Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis.

Global analysis of protein phosphorylation by mass spectrometry proteomic techniques has emerged in the last decades as a powerful tool in biological and biomedical research. However, there are several factors that make the global study of the phosphoproteome more challenging than measuring non-modified proteins. The low stoichiometry of the phosphorylated species and the need to retrieve residue specific information require particular attention on sample preparation, data acquisition and processing to ensure reproducibility, qualitative and quantitative robustness and ample phosphoproteome coverage in phosphoproteomic workflows.

Extra-plastidial degradation of chlorophyll and photosystem I in tobacco leaves involving 'senescence-associated vacuoles'.

Chlorophyll (Chl) loss is the main visible symptom of senescence in leaves. The initial steps of Chl degradation operate within the chloroplast, but the observation that 'senescence-associated vacuoles' (SAVs) contain Chl raises the question of whether SAVs might also contribute to Chl breakdown. Previous confocal microscope observations (Martínez et al.

Proteomic analysis of the phycobiliprotein antenna of the cryptophyte alga Guillardia theta cultured under different light intensities.

Plants and algae have developed various light-harvesting mechanisms for optimal delivery of excitation energy to the photosystems. Cryptophyte algae have evolved a novel soluble light-harvesting antenna utilizing phycobilin pigments to complement the membrane-intrinsic Chl a/c-binding LHC antenna. This new antenna consists of the plastid-encoded β-subunit, a relic of the ancestral phycobilisome, and a novel nuclear-encoded α-subunit unique to cryptophytes.

Dataset of the proteome of purified outer membrane vesicles from the human pathogen .

The Gram-negative bacterium is an oral and systemic pathogen, which is linked to aggressive forms of periodontitis and can be associated with endocarditis. The outer membrane vesicles (OMVs) of this species contain effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA), which they can deliver into human host cells. The OMVs can also activate innate immunity through NOD1- and NOD2-active pathogen-associated molecular patterns.

Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A.

Isolation of monomeric photosystem II that retains the subunit PsbS.

Photosystem II has been purified from a transplastomic strain of Nicotiana tabacum according to two different protocols. Using the procedure described in Piano et al. (Photosynth Res 106:221-226, 2010) it was possible to isolate highly active PSII composed of monomers and dimers but depleted in their PsbS protein content.

Oxidative folding in chloroplasts.

Significance: Disulfide-bonded proteins in chloroplasts from green plants exist in the envelope and the thylakoid membrane, and in the stroma and the lumen. The formation of disulfide bonds in proteins is referred to as oxidative folding and is linked to the import and folding of chloroplast proteins as well as the assembly and repair of thylakoid complexes. It is also important in the redox regulation of enzymes and signal transfer.

Degradation of PsbO by the Deg protease HhoA Is thioredoxin dependent.

The widely distributed members of the Deg/HtrA protease family play an important role in the proteolysis of misfolded and damaged proteins. Here we show that the Deg protease rHhoA is able to degrade PsbO, the extrinsic protein of the Photosystem II (PSII) oxygen-evolving complex in Synechocystis sp. PCC 6803 and in spinach.

Proteomics of protein secretion by Aggregatibacter actinomycetemcomitans.

The extracellular proteome (secretome) of periodontitis-associated bacteria may constitute a major link between periodontitis and systemic diseases. To obtain an overview of the virulence potential of Aggregatibacter actinomycetemcomitans, an oral and systemic human pathogen implicated in aggressive periodontitis, we used a combined LC-MS/MS and bioinformatics approach to characterize the secretome and protein secretion pathways of the rough-colony serotype a strain D7S. LC-MS/MS revealed 179 proteins secreted during biofilm growth.

Comparative proteome analysis of Saccharomyces cerevisiae: a global overview of in vivo targets of the yeast activator protein 1.

