The state of the art in the analysis of two-dimensional gel electrophoresis images

Software-based image analysis is a crucial step in the biological interpretation of two-dimensional gel electrophoresis experiments. Recent significant advances in image processing methods combined with powerful computing hardware have enabled the routine analysis of large experiments. We cover the process starting with the imaging of 2-D gels, quantitation of spots, creation of expression profiles to statistical expression analysis followed by the presentation of results. Challenges for analysis software as well as good practices are highlighted. We emphasize image warping and related methods that are able to overcome the difficulties that are due to varying migration positions of spots between gels. Spot detection, quantitation, normalization, and the creation of expression profiles are described in detail. The recent development of consensus spot patterns and complete expression profiles enables one to take full advantage of statistical methods for expression analysis that are well established for the analysis of DNA microarray experiments. We close with an overview of visualization and presentation methods (proteome maps) and current challenges in the field. An erratum to this article can be found at     

Proteome analysis of embryogenic cell suspensions of cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis>)

Using a combination of two-dimensional gel electrophoresis protein mapping and mass spectrometry analysis, we have established proteome reference maps of embryogenic cell suspensions of cowpea (Vigna unguiculata). The cell suspensions were generated from young primary leaves and contained basically pro-embryogenic masses, which enabled us to dissect their proteome composition while eliminating the complexity of too many cell types. Over 550 proteins could reproducibly be resolved over a pI range of 3–10. A total of 128 of the most abundant protein spots were excised, digested in-gel with trypsin and analyzed by tandem mass spectrometry. This enabled the identification of 67 protein spots. Two of the most abundant proteins were identified as a chitinase and as a ribonuclease belonging to the family of PR-4 and PR-10 proteins, respectively. The expression of the respective genes was confirmed by RT-PCR and the pattern of deposition of the PR-10 protein in cell suspensions as well as in developing cowpea seeds, roots, shoots and flowers were determined by Western blot experiments, using synthetic antibodies raised against a 14-amino acid synthetic peptide located close to the C-terminal region of the PR-10 protein.     

Chlorobenzoate inhibits growth and induces stress proteins in the PCB-degrading bacterium <Emphasis Type="Italic">Burkholderia xenovorans</Emphasis> LB400

Aerobic bacteria, such as Burkholderia xenovorans LB400, are able to degrade a wide range of polychlorobiphenyls (PCBs). Generally, these bacteria are not able to transform chlorobenzoates (CBAs), which accumulate during PCB degradation. In this study, the effects of CBAs on the growth, the morphology and the proteome of Burkholderia xenovorans LB400 were analysed. 4-CBA and 2-CBA were observed to inhibit the growth of strain LB400 on glucose. Strain LB400 exposed to 4-CBA exhibited increased number and size of electron-dense granules in the cytoplasm, which could be polyphosphates. Two-dimensional (2-D) polyacrylamide gel electrophoresis was used to characterise the molecular response of strain LB400 to 4-CBA. This compound induced the enzymes BenD and CatA of benzoate and catechol catabolic pathways. The induction of molecular chaperones DnaK and HtpG by 4-CBA indicated that the exposure to this compound constitutes a stressful condition for this bacterium. Additionally, the induction of some Krebs cycle enzymes was observed, probably as response to cellular energy requirements. This study contributes to the knowledge on the effects of CBA on the PCB-degrader Burkholderia xenovorans LB400.     

Differential protein expression in ovaries of uninfected and Babesia-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus

We used gel electrophoresis and mass spectrometry to investigate differences in protein expression in ovarian tissues from Babesia bovis-infected and uninfected southern cattle tick, Rhipicephalus (Boophilus) microplus. Soluble and membrane proteins were extracted from ovaries of adult female ticks, and analyzed by isoelectric focusing (IEF) and one-dimensional or two-dimensional (2-D) gel electrophoresis. Protein patterns were analyzed for differences in expression between infected and uninfected ticks. 2-D separation of proteins revealed a number of proteins that appeared to be up- or down-regulated in response to infection with Babesia, in particular membrane/membrane-associated proteins and proteins in a low molecular mass range between 6 and 36 kDa. A selection of differentially expressed proteins was subjected to analysis by capillary-HPLC-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Among the ovarian proteins that were up-regulated in infected ticks were calreticulin, two myosin subunits, an endoplasmic reticulum protein, a peptidyl-prolyl cis–trans isomerase (PPIase), a cytochrome c oxidase subunit, a glutamine synthetase, and a family of Kunitz-type serine protease inhibitors. Among the down-regulated ovarian proteins were another PPIase, a hemoglobin subunit, and a lysozyme. This study is part of an ongoing effort to establish a proteome database that can be utilized to investigate specific proteins involved in successful pathogen transmission.     

Proteomic analysis of <Emphasis Type="Italic">Psychrobacter</Emphasis><Emphasis Type="Italic"> cryohalolentis</Emphasis> K5 during growth at subzero temperatures

It is crucial to examine the physiological processes of psychrophiles at temperatures below 4°C, particularly to facilitate extrapolation of laboratory results to in situ activity. Using two dimensional electrophoresis, we examined patterns of protein abundance during growth at 16, 4, and −4°C of the eurypsychrophile Psychrobacter cryohalolentis K5 and report the first identification of cold inducible proteins (CIPs) present during growth at subzero temperatures. Growth temperature substantially reprogrammed the proteome; the relative abundance of 303 of the 618 protein spots detected (∼31% of the proteins at each growth temperature) varied significantly with temperature. Five CIPs were detected specifically at −4°C; their identities (AtpF, EF-Ts, TolC, Pcryo_1988, and FecA) suggested specific stress on energy production, protein synthesis, and transport during growth at subzero temperatures. The need for continual relief of low-temperature stress on these cellular processes was confirmed via identification of 22 additional CIPs whose abundance increased during growth at −4°C (relative to higher temperatures). Our data suggested that iron may be limiting during growth at subzero temperatures and that a cold-adapted allele was employed at −4°C for transport of iron. In summary, these data suggest that low-temperature stresses continue to intensify as growth temperatures decrease to −4°C.     

