Two-dimensional electrophoresis in proteome expression analysis

Cell proteomes are complex, given they consist of several thousand proteins. Two-dimensional electrophoresis (2DE) is unique not only for its ability to simultaneously separate thousands of proteins but also for detecting post- and co-translational modifications, which cannot be predicted from genome sequences. This review will describe the protocols applied to prepare 2D gels properly, and analyse and summarise the major challenges for successful proteome analysis using 2DE, i.e. the ability to analyse very alkaline, hydrophobic and/or low or high M(r) proteins with high resolution and the ability to detect minor components. Challenges involving sample preparation and solubilisation prior to the first dimension IEF/IPG step will be studied in depth. Sample preparation is crucial in 2DE studies and greatly influences other stages of the technique. It is the aim of this review not only to describe the challenges and limitations of 2DE but also to suggest the avenues, the evolution, the potential and the future of 2DE in proteomics.     

Development of a Listeria monocytogenes EGDe partial proteome reference map and comparison with the protein profiles of food isolates

A partially annotated proteome reference map of the food pathogen Listeria monocytogenes was developed for exponentially growing cells under standardized, optimal conditions by using the sequenced strain EGDe (serotype 1/2a) as a model organism. The map was developed by using a reproducible total protein extraction and two-dimensional (2-D) polyacrylamide gel electrophoresis analysis procedure, and it contained 33 identified proteins representing the four main protein functional classes. In order to facilitate analysis of membrane proteins, a protein compartmentalization procedure was assessed. The method used provided partial fractionation of membrane and cytosolic proteins. The total protein 2-D profiles of three serotype 1/2a strains and one serotype 1/2b strain isolated from food were compared to the L. monocytogenes EGDe proteome. An average of 13% of the major protein spots in the food strain proteomes were not matched in the strain EGDe proteome. The variation was greater for the less intense spots, and on average 28% of these spots were not matched. Two of the proteins identified in L. monocytogenes EGDe were missing in one or more of the food isolates. These two proteins were proteins involved in the main glycolytic pathway and in metabolism of coenzymes and prosthetic groups. The two corresponding genes were found by PCR amplification to be present in the four food isolates. Our results show that the L. monocytogenes EGDe reference map is a valuable starting point for analyses of strains having various origins and could be useful for analyzing the proteomes of different isolates of this pathogen.     

Comparative proteomic analysis of Listeria monocytogenes tolerance to bile stress

The ability of the food-borne pathogen Listeria monocytogenes to tolerate bile is critical to its successful infection and colonization in the human gastrointestinal tract. Using comparative proteomics, a total of 48 proteins were identified in this study in the presence of moderate (0.3 %) or high (3 %) level of bile salts in the wild-type strain EGD. Identified proteins fell into 14 functional categories covering most of the biochemical functions of bacterial cells, indicating that there were complex physiological mechanisms involved in L. monocytogenes tolerance of bile stress. Among them, 16, 14, and 18 proteins were expressed differently in the isogenic deletion mutants of L. monocytogenes EGDΔsigB, EGDΔprfA, and EGDΔprfAΔsigB, respectively, compared with their parent strain EGD at corresponding concentrations of bile salts. All proteins identified in EGDΔsigB and EGDΔprfAΔsigB were all down-expressed in the presence of bile salts, whereas several proteins were up-expressed in EGDΔprfA, in particular at the high level of bile (3 %), indicating that SigB plays an essential positive role in L. monocytogenes tolerance of bile stress and that the negative effect of PrfA may facilitate its survival in bile in the gastrointestinal tract before its successful colonization and invasion.     

Proteomic analysis of log to stationary growth phase Lactobacillus plantarum cells and a 2-DE database

Lactobacillus plantarum is part of the natural microbiota of many food fermentations as well as the human gastro-intestinal tract. The cytosolic fraction of the proteome of L. plantarum WCFS1, whose genome has been sequenced, was studied. 2-DE was used to investigate the proteins from the cytosolic fraction isolated from mid- and late-log, early- and late-stationary phase cells to generate reference maps of different growth conditions offering more knowledge of the metabolic behavior of this bacterium. From this fraction, a total of 200 protein spots were identified by MALDI-MS and a proteome production map was constructed to facilitate further studies such as detection of suitable biomarkers for specific growth conditions. More than half (57%) of the identified proteins were predicted to be involved in metabolic pathways of the bacterium. The protein profile changed during the growth of the bacteria such that 29% of the identified proteins involved in anabolic pathways were at least twofold up-regulated throughout the mid- and late-exponential and early-stationary phases. In the late-stationary phase, six proteins involved in stress or with a potential role for survival during starvation were up-regulated significantly.     