Background: The activity of the yeast activator protein 1 (Yap1p) increases under stress conditions, which leads to enhanced transcription of a number of genes encoding protective enzymes or other proteins. To obtain a global overview of changes in expression of Yap1p-targeted proteins, we compared a Yap1p-overexpressing transformant with a control transformant by triplicate analysis of the proteome using two-dimensional gel electrophoresis (2-DE). Proteins of interest were identified using MALDI-MS or LC-MS/MS.

Purification, crystallization and preliminary X-ray analysis of PPD6, a PsbP-domain protein from Arabidopsis thaliana.

The PsbP protein is an extrinsic component of photosystem II that together with PsbO and PsbQ forms the thylakoid lumenal part of the oxygen-evolving complex in higher plants. In addition to PsbP, the thylakoid lumen contains two PsbP-like proteins (PPLs) and six PsbP-domain proteins (PPDs). While the functions of the PsbP-like proteins PPL1 and PPL2 are currently under investigation, the function of the PsbP-domain proteins still remains completely unknown.

Boosting the globalization of plant proteomics through INPPO: current developments and future prospects.

The International Plant Proteomics Organization (INPPO) is a non-profit-organization consisting of people who are involved or interested in plant proteomics. INPPO is constantly growing in volume and activity, which is mostly due to the realization among plant proteomics researchers worldwide for the need of such a global platform. Their active participation resulted in the rapid growth within the first year of INPPO's official launch in 2011 via its website (www.

Phosphorylated CpxR restricts production of the RovA global regulator in Yersinia pseudotuberculosis.

Background: RovA is a global transcriptional regulator of gene expression in pathogenic Yersinia. RovA levels are kept in check by a sophisticated layering of distinct transcriptional and post-transcriptional regulatory mechanisms. In the enteropathogen Y.

Enzyme production by filamentous fungi: analysis of the secretome of Trichoderma reesei grown on unconventional carbon source.

Background: Spent hydrolysates from bioethanolic fermentation processes based on agricultural residues have potential as an abundant and inexpensive source of pentose sugars and acids that could serve as nutrients for industrial enzyme-producing microorganisms, especially filamentous fungi. However, the enzyme mixtures produced in such media are poorly defined. In this study, the secretome of Trichoderma reesei Rut C-30 grown either on a spent hydrolysate model medium (SHMM) or on a lactose-based standard medium (LBSM) was explored using proteomics.

Protein differences between human trapezius and vastus lateralis muscles determined with a proteomic approach.

Background: The trapezius muscle is a neck muscle that is susceptible to chronic pain conditions associated with repetitive tasks, commonly referred to as chronic work-related myalgia, hence making the trapezius a muscle of clinical interest. To provide a basis for further investigations of the proteomic traits of the trapezius muscle in disease, two-dimensional difference gel electrophoresis (2D-DIGE) was performed on the healthy trapezius using vastus lateralis as a reference. To obtain as much information as possible from the vast proteomic data set, both one-way ANOVA, with and without false discovery rate (FDR) correlation, and partial least square projection to latent structures with discriminant analysis (PLS-DA) were combined to compare the outcome of the analysis.

Drosophila Nora virus capsid proteins differ from those of other picorna-like viruses.

The recently discovered Nora virus from Drosophila melanogaster is a single-stranded RNA virus. Its published genomic sequence encodes a typical picorna-like cassette of replicative enzymes, but no capsid proteins similar to those in other picorna-like viruses. We have now done additional sequencing at the termini of the viral genome, extending it by 455 nucleotides at the 5' end, but no more coding sequence was found.

Clustering of MS spectra for improved protein identification rate and screening for protein variants and modifications by MALDI-MS/MS.

It is an established fact that allelic variation and post-translational modifications create different variants of proteins, which are observed as isoelectric and size subspecies in two-dimensional gel based proteomics. Here we explore the stromal proteome of spinach and Arabidopsis chloroplast and show that clustering of mass spectra is a useful tool for investigating such variants and detecting modified peptides with amino acid substitutions or post-translational modifications. This study employs data mining by hierarchical clustering of MALDI-MS spectra, using the web version of the SPECLUST program (http://bioinfo.

