Mass Spectrometry-Based Identification of Phospho-Tyr in Plant Proteomics.

O-Phosphorylation (phosphorylation of the hydroxyl-group of S, T, and Y residues) is among the first described and most thoroughly studied posttranslational modification (PTM). Y-Phosphorylation, catalyzed by Y-kinases, is a key step in both signal transduction and regulation of enzymatic activity in mammalian systems. Canonical Y-kinase sequences are absent from plant genomes/kinomes, often leading to the assumption that plant cells lack -phospho-l-tyrosine (pY).

Getting into mitochondria.

The human mitochondrial glutamate dehydrogenase isoenzymes (hGDH1 and hGDH2) are abundant matrix-localized proteins encoded by nuclear genes. The proteins are synthesized in the cytoplasm, with an atypically long N-terminal mitochondrial targeting sequence (MTS). The results of secondary structure predictions suggest the presence of two α-helices within the N-terminal region of the MTS.

Do Cupins Have a Function Beyond Being Seed Storage Proteins?

Plants continue to flourish around the site of the Chernobyl Nuclear Power Plant disaster. The ability of plants to transcend the radio-contaminated environment was not anticipated and is not well understood. The aim of this study was to evaluate the proteome of flax (Linum usitatissimum L.

Analysis of soybean tissue culture protein dynamics using difference gel electrophoresis.

Unlabelled: Excised hypocotyls from developing soybean (Glycine max (L.) merr. cv.

Establishing a leaf proteome reference map for Ginkgo biloba provides insight into potential ethnobotanical uses.

Although ginkgo (Maidenhair tree, Ginkgo biloba L.) is an ancient medicinal and ornamental tree, there has not previously been any systematic proteomic study of the leaves. Herein we describe results from the initial study identifying abundant ginkgo leaf proteins and present a gel reference map.

Convergent signaling pathways--interaction between methionine oxidation and serine/threonine/tyrosine O-phosphorylation.

Oxidation of methionine (Met) to Met sulfoxide (MetSO) is a frequently found reversible posttranslational modification. It has been presumed that the major functional role for oxidation-labile Met residues is to protect proteins/cells from oxidative stress. However, Met oxidation has been established as a key mechanism for direct regulation of a wide range of protein functions and cellular processes.

New insights into the targeting of a subset of tail-anchored proteins to the outer mitochondrial membrane.

Tail-anchored (TA) proteins are a unique class of functionally diverse membrane proteins defined by their single C-terminal membrane-spanning domain and their ability to insert post-translationally into specific organelles with an Ncytoplasm-Corganelle interior orientation. The molecular mechanisms by which TA proteins are sorted to the proper organelles are not well-understood. Herein we present results indicating that a dibasic targeting motif (i.

In silico analysis of protein Lys-N(𝜀)-acetylation in plants.

Among post-translational modifications, there are some conceptual similarities between Lys-N(𝜀)-acetylation and Ser/Thr/Tyr O-phosphorylation. Herein we present a bioinformatics-based overview of reversible protein Lys-acetylation, including some comparisons with reversible protein phosphorylation. The study of Lys-acetylation of plant proteins has lagged behind studies of mammalian and microbial cells; 1000s of acetylation sites have been identified in mammalian proteins compared with only hundreds of sites in plant proteins.

Identification of Coxiella burnetii surface-exposed and cell envelope associated proteins using a combined bioinformatics plus proteomics strategy.

The Gram-negative pathogen Coxiella burnetii is an intracellular bacterium that replicates within the phagolysosomal vacuoles of eukaryotic cells. This pathogen can infect a wide range of hosts, and is the causative agent of Q fever in humans. Surface-exposed and cell envelope associated proteins are thought to be important for both pathogenesis and protective immunity.

Is Lys-Nɛ-acetylation the next big thing in post-translational modifications?

Lys-N(ɛ)-acetylation (PKA) has recently ascended from a post-translational modification (PTM) of limited distribution to one approaching the abundance of O-phosphorylation. Thousands of KAC proteins have been identified in Archaea, bacteria, and Eukarya, and the KAC system of acetyltransferases, deacetylases, and binding proteins is superficially comparable with the kinases, phosphatases, and phospho- (P-)protein binding-proteins of O-phosphorylation. Herein, we describe recent results and compare several aspects of these two major systems of PTM in plants.

A novel regulatory mechanism based upon a dynamic core structure for the mitochondrial pyruvate dehydrogenase complex?

The Arabidopsis thaliana genome includes three genes for mitochondrial dihydrolipoamide acetyltransferase, the E2-component of the mitochondrial pyruvate dehydrogenase complex (PDC). Two genes encode E2-proteins with a single lipoyl domain, while the third has a two-lipoyl domain structure. Transcripts for each E2 protein were expressed in all plant organs.

The pea seedling mitochondrial Nε-lysine acetylome.

Posttranslational lysine acetylation is believed to occur in all taxa and to affect thousands of proteins. In contrast to the hundreds of mitochondrial proteins reported to be lysine-acetylated in non-plant species, only a handful have been reported from the plant taxa previously examined. To investigate whether this reflects a biologically significant difference or merely a peculiarity of the samples thus far examined, we immunoenriched and analyzed acetylated peptides from highly purified pea seedling mitochondria using mass spectrometry.

Circles within circles: crosstalk between protein Ser/Thr/Tyr-phosphorylation and Met oxidation.

Background: Reversible posttranslational protein modifications such as phosphorylation of Ser/Thr/Tyr and Met oxidation are critical for both metabolic regulation and cellular signalling. Although these modifications are typically studied individually, herein we describe the potential for cross-talk and hierarchical regulation.

