Orosensory stimulation effects on human saliva proteome.

Saliva flow induced by 6-gingerol (pungent), hydroxy-α/ß-sanshools (tingling), and citric acid (sour) was measured, and the time-dependent changes in the whole saliva proteome were analyzed by means of 2D-PAGE, followed by tryptic in-gel digestion and MALDI-TOF-MS peptide mass fingerprint analysis. The proteins showing significantly decreased abundance after oral 6-gingerol stimulation were identified as glutathione S-transferase P, the heat shock protein ß-1, the heat shock 70 kDa protein 1, annexin A1, and cytoplasmic ß-actin, whereas prolactin inducible proteins (PIP), short palate, lung and nasal epithelium carcinoma-associated protein 2 (SPLUNC2), zinc-α-2-glycoproteins (Zn-α-GP), and carbonic anhydrase VI (CAVI) were found with increased abundance. As the effects of this study were observed instantaneously upon stimulation, any proteome modulation is very likely to result from the release of proteins from preformed vesicles and not from de novo synthesis. The elevated levels of SPLUNC2, Zn-α-GP, and CAVI might be interpreted to trigger innate protective mechanisms in mucosal immunity and in nonimmune mucosal defense and might play an important role during the initial stage of inflammation.

Corynebacterium glutamicum as an indicator for environmental cobalt and silver stress--a proteome analysis.

Cobalt and silver are toxic for cells, but mechanisms of this toxicity are largely unknown. Analysis of Corynebacterium glutamicum proteome from cells grown in control and cobalt or silver enriched media was performed by two dimensional gel electrophoresis (2DE) followed by mass spectrometry. Our results indicate that the cell adapted to cobalt stress by inducing five defense mechanisms: Scavenging of free radicals, promotion of the generation of energy, reparation of DNA, reparation and biogenesis of Fe-S cluster proteins and supporting and reparation of cell wall. In response to the detoxification of Ag+ many proteins were up-regulated, which involved reparation of damaged DNA, minimizing the toxic effect of reactive oxygen species (ROS) and energy generation. Overexpression of proteins involved in cell wall biosynthesis (1,4-alpha-glucan branching enzyme and nucleoside-diphosphate-sugar epimerase) upon cobalt stress and induction of proteins involved in energy metabolism (2-methylcitrate dehydratase and 1, 2-methylcitrate synthase) upon silver demonstrate the potential of these enzymes as biomarkers of sub-lethal Ag+ and Co toxicity.

High-resolution two-dimensional electrophoresis.

Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry is currently the workhorse for the majority of ongoing proteome projects. Although alternative/complementary technologies, such as MudPIT, ICAT, or protein arrays, have emerged recently, there is up to now no technology that matches 2-DE in its ability for routine parallel expression profiling of large sets of complex protein mixtures. 2-DE delivers a map of intact proteins, which reflects changes in protein expression level, isoforms, or post-translational modifications. High-resolution 2-DE can resolve up to 5,000 proteins simultaneously ( approximately 2,000 proteins routinely), and detect and quantify <1 ng of protein per spot. Today's 2-DE technology with IPGs has largely overcome the former limitations of carrier ampholyte-based 2-DE with respect to reproducibility, handling, resolution, and separation of very acidic or basic proteins. Current research to further advance 2-DE technology has focused on improved solubilization/separation of hydrophobic proteins, display of low abundance proteins, and reliable protein quantitation by fluorescent dye technologies. Here, we provide a comprehensive protocol of the current high-resolution 2-DE technology with IPGs for proteome analysis and describe in detail the individual steps of this technique, i.e., sample preparation and protein solubilization, isoelectric focusing in IPG strips, IPG strip equilibration, and casting and running of multiple SDS gels. Last but not the least, a section on how to circumvent the major pitfalls is included.

Protein detection and quantitation technologies for gel-based proteome analysis.