The disulfide proteome and other reactive cysteine proteomes: analysis and functional significance.

Ten years ago, proteomics techniques designed for large-scale investigations of redox-sensitive proteins started to emerge. The proteomes, defined as sets of proteins containing reactive cysteines that undergo oxidative post-translational modifications, have had a particular impact on research concerning the redox regulation of cellular processes. These proteomes, which are hereafter termed "disulfide proteomes," have been studied in nearly all kingdoms of life, including animals, plants, fungi, and bacteria.

A novel proteomic approach reveals a role for Mg-protoporphyrin IX in response to oxidative stress.

The presence of genes encoding organellar proteins in different cellular compartments necessitates a tight coordination of expression by the different genomes of the eukaryotic cell. This coordination of gene expression is achieved by organelle-to-nucleus communication. Stress-induced perturbations of the tetrapyrrole pathway trigger large changes in nuclear gene expression.

Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function.

The light-dependent regulation of stromal enzymes by thioredoxin (Trx)-catalysed disulphide/dithiol exchange is known as a classical mechanism for control of chloroplast metabolism. Recent proteome studies show that Trx targets are present not only in the stroma but in all chloroplast compartments, from the envelope to the thylakoid lumen. Trx-mediated redox control appears to be a common feature of important pathways, such as the Calvin cycle, starch synthesis and tetrapyrrole biosynthesis.

Peptidyl-prolyl isomerase activity in chloroplast thylakoid lumen is a dispensable function of immunophilins in Arabidopsis thaliana.

Chloroplast thylakoid lumen of Arabidopsis thaliana contains 16 immunophilins, five cyclophilins and 11 FK506-binding proteins (FKBPs), which are considered protein folding catalysts, although only two of them, AtFKBP13 and AtCYP20-2, possess peptidyl-prolyl cis/trans isomerase (PPIase) activity. To address the question of the physiological significance of this activity, we obtained and characterized Arabidopsis mutants deficient in the most active PPIase, AtFKBP13, and a double mutant deficient in both AtFKBP13 and AtCYP20-2. Two-dimensional gel electrophoresis of isolated thylakoid lumen, as well as immunoblotting analyses of major photosynthetic membrane protein complexes did not reveal differences in protein composition between the mutants and the wild type.

Light induced changes in protein expression and uniform regulation of transcription in the thylakoid lumen of Arabidopsis thaliana.

In plants oxygenic photosynthesis is performed by large protein complexes found in the thylakoid membranes of chloroplasts. The soluble thylakoid lumen space is a narrow and compressed region within the thylakoid membrane which contains 80-200 proteins. Because the thylakoid lumen proteins are in close proximity to the protein complexes of photosynthesis, it is reasonable to assume that the lumen proteins are highly influenced by the presence of light.

Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance.

The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157.

A novel extended family of stromal thioredoxins.

Thioredoxins play key regulatory roles in chloroplasts by linking photosynthetic light reactions to a series of plastid functions. In addition to the established groups of thioredoxins, f, m, x, and y, novel plant thioredoxins were also considered to include WCRKC motif proteins, CDSP32, the APR proteins, the lilium proteins and HCF164. Despite their important roles, the subcellular locations of many novel thioredoxins has remained unknown.

Disulphide proteomes and interactions with thioredoxin on the track towards understanding redox regulation in chloroplasts and cyanobacteria.

Light-dependent disulphide/dithiol exchange catalysed by thioredoxin is a classical example of redox regulation of chloroplast enzymes. Recent proteome studies have mapped thioredoxin target proteins in all chloroplast compartments ranging from the envelope to the thylakoid lumen. Progress in the methodologies has made it possible to identify which cysteine residues interact with thioredoxin and to tackle membrane-bound thioredoxin targets.