Multiplex detection and identification of proteins on a PVDF membrane blocked with a synthetic polymer-based reagent

2-DE is one of the most powerful methods for analyzing proteins expressed in cells and tissues. Immunodetection of proteins blotted on a polymer membrane is the method of choice for detecting specific proteins in 2-D gels. To precisely locate spots of immunoreactive proteins in 2-D gels, both dye staining and immunodetection were performed on the same PVDF membrane. Prior to immunodetection, nonspecific adsorption of the antibodies to the membrane was blocked with a synthetic polymer-based reagent (N-102) after protein transfer. The protein was then stained with colloidal gold or CBB followed by protein spot identification by LC-MS. Described herein is a method for multiplex analysis of proteins transferred to a PVDF membrane. Proteins that were phosphorylated at tyrosine in the phosphoproteome of rice callus or human ovarian cancer cells were detected by immunoblotting and subsequently identified with high precision.     

Has immunoblotting replaced electroimmunoprecipitation? Examples from the analysis of autoantigens and transglutaminase-induced polymers of the human erythrocyte membrane

The virtues and drawbacks of immunoblotting and electroimmunoprecipitation in the characterization of macromolecules in crude mixtures are presented. Interactions between autoantibodies and human erythrocyte membrane proteins were studied by means of crossed-affinoimmunoelectrophoresis with autologous immunoglobulins incorporated into the first dimension gel and by immunoblotting of sodium dodecyl sulphate-polyacrylamide gel electrophoresis separated erythrocyte membrane proteins with autologous immunoglobulins as primary antibodies. Substrates for transglutaminase in calcium-activated human erythrocyte membranes were examined by immunoelectrophoretic and immunoblotting methods. The experiments concerning autoantibodies complemented each other and showed that epitopes on Band 3 protein, spectrin and ankyrin are recognized by circulating immunoglobulin autoantibodies in normal individuals. The polymer experiments showed the presence of spectrin, ankyrin, Band 3, Band 4.1, glucose transporter, actin and haemoglobin epitopes in the polymer (M_r 3 · 10⁶-5 · 10⁶). It is concluded that the two techniques complement each other. The most evident advantage of immunoblotting is its sensitivity and applicability while electroimmunoprecipitation in some instances allows an easier identification of distinct protein species and still has a rôle for quantification and certain monitoring purposes.     

A proteomic approach to investigate potential biomarkers directed against membrane-associated breast cancer proteins

The identification of specific protein markers for breast cancer would provide the basis for early diagnosis. Particularly, membrane and membrane-associated proteins are rich in targets for antibodies that may constitute suitable biomarkers of carcinogenesis. However, membrane proteins separation using 2-DE remains difficult. In this work, the breast cancer cell line MCF7 was used as source of proteins for the screening of potential cell membrane-associated antigens recognized by autoantibodies in patients with breast cancer and healthy volunteers. The protein extract obtained using trifluoroethanol (TFE) as cosolvent was compared to a total cell lysate protein extract prepared by a current technique. After 2-DE separation of the two extracts, their protein patterns clearly differed. About 63% of the proteins identified in the TFE-extract were predicted to possess at least one transmembrane domain. 2-D blots probed with sera from cancer patients or from healthy volunteers showed that, as expected, additional antigens were provided in the TFE-extract. Thus, the method described here appeared well suited for proteomic investigation of potential biomarkers undetected by current techniques.     

Usefulness of autoantigens depletion to detect autoantibody signatures by multiple affinity protein profiling

Patients with cancer produce specific autoantibodies against protein antigens present in limited amount among a large background of immunoglobulins (Igs), nonrelevant as biomarkers, including natural antibodies. Multiple affinity protein profiling (MAPPing) that combines 2-D immunoaffinity chromatography, enzymatic digestion of the isolated proteins, and identification by MS/MS, may facilitate the identification of these so far unknown patient antibodies. The first immunoaffinity chromatography is crucial, as it is used for selectively removing proteins (autoantigens) recognized by natural antibodies. Application of this depletion step to colon cancer cell proteins is specifically described along with the identification of the natural autoantigens, as well as the coupling of this depletion step with the next steps. By enabling to separate antibody-binding proteins recognized by either natural autoantibodies or patient-specific antibodies this approach may contribute significantly towards the definition of autoantibody signatures.     

