Michalis Aivaliotis

Comprehensive human urine standards for comparability and standardization in clinical proteome analysis

Purpose: Urine proteomics is emerging as a powerful tool for biomarker discovery. The purpose of this study is the development of a well‐characterized “real life” sample that can be used as reference standard in urine clinical proteomics studies.

Ser/Thr/Tyr Protein Phosphorylation in the Archaeon Halobacterium salinarum—A Representative of the Third Domain of Life

In the quest for the origin and evolution of protein phosphorylation, the major regulatory post-translational modification in eukaryotes, the members of archaea, the “third domain of life”, play a protagonistic role. A plethora of studies have demonstrated that archaeal proteins are subject to post-translational modification by covalent phosphorylation, but little is known concerning the identities of the proteins affected, the impact on their functionality, the physiological roles of archaeal protein phosphorylation/dephosphorylation, and the protein kinases/phosphatases involved. These limited studies led to the initial hypothesis that archaea, similarly to other prokaryotes, use mainly histidine/aspartate phosphorylation, in their two-component systems representing a paradigm of prokaryotic signal transduction, while eukaryotes mostly use Ser/Thr/Tyr phosphorylation for creating highly sophisticated regulatory networks. In antithesis to the above hypothesis, several studies showed that Ser/Thr/Tyr phosphorylation is also common in the bacterial cell, and here we present the first genome-wide phosphoproteomic analysis of the model organism of archaea, Halobacterium salinarum, proving the existence/conservation of Ser/Thr/Tyr phosphorylation in the “third domain” of life, allowing a better understanding of the origin and evolution of the so-called “Natures premier” mechanism for regulating the functional properties of proteins.

The Escherichia coli Peripheral Inner Membrane Proteome

Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher.

Analysis of the urine proteome via a combination of multi‐dimensional approaches

Urine is a biological fluid that is non‐invasively and easily harvested, and exhibits high stability from the proteomics point of view. At the downside, the overall low protein content of urine as well as the presence of low‐ and high‐abundance proteins underscores the need for protein enrichment. As a continuation of previous efforts towards the comprehensive characterization of the urine proteome, the current study targeted the mining of urine proteins through the combined application of different protein separation methodologies, specifically, liquid chromatography and preparative electrophoresis along with 1D gel electrophoresis and protein identification by mass spectrometry. In order to enhance comparison and integration of different experimental data sets, the “standard” urine sample developed within the European Kidney and Urine Proteomics (EuroKUP) COST Action, was employed. As a contribution to the existing knowledge, we focused on maintaining and providing information about experimental mass of the identified proteins as well as information pertaining to their relative abundance – as allowed by technical limitations – thus providing an initial view of different isoforms representation and facilitating their future characterization. The difficulties in comparing proteome mining data sets become once more evident, underscoring the need for adopting standardized ways for data reporting as well as for potential new approaches for data analysis involving a thorough investigation of received information at the peptide level.

The N-terminal shuttle domain of Erv1 determines the affinity for Mia40 and mediates electron transfer to the catalytic Erv1 core in yeast mitochondria.

Erv1 and Mia40 constitute the two important components of the disulfide relay system that mediates oxidative protein folding in the mitochondrial intermembrane space. Mia40 is the import receptor that recognizes the substrates introducing disulfide bonds while it is reduced. A key function of Erv1 is to recycle Mia40 to its active oxidative state. Our aims here were to dissect the domain of Erv1 that mediates the protein-protein interaction with Mia40 and to investigate the interactions between the shuttle domain of Erv1 and its catalytic core and their relevance for the interaction with Mia40. We purified these domains separately as well as cysteine mutants in the shuttle and the active core domains. The noncovalent interaction of Mia40 with Erv1 was measured by isothermal titration calorimetry, whereas their covalent mixed disulfide intermediate was analyzed in reconstitution experiments in vitro and in organello. We established that the N-terminal shuttle domain of Erv1 is necessary and sufficient for interaction to occur. Furthermore, we provide direct evidence for the intramolecular electron transfer from the shuttle cysteine pair of Erv1 to the core domain. Finally, we reconstituted the system by adding in trans the N- and C- terminal domains of Erv1 together with its substrate Mia40.

Molecular size determination of a membrane protein in surfactants by light scattering

The molecular size of an outer surface protein from the photosynthetic bacterium Chlorobium tepidum was studied by dynamic light scattering (DLS) and HPLC gel filtration. For that purpose, the membrane protein was isolated and studied in four different nonionic surfactants, namely t-octylphenoxypolyethenoxyethanol (Triton X-100), (methyl-6-O-(N)-heptyl-carbamoyl)-alpha-D-glucopyranoside (Hecameg), dodecyl-beta-D-maltoside (DDM) and n-octyl-oligo-oxyethylene (Octyl-POE). The protein was isolated by solubilization of the membranes with Triton X-100. The final purification step was a gel filtration, which was also used for surfactant exchange. Light scattering reveals the simultaneous presence of particles of different sizes in the 3-6 and 20-110 nm range, respectively. The smaller size is related to the hydrodynamic radius of the individual protein/surfactant complexes, whereas the larger size is associated with the presence of complex aggregates.