Numerous protein detection and quantitation methods for gel-based proteomics have been devised that can be classified in three major categories: (1) Universal (or "general") detection techniques, which include staining with anionic dyes (e.g., Coomassie brilliant blue), reverse (or "negative") staining with metal cations (e.g., imidazole-zinc), silver staining, fluorescent staining or labeling, and radiolabeling, (2) specific staining methods for the detection of post-translational modifications (e.g., glycosylation or phosphorylation), and (3) differential display techniques for the separation of multiple, covalently tagged samples in a single two-dimensional electrophoresis (2-DE) gel, followed by consecutive and independent visualization of these proteins to minimize methodical variations in spot positions and in protein abundance, to simplify image analysis, as well as to improve protein quantitation by including an internal standard. The most important properties of protein detection methods applied in proteome analysis include high sensitivity (i.e., low detection limit), wide linear dynamic range for quantitative accuracy, reproducibility, cost-efficiency, ease of use, and compatibility with downstream protein identification or characterization technologies, such as mass spectrometry (MS). Regrettably, no single detection method meets all these requirements, albeit fluorescence-based technologies are currently favored for most applications; hence, the major focus of this chapter is on fluorescent-dye-based protein detection and quantitation techniques. Although satisfying results with respect to sensitivity and reproducibility are also obtained by methods based on radioactive labeling of proteins (which is still unsurpassed in terms of sensitivity), radiolabeling is, however, largely impractical for routine proteomic profiling because of the costs and the health and safety concerns associated with handling radioactive compounds.

2-DE with IPGs.

In order to overcome the limitations of carrier ampholyte generated pH gradients, IPGs were developed in the late 1970s. However, the 2-DE pattern we included in the first publication on IEF with IPGs [Bjellqvist et al., J. Biochem. Biophys. Methods 1982, 6, 317-339] was far from being competitive to O'Farrell's high-resolution 2-DE with carrier ampholytes. Our 2-DE pattern in this article was, more or less, only a proof of principle. It was, however, the beginning of a long journey of stepwise improved 2-DE protocols we developed in our laboratory and summarized in the reviews published in Electrophoresis 1988, 9, 531-546 and in Electrophoresis 2000, 21, 1037-1053. Milestones were the design of the IPG strip, and the "reduction-alkylation equilibration protocol" of IPG strips after IEF for the efficient transfer of proteins from first to second dimension. The protocol of 2-DE with IPGs has been constantly refined, e.g. by the generation of tailor-made IPGs with different pH intervals from the acidic to the basic extremes (pH 2.5-12), and extended separation distances for improved resolution. In the present review, a historical outline from the technical difficulties encountered during the development of 2-DE with IPGs, to the establishment of the actual "standard protocol" will be given, as well as the modified procedures for the separation of very acidic, very alkaline, low-abundance and hydrophobic proteins, followed by a brief discussion of the advantages and technical challenges of gel-based proteomic technologies.

Identification of potential substrate proteins for the periplasmic Escherichia coli chaperone Skp.

The "seventeen kilodalton protein" (Skp) is a predominant periplasmic chaperone of Escherichia coli, which is involved in the biogenesis of abundant outer membrane proteins (OMPs) such as OmpA, PhoE, and LamB. In this study the substrate profile of Skp was investigated in a proteomics approach. Skp was overexpressed in a deficient E. coli strain as a fusion protein with the Strep-tag and captured, together with any host proteins associated with it, from the periplasmic cell extract under mild conditions via one-step Strep-Tactin affinity chromatography. Copurified substrate proteins were then identified by high resolution 2-DE with immobilized pH-gradients, followed by MALDI-TOF MS. Apart from the known Skp substrates, including OmpA and LamB, more than 30 other interacting proteins were detected, especially from the outer membrane, among these FadL and BtuB, and from the periplasm such as MalE and OppA. Thus, Skp does not only serve as a specialized chaperone for a small set of OMPs, but it seems to exhibit a broader substrate spectrum, including soluble periplasmic proteins. These findings should prompt further investigation into the physiological role of Skp and may promote its use for the bacterial production of biochemically active heterologous proteins whose folding requires secretion into the oxidizing milieu of the periplasm.

A proteome analysis of the cadmium and mercury response in Corynebacterium glutamicum.

Cadmium and mercury are well-known toxic heavy metals, but the basis of their toxicity is not well understood. In this study, we analyzed the cellular response of Corynebacterium glutamicum to sublethal concentrations of cadmium and mercury ions using 2-DE and MS. Mercury induced the over-expression of 13 C. glutamicum proteins, whereas 35 proteins were induced, and 8 proteins were repressed, respectively, under cadmium stress. The principal response to these metals was protection against oxidative stress, as demonstrated by upregulation of, e.g., Mn/Zn superoxide dismutase. Thioredoxin and oxidoreductase responded most strongly to cadmium and mercury. The increased level of heat-shock proteins, enzymes involved in energy metabolism, as well as in lipoic acid and terpenoid biosynthesis after the treatment of cells with cadmium was also registered. Identification of these proteins and their mapping into specific cellular processes enable a global understanding of the way in which C. glutamicum adapts to heavy-metal stress and may help to gain deeper insight into the toxic mechanism of these metals.