Two-dimensional electrophoresis database of fluorescence-labeled proteins of colon cancer cells

We constructed a novel database of the proteome of DLD-1 colon cancer cells by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of fluorescence-labeled proteins followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis. The database consists of 258 functionally categorized proteins corresponding to 314 protein spots. The majority of the proteins are oxidoreductases, cytoskeletal proteins and nucleic acid binding proteins. Phosphatase treatment showed that 28% of the protein spots on the gel are phosphorylated, and mass spectrometric analysis identified 21 of them. Proteins of DLD-1 cells and of laser-microdissected colon cancer tissues showed similar distribution on 2D gels, suggesting the utility of our database for clinical proteomics.     

Antimicrobial effects of sanitizers against planktonic and sessile Listeria monocytogenes cells according to the growth phase

An entomopathogenic bacterium, Xenorhabdus nematophila, is known to have potent antibiotic activities to maintain monoxenic condition in its insect host for effective pathogenesis and ultimately for optimal development of its nematode symbiont, Steinernema carpocapsae. In this study we assess its antibacterial activity against plant-pathogenic bacteria and identify its unknown antibiotics. The bacterial culture broth had significant antibacterial activity that increased with development of the bacteria and reached its maximum at the stationary growth phase. The antibiotic activities were significant against five plant-pathogenic bacterial strains: Agrobacterium vitis, Pectobacterium carotovorum subsp. atrosepticum, P. carotovorum subsp. carotovorum, Pseudomonas syringae pv. tabaci, and Ralstonia solanacearum. The antibacterial factors were extracted with butanol and fractionated using column chromatography with the eluents of different hydrophobic intensities. Two active antibacterial subfractions were purified, and the higher active fraction was further fractionated and identified as a single compound of benzylideneacetone (trans-4-phenyl-3-buten-2-one). With heat stability, the synthetic compound showed equivalent antibiotic activity and spectrum to the purified compound. This study reports a new antibiotic compound synthesized by X. nematophila, which is a monoterpenoid compound and active against some Gram-negative bacteria.     

Two-dimensional gel electrophoresis database of murine R1 embryonic stem cells

Embryonic stem (ES) cell lines represent a population of undifferentiated pluripotent cells capable of multilineage differentiation in vitro. Although very useful for studying developmental processes, human ES cell lines have also been suggested as a potential and unlimited source for cellular transplantation and the treatment of human disease. The proteomic basis of embryonic stemness (pluripotentiality and multilineage differentiation) and the transitions that lead to specific cell lineages however, remain to be defined. As an important first step in defining these processes, we have performed a proteomic analysis of undifferentiated mouse R1 ES cell lines using pH 3-10, 4-7 and 6-11 two-dimensional electrophoresis gels, matrix-assisted laser desorption/ionization and tandem mass spectrometry. Of the 700 gel spots analyzed, 241 distinct protein species were identified corresponding to 218 unique proteins, with a significant proportion functionally related to protein expression.     

Minimum growth temperatures of Listeria monocytogenes and non-haemolytic Listeria

Minimum growth temperatures and those of decreased growth were determined for 100 strains of listerias. The ability of 78 strains of Listeria monocytogenes isolated from animals and 22 non-haemolytic strains to grow at low temperatures was studied, using a flooding technique, in a plate-type continuous temperature gradient incubator at temperatures between -1.6 and 14.5 degrees C. The mean minimum temperature for L. monocytogenes was +1.7 +/- 0.5 degrees C. The growth of non-haemolytic listerias was unobservable at +1.7 +/- 0.5 degrees C. The L. monocytogenes strains grew at about 0.6 degrees C lower than the non-pathogenic strains. No differences in growth temperatures were observed among L. monocytogenes strains isolated from different sources. The serovars with the OI antigen grew at lower temperatures (+1.0 +/- 0.3 degrees C) than the other common serovar 4b (+1.3 +/- 0.4 degrees C). The results indicate that L. monocytogenes grows better than non-haemolytic strains under cold conditions. The possible role of haemolysins as growth factors is also discussed.     

Global changes in gene expression observed at the transition from growth to stationary phase in Listeria monocytogenes ScottA batch culture

Listeria monocytogenes is a food-borne Gram-positive bacterium that is responsible for a variety of infections (worldwide) annually. The organism is able to survive a variety of environmental conditions and stresses, however, the mechanisms by which L. monocytogenes adapts to environmental change are yet to be fully elucidated. An understanding of the mechanism(s) by which L. monocytogenes survives unfavourable environmental conditions will aid in developing new food processing methods to control the organism in foodstuffs. We have utilized a proteomic approach to investigate the response of L. monocytogenes batch cultures to the transition from exponential to stationary growth phase. Proteomic analysis showed that batch cultures of L. monocytogenes perceived stress and began preparations for stationary phase much earlier (approximately A(600) = 0.75, mid-exponential) than predicted by growth characteristics alone. Global analysis of the proteome revealed that the expression levels of more than 50% of all proteins observed changed significantly over a 7-9 h period during this transition phase. We have highlighted ten proteins in particular whose expression levels appear to be important in the early onset of the stationary phase. The significance of these findings in terms of functionality and the mechanistic picture are discussed.     

