus, a woody perennial model organism. With the recent release and subsequent curation of the P. trichocarpa genome (2), these large-scale MS-based proteomic investigations offer the potential to introduce new biological insights into woody perennial plant biology (3, 4, 5). For example, we have recently demonstrated the ability to measure ∼17% of the Populus proteome by coupling multidimensional liquid chromatography (MudPIT)¹ with nano-electrospray tandem mass spectrometry (2D-LC-MS/MS) (6). Relative to the two-dimensional gel-based approaches (7), MudPIT provides enhanced separation and when used in conjunction with MS/MS, surpasses the throughput and number of identifiable proteins detected in complex mixtures (8). Although we have demonstrated the general effectiveness of this approach, the identification and quantitation of the proteins expressed in a plant cell or tissue are still notoriously complicated by a number of factors, including the size and complexity of plant genomes, abundance of protein variants, as well as the dynamic range of protein identification. To overcome these challenges, improvements are needed in sample preparation, MS instrumentation, and data interpretation.The architecture of plant cell walls provides resistance to chemical and biological degradation, thus requiring mechanical and detergent-based lysis for optimal proteome analysis. However, this criterion presents a major challenge for plant proteomic research using electrospray mass spectrometry, as detergent-containing solutions can impede enzymatic digestion and cause significant analyte suppression (9). Therefore, most plant proteomic studies using the “MudPIT” strategy apply mechanical disruption in conjunction with a detergent-free preparation method (10). Typically, strong chaotropic agents such as urea and guanidine hydrochloride are used for the extraction, denaturation, and digestion of proteins. In a recent study, Mann et al. (2009) introduced a filter-aided sample preparation (FASP) method that uses and effectively removes sodium dodecyl sulfate (SDS) before enzymatic digestion and electrospray analysis (11). This study demonstrated enhanced retrieval of peptides from biological materials, yielding a more accurate representation of the proteome. We developed a similar experimental approach for extraction of proteins from plant tissue to obtain a more comprehensive, unbiased proteome characterization well beyond that achievable with currently available methods. Similar to the FASP method, we demonstrate the power of SDS for proteomic sample preparation, not only in its ability to more-thoroughly lyse cells, but also its ability to better solubilize both hydrophilic and hydrophobic proteins. This powerful attribute gives proteolytic enzymes maximum opportunity to generate peptides specific to their cleavage potential so that at least a few representative peptides can be obtained for proteins that would have otherwise been discarded or lost because of insolubility, e.g. membrane-bound proteins. Rather than performing a buffer exchange with urea, depletion of SDS is achieved by precipitating proteins out of solution using trichloroacetic acid.Characterization of protein expression in plants is further complicated by the heterogeneous mixture of various cell types, each with a unique proteome signature and individualized response to environmental chemical or physical signals. This inherent complexity of plant proteomes and the large dynamic range in protein abundance overwhelms current analytical platforms (12). Moreover, biochemical regulatory networks in plants are more elaborate and dynamic than in microbial species; consequently, many biological components are left undiscovered, including modified peptides and low-abundance proteins (13, 14, 15). Recent developments in ion-trap MS instrumentation, namely the dual-pressure linear ion trap mass spectrometer (LTQ Velos), have demonstrated improved ability to comprehensively characterize complex proteomics samples (16). Featuring a newly designed ion source and a two-chamber ion trap mass analyzer, the LTQ Velos achieves greater dynamic range, sensitivity, and speed of spectral acquisition when applied to complex proteomic samples. Cumulatively, the technological advancements afford substantial increases in the detection and identification of both proteins and unique peptides when compared with existing state-of-the-art technologies. Therefore, to satisfy the need for depth of proteome characterization in plants, we apply the newly developed LTQ Velos for mass spectrometry measurements of the Populus proteome.For most terrestrial plants, life begins and ends in the same physical location. For woody perennial plants, this sedentary lifestyle may last thousands of years. One consequence of this lifestyle is that each plant typically experiences dramatic changes in its ambient environment throughout its lifetime and, at any given time, equilibrium between endogenous growth processes and exogenous constraints exerted by the environment must be tightly controlled. To survive under varying environmental conditions, temporal plastic responses evoke patterns of protein expression that progressively influence morphological, anatomical, and functional traits of three principal organs—leaf, root, and stem. Collectively and individually, these organs operate to perceive and respond to periodic and chronic environment conditions. Currently, a comprehensive understanding of the spatial variation in protein expression patterns across the organ types is lacking for woody perennial plants, in which most large-scale proteome analyses with Populus were performed on isolated organs, tissues, organelles, or subcellular structures. For this reason, we combined the state-of-the-art LTQ-Velos platform with the SDS/TCA sample preparation methodology to generate a high-coverage proteome atlas of the principal organ types from Populus.     

Sexuality Generates Diversity in the Aflatoxin Gene Cluster: Evidence on a Global Scale

Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B₁ being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the null hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B₁-dominant and G₁-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.     