Proteome approaches combined with Fourier transform infrared spectroscopy revealed a distinctive biofilm physiology in Bordetella pertussis.

Proteome analysis was combined with whole-cell metabolic fingerprinting to gain insight into the physiology of mature biofilm in Bordetella pertussis, the agent responsible for whooping cough. Recent reports indicate that B. pertussis adopts a sessile biofilm as a strategy to persistently colonize the human host. However, since research in the past mainly focused on the planktonic lifestyle of B. pertussis, knowledge on biofilm formation of this important human pathogen is still limited. Comparative studies were carried out by combining 2-DE and Fourier transform infrared (FT-IR) spectroscopy with multivariate statistical methods. These complementary approaches demonstrated that biofilm development has a distinctive impact on B. pertussis physiology. Results from MALDI-TOF/MS identification of proteins together with results from FT-IR spectroscopy revealed the biosynthesis of a putative acidic-type polysaccharide polymer as the most distinctive trait of B. pertussis life in a biofilm. Additionally, expression of proteins known to be involved in cellular regulatory circuits, cell attachment and virulence was altered in sessile cells, which strongly suggests a significant impact of biofilm development on B. pertussis pathogenesis. In summary, our work showed that the combination of proteomics and FT-IR spectroscopy with multivariate statistical analysis provides a powerful tool to gain further insight into bacterial lifestyles.

Sample solublization buffers for two-dimensional electrophoresis.

Before two-dimensional electrophoresis (2-DE), proteins of the sample must be denatured, reduced, disaggregated, and solubilized. Sample solubilization is usually carried out in a buffer containing chaotropes (typically 9.5 M urea, or 5-8 M urea and 2 M thiourea), 2-4% nonionic and/or zwitterionic detergent(s), reducing agent(s), carrier ampholytes and, depending on the type of sample, protease inhibitors. In this chapter, the major constituents of sample solubilization/lysis buffers will be briefly reviewed, some general sample preparation guidelines will be given, and the most common protein solubilization cocktails will be described.

Sample prefractionation in granulated sephadex IEF gels.

Prefractionation procedures not only aid in reducing sample complexity, but also permit loading of higher protein amounts within the separation range applied to two-dimensional electrophoresis (2-DE) gels and thus facilitate the detection of less abundant protein species. Hence we developed a simple, cheap, and fast prefractionation procedure based on flat-bed isoelectric focusing (IEF) in granulated Sephadex gels, containing chaotropes, zwitterionic detergents and carrier ampholytes. After IEF, up to ten Sephadex gel fractions alongside the pH gradient are obtained, and then applied directly onto the surface of the corresponding narrow pH range immobilized pH gradient (IPG) strips as first dimension of 2-DE. The major advantages of this technology are the highly efficient electrophoretic transfer of the prefractionated proteins from the Sephadex IEF fraction into the IPG strip without any sample dilution, and full compatibility with subsequent 2-DE, because the prefractionated samples have not to be eluted, concentrated or desalted, nor does the amount of the carrier ampholytes in the Sephadex fraction interfere with IEF in IPG strips. This sample prefractionation method has been successfully applied for the separation, detection and identification of low abundance proteins from pro- and eukaryotic samples.

A proteome analysis of Corynebacterium glutamicum after exposure to the herbicide 2,4-dichlorophenoxy acetic acid (2,4-D).