Microfluidic chip analysis of outer membrane proteins responsible for serological cross-reaction between three Gram-negative bacteria: Proteus morganii O34, Escherichia coli O111 and Salmonella Adelaide O35

Bacterial strains have complex and individual antigenic structure, which provides basis for their serological identification. However, serological cross-reaction may occur when antibodies against a certain strain recognize other strains too. The molecular basis of this phenomenon is the expression of similar or identical antigenic epitopes on the surface of different bacterial cells. Such cross-reactions might harden the serological diagnosis of pathogenic bacteria. But it can be also advantageous, when antigens of non-pathogenic strains can be used in the serological examinations. Serological cross-reaction between three taxonomically different strains--Proteus morganii O34 (8662/64), Escherichia coli O111 and Salmonella Adelaide O35--have been described. It has been proven that it is based partially on the similar lipopolysaccharide structures of these pathogens. In this study the involvement of the outer membrane proteins of these strains in the serological cross-reaction is presented. Microfluidic chip technology was applied for the detection of common proteins, which provided fast and quantitative data about the proteins that might be responsible for serological cross-reaction. Two outer membrane proteins with apparent molecular mass of 36 and 41 kDa, respectively, could be detected in the profile of each strain, while individual dominating protein peaks have also appeared in the protein profiles. The presence of common protein antigens was proven by Western blotting.     

Identification of immunoreactive proteins of Chlamydia trachomatis by Western blot analysis of a two-dimensional electrophoresis map with patient sera.

Western blots of two-dimensional electrophoretic maps of proteins from Chlamydia trachomatis were probed with sera from 17 seropositive patients with genital inflammatory disease. Immunoblot patterns (comprising 28 to 2 spots, average 14.8) were different for each patient; however, antibodies against a spot-cluster due to the chlamydia-specific antigen outer membrane protein-2 (OMP2) were observed in all sera. The next most frequent group of antibodies (15/17; 88%) recognized the hsp60 GroEL-like protein, described as immunopathogenic in chlamydial infections. Reactivity to the major surface-exposed and variable antigen major outer membrane protein (MOMP) was observed at a relatively lower frequency (13/17; 76%). The hsp70 DnaK-like protein was also frequently recognized (11/17; 64.7%) in this patient group. Besides the above confirmatory findings, the study detected several new immunoreactive proteins, with frequencies ranging from 11/17 to 1/17. Some were characterized also by N-terminal amino acid sequencing and homology searches. Amongst these were a novel outer membrane protein (OmpB) and, interestingly, five conserved bacterial proteins: four (23%) sera reacted with the RNA polymerase alpha-subunit, five (29%) recognized the ribosomal protein S1, eight (47%) the protein elongation factor EF-Tu, seven (41%) a putative stress-induced protease of the HtrA family, and seven sera (41%) the ribosomal protein L7/L12. Homologs of the last two proteins were shown to confer protective immunity in other bacterial infections. The data show that immunological sensitization processes commonly thought to play a role in chlamydial pathogenicity may be sustained not only by the hsp60 GroEl-like protein, but also by other conserved bacterial antigens, some of which may be also considered as potential vaccine candidates.     

Detection of immunogenic parasite and host-specific proteins in the sera of active and chronic individuals infected with Toxoplasma gondii

Toxoplasma gondii infection in pregnant women may result in abortion and foetal abnormalities, and may be life-threatening in immunocompromised hosts. To identify the potential infection markers of this disease, 2-DE and Western blot methods were employed to study the parasite circulating antigens and host-specific proteins in the sera of T. gondii-infected individuals. The comparisons were made between serum protein profiles of infected (n=31) and normal (n=10) subjects. Antigenic proteins were identified by immunoblotting using pooled sera and monoclonal anti-human IgM-HRP. Selected protein spots were characterised using mass spectrometry. Prominent differences were observed when serum samples of T. gondii-infected individuals and normal controls were compared. A significant up-regulation of host-specific proteins, α(2)-HS glycoprotein and α(1)-B glycoprotein, was also observed in the silver-stained gels of both active and chronic infections. However, only α(2)-HS glycoprotein and α(1)-B glycoprotein in the active infection showed immunoreactivity in Western blots. In addition, three spots of T. gondii proteins were detected, namely (i) hypothetical protein chrXII: 3984434-3 TGME 49, (ii) dual specificity protein phosphatase, catalytic domain TGME 49 and (iii) NADPH-cytochrome p450 reductase TGME 49. Thus, 2-DE approach followed by Western blotting has enabled the identification of five potential infection markers for the diagnosis of toxoplasmosis: three are parasite-specific proteins and two are host-specific proteins.     