Adenosine-Mediated Enteric Neuromuscular Function Is Affected during Herpes Simplex Virus Type 1 Infection of Rat Enteric Nervous System

Adenosine plays an important role in regulating intestinal motility and inflammatory processes. Previous studies in rodent models have demonstrated that adenosine metabolism and signalling are altered during chronic intestinal inflammatory diseases. However, the involvement of the adenosinergic system in the pathophysiology of gut dysmotility associated to a primary neurodysfunction is still unclear. Recently, we showed that the neurotropic Herpes simplex virus type-1 (HSV-1), orally inoculated to rodents, infects the rat enteric nervous system (ENS) and affects gut motor function without signs of systemic infection. In this study we examined whether changes in purinergic metabolism and signaling occur during permanent HSV-1 infection of rat ENS. Using isolated organ bath assays, we found that contraction mediated by adenosine engagement of A₁ or A_2A receptors was impaired at 1 and 6 weeks post-viral administration. Immunofluorescence studies revealed that viral infection of ENS led to a marked redistribution of adenosine receptors: A₁ and A_2B receptors were confined to the muscle layers whereas A_2A and A₃ receptors were expressed mainly in the myenteric plexus. Viral-induced ENS neurodysfunction influenced adenosine metabolism by increasing adenosine deaminase and CD73 levels in longitudinal muscle-myenteric plexus with no sign of frank inflammation. This study provides the first evidence for involvement of the adenosinergic system during HSV-1 infection of the ENS. As such, this may represent a valid therapeutic target for modulating gut contractility associated to a primary neurodysfunction.     

Gerstmann-Sträussler-Scheinker disease with P102L-V129 mutation: a case with psychiatric manifestations at onset

Gerstmann-Str?ussler-Scheinker disease (GSS) is an adult onset, rare, genetically determined autosomal dominant prion disease. Clinically, it is characterized predominantly by slowly progressive spino-cerebellar dysfunction with ataxia, absent reflexes in the legs and cognitive impairment. Onset is usually in the fifth decade and in the early phase, ataxia is predominant. Mutations in the prion protein gene (PRNP) had been identified and the most important of these is at codon 129. A genotype-phenotype relationship with genetic polymorphism at residue 129 between methionine and valine has been supposed. We describe a patient with GSS and P102L-V129 mutation in which the onset with prominent psychiatric features characterized by apathy and depression and not with cerebellar sign and the clinical course with seizures, nor observed in P102L-V129 cases, allow us to confirm observations that the GSS caused by the 102 mutation is influenced by the codon 129 polymorphism with a specific genotype-phenotype influence, but probably other additional factors might be considered as background for phenotypic variability.     

Calcium oscillations trigger focal adhesion disassembly in human U87 astrocytoma cells

Integrin-associated intracellular Ca(2+) oscillations modulate cell migration, probably by controlling integrin-mediated release of the cell rear during migration. Focal adhesion kinase (FAK), via its tyrosine phosphorylation activity, plays a key role in integrin signaling. In human U87 astrocytoma cells, expression of the dominant negative FAK-related non-kinase domain (FRNK) inhibits the Ca(2+)-sensitive component of serum-dependent migration. We investigated how integrin-associated Ca(2+) signaling might be coupled to focal adhesion (FA) dynamics by visualizing the effects of Ca(2+) spikes on FAs using green fluorescent protein (GFP)-tagged FAK and FRNK. We report that Ca(2+) spikes are temporally correlated with movement and disassembly of FAs, but not their formation. FRNK transfection did not affect generation of Ca(2+) spikes, although cell morphology was altered, with fewer FAs of larger size and having a more peripheral localization being observed. Larger sized FAs in FRNK-transfected cells were not disassembled by Ca(2+) spikes, providing a possible explanation for impaired Ca(2+)-dependent migration in these cells. Stress fiber end movements initiated by Ca(2+) spikes were visualized using GFP-tagged myosin light chain kinase (MLCK). Ca(2+)-associated movements of stress fiber ends and FAs had similar kinetics, suggesting that stress fibers and FAs move in a coordinated fashion. This indicates that increases in Ca(2+) likely trigger disassembly of adhesive structures that involves disruption of integrin-extracellular matrix interactions, supporting a key role for Ca(2+)-sensitive inside-out signaling in cell migration. A rapid increase in tyrosine phosphorylation of FAK was found in response to an elevation in Ca(2+) induced by thapsigargin, and we propose that this represents the initial triggering event linking Ca(2+) signaling and FA dynamics to cell motility.     

Preventive study in subjects at risk of fatal familial insomnia: Innovative approach to rare diseases

The text describes a preventive clinical trial with drug treatment in a very rare neurodegenerative disease (Fatal familial Insomnia, FFI) designed with the help of individuals at genetic risk of developing the disease, asymptomatic carriers, who have agreed to be exposed over a 10-year period to doxycycline, an antibiotic with anti-prion activity. At least 10 carriers of the FFI mutation over 42 y old will be treated with doxycycline (100 mg/die) and the incidence of the disease will be compared to that of an historical dataset. For ethical reasons a randomized, double-blind, placebo-controlled trial was not feasible, however the study design and the statistical analysis ensure the scientific value of the results. This approach might represent an important breakthrough in terms of potential therapy and knowledge of rare diseases that could give some hopes to these neglected patients.