Identification of substrates of the Listeria monocytogenes sortases A and B by a non-gel proteomic analysis

Sortases are enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria by cleaving a sorting motif located in the C-terminus of the protein substrate. The best-characterized motif is LPXTG, which is cleaved between the T and G residues. In this study, a non-gel proteomic approach was used to identify surface proteins recognized by the two sortases of Listeria monocytogenes, SrtA and SrtB. Material containing peptidoglycan and strongly associated proteins was purified from sortase-defective mutants, digested with trypsin, and the resulting peptide mixture analysed by two-dimensional nano-liquid chromatography coupled to ion-trap mass spectrometry. Unlike enzymes involved in peptidoglycan metabolism, other surface proteins displayed uneven distribution in the mutants. A total of 13 LPXTG-containing proteins were identified exclusively in strains having a functional SrtA. In contrast, two surface proteins, Lmo2185 and Lmo2186, were identified only when SrtB was active. The analysis of the peptides identified in these proteins suggests that SrtB of L. monocytogenes may recognize two different sorting motifs, NXZTN and NPKXZ. Taken together, these data demonstrate that non-gel proteomics is a powerful technique to rapidly identify sortase substrates and to gain insights on potential sorting motifs.     

Characterization of Listeria monocytogenes isolates by esterase electrophoresis.

Multiple bands of alpha-naphthyl-propionyl esterase (alpha NPE) activity observed following starch gel electrophoresis of cell extracts allowed 219 Listeria monocytogenes isolates from milk, nondairy foods, and clinical and veterinary sources to be assigned to 17 different alpha NPE types. Multilocus enzyme electrophoresis (MEE) analysis was used to obtain electrophoretic mobility data for alpha NPEs and nine other metabolic enzymes; on the basis of these data, the 219 strains were assigned to 59 electrophoretic types. Each of the methods separated the strains into two main groups, and there was extensive agreement on assignment of the strains to the groups. Although the method is less discriminatory than MEE, the usefulness of alpha NPE electrophoresis alone as a rapid, simple typing method for L. monocytogenes for certain purposes is indicated by agreement among alpha NPE, MEE, and restriction fragment length polymorphism types as molecular markers for persistent and transient L. monocytogenes strains found in sequential raw-milk and nondairy food samples obtained from different processors.     

The extracellular proteome of Rhizobium etli CE3 in exponential and stationary growth phase

Background

The extracellular proteome or secretome of symbiotic bacteria like Rhizobium etli is presumed to be a key element of their infection strategy and survival. Rhizobia infect the roots of leguminous plants and establish a mutually beneficial symbiosis. To find out the possible role of secreted proteins we analyzed the extracellular proteome of R. etli CE3 in the exponential and stationary growth phases in minimal medium, supplemented with succinate-ammonium.

Results

The extracellular proteins were obtained by phenol extraction and identified by LC-ESI MS/MS. We identified 192 and 191 proteins for the exponential and stationary phases respectively. Using the software Signal P, we predicted signal peptides for 12.95% and 35.60% of the proteins identified in the exponential and stationary phases, respectively, which could therefore be secreted by the Sec pathway. For the exponential growth phase, we found in abundance proteins like the ribosomal proteins, toxins and proteins belonging to the group "defence mechanisms". For the stationary growth phase, we found that the most abundant proteins were those with unknown function, and in many of these we identified characteristic domains of proteases and peptidases.

Conclusions

Our study provided the first dataset of the secretome of R. etli and its modifications, which may lead to novel insights into the adaptive response of different stages of growth. In addition, we found a high number of proteins with unknown function; these proteins could be analyzed in future research to elucidate their role in the extracellular proteome of R. etli.     

Multiple-locus variable-number tandem-repeats analysis of Listeria monocytogenes using multicolour capillary electrophoresis and comparison with pulsed-field gel electrophoresis typing

The multiple-locus variable-number tandem-repeats analysis (MLVA) method for genotyping has proven to be a fast and reliable typing tool in several bacterial species. MLVA is in our laboratory the routine typing method for Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli O157. The gram-positive bacteria Listeria monocytogenes, while not isolated as frequent as S. Typhimurium and E. coli, causes severe illness with an overall mortality rate of 30%. Thus, it is important that any outbreak of this pathogen is detected early and a fast trace to the source can be performed. In view of this, we have used the information provided by two fully sequenced L. monocytogenes strains to develop a MLVA assay coupled with high-resolution capillary electrophoresis and compared it to pulsed-field gel electrophoresis (PFGE) in two sets of isolates, one Norwegian (79 isolates) and one Swedish (61 isolates) set. The MLVA assay could resolve all of the L. monocytogenes serotypes tested, and was slightly more discriminatory than PFGE for the Norwegian isolates (28 MLVA profiles and 24 PFGE profiles) and opposite for the Swedish isolates (42 MLVA profiles and 43 PFGE profiles).     