The PhastSystem equipment used for crossed immunoelectrophoresis combined with immunoblotting of coprecipitated monoclonal antibodies as studied with platelet membrane receptor proteins

The Pharmacia PhastSystem equipment has been used for crossed immunoelectrophoresis combined with a technique for immunoblotting with monoclonal antibodies. This miniaturized gel system is compared to the conventional approach using platelet membrane receptor proteins as a model. Whereas in the conventional system the electrophoretic procedure takes place within 20 h, 3 h are adequate for the small gel system. Because of the short second-dimensional electrophoresis, and only one overnight incubation, the total electrophoretic and blotting procedure could be reduced from about 48 h to 24 h. The amount of antiserum used during the second-dimensional electrophoresis could be reduced roughly by a factor of 5. The examples with electrophoresis and immunoblotting using platelet extracts in 1% Triton X-100 demonstrate that membrane receptor proteins can be studied even when present as noncovalent complexes. The immunoblotting can be used with monoclonal antibodies that do not function in Western blotting.     

Biomarker Candidates of <Emphasis Type="Italic">Chlamydophila pneumoniae</Emphasis> Proteins and Protein Fragments Identified by Affinity-Proteomics Using FTICR-MS and LC-MS/MS

We report here an affinity-proteomics approach that combines 2D-gel electrophoresis and immunoblotting with high performance mass spectrometry to the identification of both full length protein antigens and antigenic fragments of Chlamydophila pneumoniae (C. pneumoniae). The present affinity-mass spectrometry approach effectively utilized high resolution FTICR mass spectrometry and LC-tandem-MS for protein identification, and enabled the identification of several new highly antigenic C. pneumoniae proteins that were not hitherto reported or previously detected only in other Chlamydia species, such as Chlamydia trachomatis. Moreover, high resolution affinity-MS provided the identification of several neo-antigenic protein fragments containing N- and C-terminal, and central domains such as fragments of the membrane protein Pmp21 and the secreted chlamydial proteasome-like factor (Cpaf), representing specific biomarker candidates.     

Use of short monolithic columns for isolation of low abundance membrane proteins

Convective interaction media (CIM) monoliths provide a stationary phase with a high binding capacity for large molecules and are capable of high flow rates at a very low pressure drop. Used as anion- and cation-exchangers or with affinity ligands such as antibodies, these columns have the potential for processing large volumes of complex biological mixtures within a short time. In the present report, monoclonal antibodies against several rat liver plasma membrane proteins were bound and cross-linked to protein A or protein G CIM affinity columns with a bed volume of only 60 microL. Antigens recognized by bound antibodies and co-eluting (interacting) proteins were rapidly isolated in a single step from either total plasma membrane extracts or subfractions isolated using anion-exchange CIM disk-shaped columns. The isolated antigens and co-eluting proteins were subsequently identified by immunoblot or by LC-MS/MS.     

Generation and characterization of a specific polyclonal antibody against the mouse serotonin receptor 1A: a state-of-the-art recommendation on how to characterize antibody specificity

A series of different antibodies against serotonin receptor 1A (5HT1A_R) have been reported although only limited information on the specificity of these antibodies and the antigens recognized is available. Herein, we characterized reactivity of an antibody by a gel-based proteomics method that should represent a model how antibodies may be defined in the future. An antibody against the 5HT1A_R was generated, used for immunoprecipitation and immunoblotting on blue-native gels containing a 5HT1A_R complex. The 5HT1A_R was isolated from tissue and was defined by nano-LC-ESI-MS/MS. A single band on the native gel and a single spot representing the denatured receptor in the 3rd dimensional step of gel electrophoresis was detected. Immunoprecipitation revealed a single band for the denatured 5HT1A_R. Herein, a procedure is proposed to characterize an antibody by the use of a robust method unambiguously identifying and characterizing the antigen, 5HT1A_R, from mouse whole brain.     

Overexpression,Purification and Validation of Antigenic Salmonella enterica Serovar Typhi Proteins Identified from LC-MS/MS

In our earlier study, an immunoblot analysis using sera from febrile patients revealed that a 50-kDa band from an outer membrane protein fraction of Salmonella enterica serovar Typhi was specifically recognized only by typhoid sera and not sera from other febrile illnesses. Here, we investigated the identities of the proteins contained in the immunogenic 50-kDa band to pinpoint antigens responsible for its immunogenicity. We first used LC-MS/MS for protein identification, then used the online tool ANTIGENpro for antigenicity prediction and produced recombinant proteins of the lead antigens for validation in an enzyme-linked immunosorbent assay (ELISA). We found that proteins TolC, GlpK and SucB were specific to typhoid sera but react to antibodies differently under native and denatured conditions. This difference suggests the presence of linear and conformational epitopes on these proteins.     

