Autoantibody profiling in multiple sclerosis reveals novel antigenic candidates

An important contribution of B cells and autoantibodies has been demonstrated in the pathogenesis of multiple sclerosis (MS), leading to interest in the use of such autoantibodies as diagnostic or prognostic biomarkers. The objective of this study was to identify novel Ab biomarkers for MS using "serological Ag selection". Using a phage display library derived from MS brain plaques, we applied serological Ag selection to identify antigenic targets specifically interacting with Abs present in the cerebrospinal fluid (CSF) of 10 relapsing-remitting MS patients. These antigenic targets were further evaluated on a large panel of CSF from 63 other MS patients, 30 patients with other inflammatory disorders, and 64 patients with noninflammatory neurological disorders. A panel of eight antigenic targets was identified that showed a 86% specificity and 45% sensitivity in discriminating MS patients and controls. Four of the antigenic targets showed exclusive reactivity (100% specificity; 23% sensitivity) in the MS group as compared with the control group. Detailed bio-informatic analyses revealed a novel Ag, SPAG16. Among the novel phage peptides identified, novel epitopes were generated from untranslated sequences and out-of-frame sequences. Of 10 relapsing-remitting patients used for serological Ag selection, Ab reactivity toward one of the eight antigenic targets was also demonstrated in serum of 38% CSF-positive patients. Autoantibody profiles against epitopes derived from MS brain tissue could serve as diagnostic markers or form the basis for the identification of a subgroup of MS patients.     

Identification of tumor antigens in renal cell carcinoma by serological proteome analysis

We have investigated the suitability of proteomics for identification of tumor-associated antigens. First, we compared the proteomes of nontumorous kidney and renal cell carcinoma (RCC) by two-dimensional gel electrophoresis (2-DE) and silver staining. Protein patterns were markedly different (approximately 800 spots in RCCs versus approximately 1400 spots in kidney). 2-DE immunoblotting revealed five RCC-specific spots, reproducibly reactive with RCC-patient but not healthy donor control sera. Two of these antigens were isolated by preparative 2-DE, and identified by Edman sequencing of tryptic peptides. The first antigen, smooth muscle protein 22-alpha (SM22-alpha), is an actin-binding protein of unknown function predominantly expressed in smooth muscle cells. In situ hybridization revealed that SM22-alpha is not expressed in the malignant cells but in mesenchymal cells of the tumor stroma. The second antigen represents carbonic anhydrase I (CAI), an isoform usually not expressed in kidney. Interestingly, a different isoform (CAXII) has previously been identified by serological expression cloning as an antigen overexpressed in some RCCs. In additional assays, antibodies to recombinant CAI or SM22-alpha were detected in sera from 3/11 or 5/11 RCC patients, respectively, whereas sera from 13 healthy individuals did not react. In conclusion, serological proteome analysis may be a new tool for the identification of tumor-associated antigens.     

Identification of tumor antigens in human lung squamous carcinoma by serological proteome analysis

Autoantibodies against tumor antigens are promising means for cancer diagnosis and prognosis. In this study, we applied a proteomic approach to identify proteins that commonly elicit humoral response in lung squamous carcinoma (LSC). Sera from 20 newly diagnosed patients with LSC and 20 matched healthy individuals were analyzed for antibody-based reactivity against LSC proteins separated by two-dimensional electrophoresis. Autoantibodies against triosephosphate isomerase (Tim) and superoxide dismutase [Mn] (MnSOD) were detected in sera from over 20% patients with LSC but none from the normal controls. Furthermore, the occurrence of autoantibodies against Tim and MnSOD was evaluated by ELISA in an additional 40 LSC patients, 30 other types of cancer (OTC) patients, and 50 noncancer controls (NC). Results showed that frequency of autoantibody against Tim (27.5%) in LSC patients was significantly higher than that in OTC patients (6.7%, p = 0.027) and in NC (6%, p = 0.005). Likewise, frequency of autoantibody against MnSOD in LSC (20%) patients was significantly higher than that in NC (4%, p = 0.016), however, there was no significant difference when comparing to that in OTC patients (6.7%, p = 0.115). We also observed significantly increased expression and secretion of Tim and MnSOD in LSC, which possibly account for their autoantibody development. Our results indicate that autoantibody and antigen of Tim and MnSOD may be useful for screening and diagnosis of the lung squamous carcinoma.     