The actin propulsive machinery: the proteome of Listeria monocytogenes tails

Actin-based comet tails produced by Listeria monocytogenes are considered as representative models for cellular force-producing machineries crucial for cell migration. We here present a proteomic picture of these tails formed in extracts from brain and platelets. This provides a comprehensive view, revealing high molecular complexity and novel host cell proteins as tail components, and suggests the participation of specific multicomponent regulatory complexes. This work forms a new basis to expand current models of cellular protrusion.     

An integrated proteome database for two-dimensional electrophoresis data analysis and laboratory information management system

We describe an integrated proteome database, termed Yonsei Proteome Research Center Proteome Database (YPRC-PDB) which can store, retrieve and analyze various information including two-dimensional electrophoresis (2-DE) images and associated spot information that were obtained during studies of hepatocellular carcinoma (HCC). YPRC-PDB is also designed to perform as a laboratory information management system that manages sample information, clinical background, conditions of both sample preparation and 2-DE, and entire sets of experimental results. It also features query system and data-mining applications, which are amenable to automatically analyze expression level changes of a specific protein and directly link to clinical information. The user interface is web-based, so that the results from other laboratories can be shared effectively. In particular, the master gel image query is equipped with a graphic tool that can easily identify the relationship between the specific pathological stage of HCC and expression levels of a potential marker protein on the master gel image. Thus, YPRC-PDB is a versatile integrated database suitable for subsequent analyses. The information in YPRC-PDB is updated easily and it is available to authorized users on the World Wide Web (http://yprcpdb.proteomix.org/ approximately damduck/).     

Two-dimensional fluorescence difference gel electrophoresis analysis of the urine proteome in human diabetic nephropathy

Urinary proteins may provide clues regarding pathogenesis of kidney disease as well as providing markers of disease activity. We employed two-dimensional differential in-gel electrophoretic analysis (2-D DIGE) to assess multiple urine samples in patients with diabetic nephropathy. Patient samples were collected as timed overnight collections. All the patients had longstanding diabetes, impaired renal function, and overt proteinuria. Control and patient urinary protein were analyzed by 2-D DIGE and DeCyder analysis. Ninety-nine spots were significantly regulated in the urine proteome of the diabetic samples, with 63 up- and 36 down-regulated. One spot corresponding to a pI 5-6 and a molecular weight between 45 and 66 kDa was consistently up-regulated by 19-fold across individuals in the diabetic group. Surface-enhanced laser desorption/ionization-time of flight analysis of in-gel tryptic digest of this spot identified this protein as alpha 1 antitrypsin (AAT). ELISA of urine samples from a separate group of patients and controls confirmed a marked increase of AAT in diabetic patients. Immunostaining of human diabetic kidneys revealed up-regulation of AAT in areas of renal fibrosis. In conclusion, we developed a method to analyze numerous urine samples from patients and allowed for detection and identification of regulated urine protein spots.     

Autophagy limits Listeria monocytogenes intracellular growth in the early phase of primary infection

Autophagy has been recently proposed to be a component of the innate cellular immune response against several types of intracellular microorganisms. However, other intracellular bacteria including Listeria monocytogenes have been thought to evade the autophagic cellular surveillance. Here, we show that cellular infection by L. monocytogenes induces an autophagic response, which inhibits the growth of both the wild-type and a DeltaactA mutant strain, impaired in cell-to-cell spreading. The onset of early intracellular growth is accelerated in autophagy-deficient cells, but the growth rate once bacteria begin to multiply in the cytosol does not change. Moreover, a significant fraction of the intracellular bacteria colocalize with autophagosomes at the early time-points after infection. Thus, autophagy targets L. monocytogenes during primary infection by limiting the onset of early bacterial growth. The bacterial expression of listeriolysin O but not phospholipases is necessary for the induction of autophagy, suggesting a possible role for permeabilization of the vacuole in the induction of autophagy. Interestingly, the growth of a DeltaplcA/B L. monocytogenes strain deficient for bacterial phospholipases is impaired in wild-type cells, but restored in the absence of autophagy, suggesting that bacterial phospholipases may facilitate the escape of bacteria from autophagic degradation. We conclude that L. monocytogenes are targeted for degradation by autophagy during the primary infection, in the early phase of the intracellular cycle, following listeriolysin O-dependent vacuole perforation but preceding active multiplication in the cytosol, and that expression of bacterial phospholipases is necessary for the evasion of autophagy.