High Performance Proteomics: 7th HUPO Brain Proteome Project Workshop March 7-9, 2007 Wellcome Trust Conference Centre, Hinxton, UK

The Wellcome Trust Conference Centre at Hinxton, UK, was the meeting place of the 7th HUPO Brain Proteome Project Workshop entitled "High Performance Proteomics". It started on Wednesday, March 7, 2007 with a steering committee meeting followed by a two days series of talks dealing with the standardization and handling of tissues, body fluids as well as of proteomics data. The presentation and accompanying vivid discussions created a picture of actual strategies and standards in recent proteomics.     

Proteome map of normal rat retina and comparison with the proteome of diabetic rat retina: new insight in the pathogenesis of diabetic retinopathy

We have employed proteomics to establish a proteome map of the normal rat retina. This baseline map was then used for comparison with the early diabetic rat retinal proteome. Diabetic rat retinae were obtained from Dark Agouti rats after 10 wk of streptozotocin-induced hyperglycaemia. Extracted proteins from normal and diabetic rat retinae were separated and compared using 2-DE. A total of 145 protein spots were identified in the normal rat retina using MALDI-MS and database matching. LC-coupled ESI-MS increased the repertoire of identified proteins by 23 from 145 to 168. Comparison with early diabetic rat retinae revealed 24 proteins unique to the diabetic gels, and 37 proteins absent from diabetic gels. Uniquely expressed proteins identified included the HSPs 70.1A and 8, and platelet activating factor. There were eight spots with increased expression and 27 with decreased expression on diabetic gels. Beta catenin, phosducin and aldehyde reductase were increased in expression in diabetes whilst succinyl coA ligase and dihydropyrimidase-related protein were decreased. Identification of such changes in protein expression has given new insights and a more comprehensive understanding of the pathogenesis of diabetic retinopathy, widening the scope of potential avenues for new therapies for this common cause of blindness.     

Recent progress in liquid chromatography-based separation and label-free quantitative plant proteomics

Recent innovations in liquid chromatography-mass spectrometry (LC-MS)-based methods have facilitated quantitative and functional proteomic analyses of large numbers of proteins derived from complex samples without any need for protein or peptide labelling. Regardless of its great potential, the application of these proteomics techniques to plant science started only recently. Here we present an overview of label-free quantitative proteomics features and their employment for analysing plants. Recent methods used for quantitative protein analyses by MS techniques are summarized and major challenges associated with label-free LC-MS-based approaches, including sample preparation, peptide separation, quantification and kinetic studies, are discussed. Database search algorithms and specific aspects regarding protein identification of non-sequenced organisms are also addressed. So far, label-free LC-MS in plant science has been used to establish cellular or subcellular proteome maps, characterize plant-pathogen interactions or stress defence reactions, and for profiling protein patterns during developmental processes. Improvements in both, analytical platforms (separation technology and bioinformatics/statistical analysis) and high throughput nucleotide sequencing technologies will enhance the power of this method.     

Zygocotyle lunata: proteomic analysis of the adult stage

The somatic extract of Zygocotyle lunata (Trematoda: Paramphistomidae) adults collected from experimentally infected mice was investigated using a proteomic approach to separate and identify tryptic peptides from the somatic extract of Z. lunata adult worms. A shot-gun liquid chromatography/tandem mass spectrometry procedure was used. We used the MASCOT search engine (Matrix-Science) and ProteinPilot software v2.0 (Applied Biosystems) for the database search. A total of 36 proteins were accurately identified from the worms. The largest protein family consisted of metabolic enzymes. Structural, motor and receptor binding proteins and proteins related to oxygen transport were identified in the somatic extract of Z. lunata. This is the first study that attempts to identify the proteome of Z. lunata. However, more work is needed to improve our knowledge of trematodiasis in general and more specifically to have a better understanding about host–parasite relationships in infections with paramphistomes.     

Metal complexes as artificial proteases in proteomics: a palladium(II) complex cleaves various proteins in solutions containing detergents

Most popular agents for site-specific protein cleavage are proteolytic enzymes. Because they become denatured and inactivated by detergents, enzymes are inconvenient for proteomic analysis of hydrophobic proteins which require detergents as solubilizing agents. We used cis-[Pd(en)(H₂O)₂]²⁺ (in which en represents ethylenediamine) as an artificial protease to effect cleavage of three bovine proteins, namely ubiquitin, β-casein, and serum albumin, in separate experiments. Cleavage took place in aqueous solutions containing 1.0% wt./vol. of either 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Zwittergent 3-14 at 2.5 < pH < 2.9 and 55-60 °C for 3-72 h. Digests were separated by HPLC and analyzed by tandem mass spectrometry. Peptides were identified by de novo sequencing and matched against the bovine genome. Because cleavage by Pd(II) complexes is rather selective and therefore infrequent, 72% of the identified peptides in the digests contained more than 10 amino acid. Palladium(II) complexes hold promise as cleavage agents in proteomics studies of membrane proteins.