Identification of vaccine candidate antigens of Staphylococcus aureus by serological proteome analysis

In this study we applied serological proteome analysis (Klade, C. S. et al. Proteomics 2001, 1, 890-898) for identification of bacterial vaccine candidate antigens. First, approximately one hundred sera from healthy individuals and patients suffering from Staphylococcus aureus infections were screened for antibodies against staphylococcal lysates and recombinant proteins representing surface antigens. Two pools (healthy donors, patients) each consisting of five sera with the highest antiproteinaceous IgG reactivity were selected. Second, S. aureus COL was grown under different conditions and the number of antigens expressed was monitored by Western blot analysis. Third, surface proteins were enriched by digesting the bacterial cell wall under isotonic conditions and subsequent removal of protoplasts. These protein preparations were resolved by two-dimensional electrophoresis (2-DE) (pI 4-7). 2-DE immunoblotting using the preselected serum pools at 1:10 000-1:100 000 dilutions revealed a number of highly immunogenic staphylococcal proteins. Twenty-one spots were isolated by preparative 2-DE, and analysed by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry sequencing of tryptic peptides. This led to the identification of 15 proteins including known and novel vaccine candidates. Seroreactivity of several antigens including serine-aspartate repeat containing protein D, immuno-dominant staphylococcal antigen and a novel 309 amino acid lipoprotein was independently confirmed by enzyme-linked immunosorbent assay and Western blot analysis of purified recombinant proteins. In conclusion, serological proteome analysis proved to be a powerful tool for the identification of novel staphylococcal antigens, which provide a basis for rational vaccine design.     

Predominant identification of RNA-binding proteins in Fas-induced apoptosis by proteome analysis

Proteome analysis of Jurkat T cells was performed in order to identify proteins that are modified during apoptosis. Subtractive analysis of two-dimensional gel patterns of apoptotic and nonapoptotic cells revealed differences in 45 protein spots. 37 protein spots of 21 different proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. The hnRNPs A0, A2/B1, A3, K, and R; the splicing factors p54(nrb), SRp30c, ASF-2, and KH-type splicing regulatory protein (FUSE-binding protein 2); and alpha NAC, NS1-associated protein 1, and poly(A)-binding protein 4 were hitherto unknown to be involved in apoptosis. The putative cleavage sites of the majority of the proteins could be calculated by the molecular masses and isoelectric points in the two-dimensional electrophoresis gel, the peptide mass fingerprints, and after translation by treatment with recombinant caspase-3. Remarkably, 15 of the 21 identified proteins contained the RNP or KH motif, the best characterized RNA-binding motifs.     

Surface proteome mining for identification of potential vaccine candidates against Campylobacter jejuni: an in silico approach

Functional & Integrative Genomics - Campylobacter jejuni remains a major cause of human gastroenteritis with estimated annual incidence rate of 450 million infections worldwide. C. jejuni is a...     