聚乙二醇修饰蛋白体系的SDS-聚丙烯酰胺凝胶电泳染色方法新探

比较了聚乙二醇修饰蛋白体系的SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)银染、考染、碘化钡染色3种染色方法;提出和比较了银染-碘化钡复染和考染-碘化钡复染2种复染方法.结果表明,银染-碘化钡复染的凝胶中,未修饰蛋白条带消失,PEG修饰蛋白条带保留,游离PEG条带显色;而考染-碘化钡复染的凝胶中,未修饰蛋白、修饰蛋白和游离的PEG条带可同时显色.两种复染方法中,PEG组分的检测限均达到了0.01μg.因此,对PEG修饰蛋白体系的SDS-PAGE可先用考染或银染后再用碘化钡复染,便可在同一块凝胶上先后或同时观察到未修饰蛋白、修饰蛋白和游离PEG的情况,简化了实验操作,方便了实验结果的比较分析.     

Glucose/Ribitol Dehydrogenase and 16.9 kDa Class I Heat Shock Protein 1 as Novel Wheat Allergens in Baker’s Respiratory Allergy

Wheat allergens are responsible for symptoms in 60–70% of bakers with work-related allergy, and knowledge, at the molecular level, of this disorder is progressively accumulating. The aim of the present study is to investigate the panel of wheat IgE positivity in allergic Italian bakers, evaluating a possible contribution of novel wheat allergens included in the water/salt soluble fraction. The water/salt-soluble wheat flour proteins from the Italian wheat cultivar Bolero were separated by using 1-DE and 2-DE gel electrophoresis. IgE-binding proteins were detected using the pooled sera of 26 wheat allergic bakers by immunoblotting and directly recognized in Coomassie stained gel. After a preparative electrophoretic step, two enriched fractions were furtherly separated in 2-DE allowing for detection, by Coomassie, of three different proteins in the range of 21–27 kDa that were recognized by the pooled baker’s IgE. Recovered spots were analyzed by nanoHPLC Chip tandem mass spectrometry (MS/MS). The immunodetected spots in 2D were subjected to mass spectrometry (MS) analysis identifying two new allergenic proteins: a glucose/ribitol dehydrogenase and a 16.9 kDa class I heat shock protein 1. Mass spectrometer testing of flour proteins of the wheat cultivars utilized by allergic bakers improves the identification of until now unknown occupational wheat allergens.     

Mass spectrometric approaches for elucidation of antigenantibody recognition structures in molecular immunology

Mass spectrometric approaches have recently gained increasing access to molecular immunology and several methods have been developed that enable detailed chemical structure identification of antigen-antibody interactions. Selective proteolytic digestion and MS-peptide mapping (epitope excision) has been successfully employed for epitope identification of protein antigens. In addition, "affinity proteomics" using partial epitope excision has been developed as an approach with unprecedented selectivity for direct protein identification from biological material. The potential of these methods is illustrated by the elucidation of a beta-amyloid plaque-specific epitope recognized by therapeutic antibodies from transgenic mouse models of Alzheimer's disease. Using an immobilized antigen and antibody-proteolytic digestion and analysis by high resolution Fourier transform ion cyclotron resonance mass spectrometry has lead to a new approach for the identification of antibody paratope structures (paratope-excision; "parex-prot"). In this method, high resolution MS-peptide data at the low ppm level are required for direct identification of paratopes using protein databases. Mass spectrometric epitope mapping and determination of "molecular antibody-recognition signatures" offer high potential, especially for the development of new molecular diagnostics and the evaluation of new vaccine lead structures.     

Mass spectrometrical analysis of bilin-binding protein from the wing of Hebomoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae)

Bilin-binding protein (BBP) is a member of the lipocalin superfamily and a pigment protein in Lepidoptera. It is binding to a series of lipidic compounds but its functions remain to be elucidated. Working on wing proteins in Hebomoia glaucippe, we observed this protein on gels and decided to characterize BBP. A gel-based mass spectrometrical method using two-dimensional gel electrophoresis followed by in-gel digestion of protein spots followed by nano-LC-ESI-MS/MS (ion trap, HCT) identification and characterization of proteins was applied. An antibody was generated against the protein and immunoblotting in the butterfly and mouse brain was carried out. Two spots were identified from the butterfly wing as BBP (P09464) with high sequence coverage. Nitrotyrosination (Y163; as aminotyrosine) was observed and nitration was verified using immunoblotting. Additional posttranslational modifications (PTMs) as hypusine, carboxylation, kynurenine, aminoadipic acid, were proposed. The presence of BBP-immunoreactive protein was also observed in mouse brain. The characterization of BBP showed high sequence similarity with mouse apolipoprotein D and the findings suggest a tentative function of BBP comparable to apolipoproteins. The role of the PTMs remains elusive but nitration, in analogy to nitration effects reported in literature, proposes a role for mechanoelastic proteins and protein-protein interactions.     