Results: The proximity of Met to Ser/Thr/Tyr within the proteome has not previously been addressed.

Initial description of the developing soybean seed protein Lys-N(ε)-acetylome.

Unlabelled: Characterization of the myriad protein posttranslational modifications (PTM) is a key aspect of proteome profiling. While there have been previous studies of the developing soybean seed phospho-proteome, herein we present the first analysis of non-histone lysine-N(Ɛ)-acetylation in this system. In recent years there have been reports that lysine acetylation is widespread, affecting thousands of proteins in diverse species from bacteria to mammals.

Seed proteomics.

Rather than providing a single specific protocol, the inclusive area of seed proteomics is reviewed; methods are described and compared and primary literature citations are provided. The limitations and challenges of proteomics as an approach to study seed biology are emphasized. The proteomic analysis of seeds encounters some specific problems that do not impinge on analyses of other plant cells, tissues, or organs.

A functional genomic analysis of Arabidopsis thaliana PP2C clade D.

In the reference dicot plant Arabidopsis thaliana, the PP2C family of P-protein phosphatases includes the products of 80 genes that have been separated into ten multi-protein clades plus six singletons. Clade D includes the products of nine genes distributed among three chromosomes (APD1, At3g12620; APD2, At3g17090; APD3, At3g51370; APD4, At3g55050; APD5, At4g33920; APD6, At4g38520; APD7, At5g02760; APD8, At5g06750; and APD9, At5g66080). As part of a functional genomics analysis of protein phosphorylation, we retrieved expression data from public databases and determined the subcellular protein localization of the members of clade D.

Proteomic analysis of the testa from developing soybean seeds.

Unlabelled: Soybean (Glycine max (L.) Merr. cv Jack) seed development was separated into nine defined stages (S1 to S9).

A versatile mass spectrometry-based method to both identify kinase client-relationships and characterize signaling network topology.

While more than a thousand protein kinases (PK) have been identified in the Arabidopsis thaliana genome, relatively little progress has been made toward identifying their individual client proteins. Herein we describe the use of a mass spectrometry-based in vitro phosphorylation strategy, termed Kinase Client assay (KiC assay), to study a targeted-aspect of signaling. A synthetic peptide library comprising 377 in vivo phosphorylation sequences from developing seed was screened using 71 recombinant A.

In silico biosynthesis of virenose, a methylated deoxy-sugar unique to Coxiella burnetii lipopolysaccharide.

Background: Coxiella burnetii is Gram-negative bacterium responsible for the zoonosis Q-fever. While it has an obligate intracellular growth habit, it is able to persist for extended periods outside of a host cell and can resist environmental conditions that would be lethal to most prokaryotes. It is these extracellular bacteria that are the infectious stage encountered by eukaryotic hosts.

"Scanning mutagenesis" of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex.

The mitochondrial pyruvate dehydrogenase complex (mtPDC) is regulated by reversible seryl-phosphorylation of the E1α subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated Ser-residue and the sequences of the flanking amino acids are highly conserved.

The proteomic future: where mass spectrometry should be taking us.

A newcomer to the -omics era, proteomics, is a broad instrument-intensive research area that has advanced rapidly since its inception less than 20 years ago. Although the 'wet-bench' aspects of proteomics have undergone a renaissance with the improvement in protein and peptide separation techniques, including various improvements in two-dimensional gel electrophoresis and gel-free or off-gel protein focusing, it has been the seminal advances in MS that have led to the ascension of this field. Recent improvements in sensitivity, mass accuracy and fragmentation have led to achievements previously only dreamed of, including whole-proteome identification, and quantification and extensive mapping of specific PTMs (post-translational modifications).

Mapping the lipoylation site of Arabidopsis thaliana plastidial dihydrolipoamide S-acetyltransferase using mass spectrometry and site-directed mutagenesis.

Catalytic enhancement achieved by the pyruvate dehydrogenase complex (PDC) results from a combination of substrate channeling plus active-site coupling. The mechanism for active-site coupling involves lipoic acid prosthetic groups covalently attached to Lys in the primary sequence of the dihydrolipoyl S-acetyltransferase (E2) component. Arabidopsis thaliana plastidial E2 (AtplE2-1A-His(6)) was expressed in Escherichia coli.

Proteomic comparison of virulent phase I and avirulent phase II of Coxiella burnetii, the causative agent of Q fever.

Coxiella burnetii, a category B biological warfare agent, causes multiple outbreaks of the zoonotic disease Q fever world-wide, each year. The virulent phase I and avirulent phase II variants of the Nine Mile RSA 493 and 439 strains of C. burnetii were propagated in embryonated hen eggs and then purified by centrifugation through Renografin gradients.

Using multiplex-staining to study changes in the maize leaf phosphoproteome in response to mechanical wounding.

Mechanical wounding of 2-week-old maize (Zea mays L.) leaves, one of the first steps in both pathogen infection and herbivore attack, stimulates metabolism and activates signal transduction pathways dedicated to defense and recovery. The signaling pathways include reversible protein phosphorylation which can modulate protein activities, and transmit signals within cellular pathways and networks.

Asp295 stabilizes the active-site loop structure of pyruvate dehydrogenase, facilitating phosphorylation of ser292 by pyruvate dehydrogenase-kinase.

We have developed an in vitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thalianaα2β2-heterotetrameric pyruvate dehydrogenase (E1) plus A. thaliana E1-kinase (AtPDK). Upon addition of MgATP, Ser292, which is located within the active-site loop structure of E1α, is phosphorylated.