The herbicide 2,4-dichlorophenoxy acetic acid (2,4-D) induces a wide spectrum of toxic responses in living organisms. In this study, we analyzed the stress-induced responses of Corynebacterium glutamicum cells on protein level upon treatment with 2,4-D. For this, growing C. glutamicum cells were exposed to sublethal concentrations of 2,4-D, and changes of the gene expression profiles in comparison to non-exposed organisms were analyzed by two-dimensional gel electrophoresis and mass spectrometry. 2,4-D induced the over-expression of at least six C. glutamicum proteins, four of which could be identified by MALDI-TOF-MS. One protein (Cg2521; long-chain acyl-CoA synthetase) was related to the energy metabolism, and two proteins were involved in cell envelope synthesis (Cg2410; glutamine-dependent amidotransferase, and Cg1672; glycosyltransferase). The last induced protein was the ABC type transport system (Cg2695, ATPase component). The newly observed proteins, except for the ABC transport system, were not in general stress-related proteins, but were specifically expressed upon 2,4-D exposure and, therefore, can be used as respective biomarkers. Moreover, since these proteins seem to play a pivotal role in the adaptation of the cell to 2,4-D, they may help to gain deeper insight into the damage mechanisms of 2,4-D induced in the living cell.

Dual specificities of the glyoxysomal/peroxisomal processing protease Deg15 in higher plants.

Glyoxysomes are a subclass of peroxisomes involved in lipid mobilization. Two distinct peroxisomal targeting signals (PTSs), the C-terminal PTS1 and the N-terminal PTS2, are defined. Processing of the PTS2 on protein import is conserved in higher eukaryotes. The cleavage site typically contains a Cys at P1 or P2. We purified the glyoxysomal processing protease (GPP) from the fat-storing cotyledons of watermelon (Citrullus vulgaris) by column chromatography, preparative native isoelectric focusing, and 2D PAGE. The GPP appears in two forms, a 72-kDa monomer and a 144-kDa dimer, which are in equilibrium with one another. The equilibrium is shifted on Ca(2+) removal toward the monomer and on Ca(2+) addition toward the dimer. The monomer is a general degrading protease and is activated by denatured proteins. The dimer constitutes the processing protease because the substrate specificity proven for the monomer (Phi-Arg/Lys downward arrow) is different from the processing substrate specificity (Cys-Xxx downward arrow/Xxx-Cys downward arrow) found with the mixture of monomer and dimer. The Arabidopsis genome analysis disclosed three proteases predicted to be in peroxisomes, a Deg-protease, a pitrilysin-like metallopeptidase, and a Lon-protease. Specific antibodies against the peroxisomal Deg-protease from Arabidopsis (Deg15) identify the watermelon GPP as a Deg15. A knockout mutation in the DEG15 gene of Arabidopsis (At1g28320) prevents processing of the glyoxysomal malate dehydrogenase precursor to the mature form. Thus, the GPP/Deg15 belongs to a group of trypsin-like serine proteases with Escherichia coli DegP as a prototype. Nevertheless, the GPP/Deg15 possesses specific characteristics and is therefore a new subgroup within the Deg proteases.

Two-dimensional electrophoresis for plant proteomics.

Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteome analysis. 2-DE allows separation of highly complex mixtures of proteins according to isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance and delivers a map of intact proteins, which reflects changes in protein expression level, isoforms, or posttranslational modifications. 2-DE can resolve more than 5000 proteins simultaneously (approx 2000 proteins routinely) and can detect and quantify <1 ng of protein per spot. Today's 2-DE technology with IPGs has overcome the former limitations of carrier ampholyte-based 2-DE with respect to reproducibility, handling, resolution, and separation of very acidic and/or basic proteins. The development of IPGs between pH 2.5 and 12 has allowed the analysis of very alkaline proteins and the construction of the corresponding databases. Narrow pH range IPGs provide increased resolution (delta pI = 0.001) and, in combination with prefractionation methods, permit the detection of low abundance proteins. In this article we provide a comprehensive protocol of the current 2-DE technology for plant proteome analysis and describe in detail the individual steps of this technique.

Guidelines for the next 10 years of proteomics.

In the last ten years, the field of proteomics has expanded at a rapid rate. A range of exciting new technology has been developed and enthusiastically applied to an enormous variety of biological questions. However, the degree of stringency required in proteomic data generation and analysis appears to have been underestimated. As a result, there are likely to be numerous published findings that are of questionable quality, requiring further confirmation and/or validation. This manuscript outlines a number of key issues in proteomic research, including those associated with experimental design, differential display and biomarker discovery, protein identification and analytical incompleteness. In an effort to set a standard that reflects current thinking on the necessary and desirable characteristics of publishable manuscripts in the field, a minimal set of guidelines for proteomics research is then described. These guidelines will serve as a set of criteria which editors of PROTEOMICS will use for assessment of future submissions to the Journal.