An optimized procedure for solubilization, reduction, and transfer of human breast cancer membrane-enriched fraction by 2-DE

The separation of integral and peripheral membrane proteins is still a challenge, although many achievements have been made in the 2-DE-based membrane proteomics. Using a human breast cancer cell line, MCF-7, we investigated the influences of Tris, reducing reagents, cup loading, and SDS on membrane protein solubilization and separation by 2-DE. The addition of Tris to the sample solution improved the solubilization of the membrane-enriched fraction, and the best-quality gel patterns were obtained at 20 mM Tris. Tributylphosphine (TBP), a reducing agent, was not optimum in the 2-DE process because it not only decreased the solubilization of hydrophobic proteins but also caused some proteins, such as hsp60, prohibitin, and actin, to be resolved to a string of spots. However, when combined with DTT, TBP could improve the resolution of 2-DE patterns. Cup loading significantly facilitated the entrance of membrane proteins into IPG strips and over 1000 protein spots with high resolution were visualized. Adopting this strategy, an ATP synthase alpha chain was resolved into two adjacent spots for the first time in 2-DE gel patterns through the adding DTT in the middle of the IEF. A high SDS concentration in the equilibration buffer enhanced the transfer and increased the staining intensity of 50% of the protein spots in the gels, but also resulted in losses of some spots.     

The biological activity of radioiodinated anti-T antibodies

Rabit anti-T (IgG) was radioiodinated by iodine monochloride using three different methods: Direct, Indirect and Protected. Although protection of antigen combining sites during radiolabelling (Protected method) did not significantly increase the binding of the rabbit anti-T to an artificial hapten, binding of the antibody to the natural antigen on the cell membrane was considerably improved when the antibody was labelled by this Protected method. We propose that radiolabelled anti-T may be useful in tumour localizing studies in man, but that binding efficiency of radiolabelled antibodies to isolated antigens may not reflect binding efficiency to cell bound antigens.     

Two-dimensional Blue Native/sodium dodecyl sulfate gel electrophoresis for analysis of multimeric proteins in platelets

Two-dimensional (2-D) Blue Native/SDS gel electrophoresis combines a first-dimensional separation of monomeric and multimeric proteins in their native state with a second denaturing dimension. These high-resolution 2-D gels aim at identifying multiprotein complexes with respect to their subunit composition. We applied this method for the first time to analyze two human platelet subproteomes: the cytosolic and the microsomal membrane protein fraction. Solubilization of platelet membrane proteins was achieved with the nondenaturing detergent n-dodecyl-beta-D-maltoside. To validate native solubilization conditions, we demonstrated the correct assembly of the Na,K-ATPase, a functional multimeric transmembrane protein, when expressed in Xenopus oocytes. We identified 63 platelet proteins after in-gel tryptic digestion of 58 selected protein spots and liquid chromatography-coupled tandem mass spectrometry. Nine proteins were detected for the first time in platelets by a proteomic approach. We also show that this technology efficiently resolves several known membrane and cytosolic multiprotein complexes. Blue Native/SDS gel electrophoresis is thus a valuable procedure to analyze specific platelet subproteomes, like the membrane(-bound) protein fraction, by mass spectrometry and immunoblotting and could be relevant for the study of protein-protein interactions generated following platelet activation.     

The proteome of maize leaves: use of gene sequences and expressed sequence tag data for identification of proteins with peptide mass fingerprints.

As a first step in establishing a proteome database for maize, we have embarked on the identification of the leaf proteins resolved on two-dimensional (2-D) gels. We detected nearly 900 spots on the gels with a pH 4-7 gradient and over 200 spots on the gels with a pH 6-11 gradient when the proteins were visualized with colloidal Coomassie blue. Peptide mass fingerprints for 300 protein spots were obtained with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer and 149 protein spots were identified using the protein databases. We also searched the pdbEST databases to identify the leaf proteins and verified 66% of the protein spots that had been identified using the protein databases. Sixty-seven additional protein spots were identified from expressed sequence tags (ESTs). Many abundant leaf proteins are present in multiple spots. Functions of over 50% of the abundant leaf proteins are either unknown or hypothetical. Our results show that EST databases in conjunction with peptide mass fingerprints can be used for identifying proteins from organisms with incomplete genome sequence information.