Identification of inducible protein complexes in the phenol degrader Pseudomonas sp. strain phDV1 by blue native gel electrophoresis and mass spectrometry

Summary.Pseudomonas sp. strain phDV1, being a phenol degrading bacterium, has been found to utilize phenol as sole carbon source via the meta pathway. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes involved in the aromatic degradation pathway. In particular, the Pseudomonas sp. strain phDV1 proteome was monitored under two different growth substrate conditions, using glucose or phenol as sole carbon source. The protein complexes map was compared by BN-PAGE after fractionation by sucrose density centrifugation of the cell extracts. Multiple differences were detected. Further, analysis and identification of the subunit composition of these complexes was carried out using MALDI-TOF MS, allowing the identification of 49 proteins. Additionally, functional information regarding protein–protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. Application of this functional proteomics method resulted in an higher number of the identified proteins.

Using nanoelectrospray ion mobility spectrometry (GEMMA) to determine the size and relative molecular mass of proteins and protein assemblies: a comparison with MALLS and QELS

AbstractThe determination of protein assembly size and relative molecular mass is currently of great importance in biochemical analysis. In particular, the technique of nanoelectrospray (nES) with a gas-phase electrophoretic mobility molecular analyzer (GEMMA) has received increased attention for such measurements. However, in order for the GEMMA technique to gain broader acceptance in protein analysis, it must be further evaluated and compared with other established bioanalytical techniques. In the present study, nES-GEMMA was evaluated for the analysis of a set of protein and protein complexes involved in the Sec and the bacterial type III secretion pathway of enteropathogenic Escherichia coli bacteria. The same set of proteins, isolated and purified using standard biochemical protocols, were also analyzed using multi-angle laser light scattering (MALLS) and quasi-elastic light scattering (QELS), following size exclusion chromatography. This allowed for direct comparisons between the three techniques. It was found that nES-GEMMA, in comparison to the more established MALLS and QELS techniques, offers several complementary advantages. It requires considerably less amount of material, i.e., nanogram vs. milligram amounts, and time per sample analysis, i.e., few minutes vs. tens of minutes. Whereas the determined size and relative molecular mass are similar between the compared methods, the electrophoretic diameters determined using nES-GEMMA seem to be systematically smaller compared to the hydrodynamic diameter derived by QELS. Some of the GEMMA technique disadvantages include its narrow dynamic range, limited by the fact that at elevated protein concentrations there is increased potential for the occurrence of nES-induced oligomers. Thus, it is preferred to analyze dilute protein solutions because non-specific oligomers are less likely to occur whereas biospecific oligomers remain detected. To further understand the formation of nES-oligomers, the effect of buffer concentration on their formation was evaluated. Also, nES-GEMMA is not compatible with all the buffers commonly used with MALLS and QELS. Overall, however, the nES-GEMMA technique shows promise as a high-throughput proteomics/protein structure tool. FigureNanoES-GEMMA electropherogram of SecA protein dimer

High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of cytoplasmatic protein complexes from Chlorobium tepidum

Chl. tepidum is a Gram-negative green-sulfur bacterium, which is strict by anaerobic and grows by utilizing sulfide or thiosulfate as an electron source. Blue native-polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes. In particular, the Chl. tepidum-soluble proteome was monitored under native condition by using BN-PAGE. The BN-PAGE protein complexes map was analyzed by MALDI-TOF MS after trypsin treatment and from 42 BN proteins bands, 62 different proteins were identified. Additionally, functional information regarding protein–protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. One-hundred and seventy gel bands were spotted, out of which 187 different proteins were identified. The identified proteins belong to various functional categories like energy metabolism, protein synthesis, amino acid biosynthesis, central intermediate metabolism, and biosynthesis of cofactors indicating the potential of the method for elucidation of functional proteomes.

The protein interaction network of a taxis signal transduction system in a Halophilic Archaeon

BackgroundThe taxis signaling system of the extreme halophilic archaeon Halobacterium (Hbt.) salinarum differs in several aspects from its model bacterial counterparts Escherichia coli and Bacillus subtilis. We studied the protein interactions in the Hbt. salinarum taxis signaling system to gain an understanding of its structure, to gain knowledge about its known components and to search for new members.ResultsThe interaction analysis revealed that the core signaling proteins are involved in different protein complexes and our data provide evidence for dynamic interchanges between them. Fifteen of the eighteen taxis receptors (halobacterial transducers, Htrs) can be assigned to four different groups depending on their interactions with the core signaling proteins. Only one of these groups, which contains six of the eight Htrs with known signals, shows the composition expected for signaling complexes (receptor, kinase CheA, adaptor CheW, response regulator CheY). From the two Hbt. salinarum CheW proteins, only CheW1 is engaged in signaling complexes with Htrs and CheA, whereas CheW2 interacts with Htrs but not with CheA. CheY connects the core signaling structure to a subnetwork consisting of the two CheF proteins (which build a link to the flagellar apparatus), CheD (the hub of the subnetwork), two CheC complexes and the receptor methylesterase CheB.ConclusionsBased on our findings, we propose two hypotheses. First, Hbt. salinarum might have the capability to dynamically adjust the impact of certain Htrs or Htr clusters depending on its current needs or environmental conditions. Secondly, we propose a hypothetical feedback loop from the response regulator to Htr methylation made from the CheC proteins, CheD and CheB, which might contribute to adaptation analogous to the CheC/CheD system of B. subtilis.