Lumbar cerebrospinal fluid proteome in multiple sclerosis: characterization by ultrafiltration, liquid chromatography, and mass spectrometry

Neurological diseases, including multiple sclerosis (M.S.), often provoke changes in the functioning of the endothelial and epithelial brain barriers and give rise to disease-associated alterations of the cerebrospinal fluid (CSF) proteome. In the present study, pooled and ultrafiltered CSF of M.S. and non-M.S. patients was digested with trypsin and analyzed by off-line strong cation-exchange chromatography (SCX) coupled to on-line reversed-phase LC-ESI-MS/MS. In an alternative approach, the trypsin-treated subproteomes were analyzed directly by LC-ESI-MS/MS and gas-phase fractionation in the mass spectrometer. Taken together, both proteomic approaches in combination with a three-step evaluation process including the search engines Sequest and Mascot, and the validation software Scaffold, resulted in the identification of 148 proteins. Sixty proteins were identified in CSF for the first time by mass spectrometry. For validation purposes, the concentration of cystatin A was determined in individual CSF and serum samples of M.S. and non-M.S. patients using ELISA.     

Identification of hepatocellular-carcinoma-associated antigens and autoantibodies by serological proteome analysis combined with protein microarray

To comprehensively study autoantibodies in patients with hepatocellular carcinoma (HCC), we used an approach-based serology and proteomics technologies. Total proteins extracted from HepG2 cells and HepG2.2.15 cells were separated by two-dimensional gel electrophoresis (2DE) and then transferred onto polyvinylidene difluoride (PVDF) membranes, which were subsequently incubated with sera from HCC patients or from normal controls. As a result, 13 HCC-associated antigens were identified. Antigenicity of eight proteins was further confirmed using recombinant proteins by Western blotting (WB) and protein microarray. The results of antigen microarray analysis showed strong signals of keratin 8 and lamin A/C in chronic hepatitis controls; therefore, the autoantibodies to keratin 8 and lamin A/C may not be HCC-specific. These two antigens were removed from subsequent analyses. The frequencies of positive reactions to DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, eukaryotic translation elongation factor 2 (eEF2), apoptosis-inducing factor (AIF), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2), prostatic binding protein, and triosephosphate isomerase (TIM) were significantly higher in HCC than in chronic hepatitis and normal individuals. Positive reactions to DEAD box polypeptide 3, eEF2, AIF, and prostatic binding protein were significantly more frequent in HCC than in any other cancer. The sensitivity of any individual antigen in HCC at stage I ranged from 50 to 85%. When the combinations of six antigens were analyzed, the sensitivity increased to 90%. We conclude that the detection of autoantibodies against the six antigens may have value on early diagnosis of HCC.     

Psoroptes ovis: identification of vaccine candidates by immunoscreening

Serum from successful vaccine trials against the sheep scab mite, Psoroptes ovis, was used to immunoscreen a cDNA library constructed from mixed-stage and gender P. ovis to identify potential recombinant vaccine candidates. Immunodominant recombinant proteins recognised by IgG in these sera were selected for further analysis. Two candidates were identified in this way; a catchin-like protein (CLP) and a novel mu class glutathione S-transferase (GST). Both candidates were expressed in bacteria as recombinant proteins, the GST as an active enzyme, and combined with four other recombinant allergens in a multi-component recombinant vaccine. Strong serum IgG responses were induced in sheep against each of the components of the recombinant vaccine, however, the protective efficacy of the vaccine could not be determined because of variability in the establishment of a challenge infection.     

Rapid identification of yeasts by serological methods: a combined serological and biological method.

A total of 387 yeasts from the contents of the digestive tracts of domestic animals and poultry were identified by slide agglutination tests using factor antisera and urease tests. The results of this serological test were very satisfactory with respect to accuracy and rapidity, particularly when performed in combination with concomitant physiological tests only for assimilation of inositol and potassium nitrate. It may be concluded that such a combination of serological and biological tests is very useful for identifying yeast strains from various sources.     

A branched-chain organic acid linked to multiple sclerosis: first identification by NMR spectroscopy of CSF

(1)H NMR spectroscopy of cerebrospinal fluid (CSF) is currently being used to study metabolic profiles characteristic of distinct multiple sclerosis (MS) manifestations. For select MS patient groups, we have previously detected significantly increased concentrations of several identified metabolites and one unidentified compound. We now present, for the first time, the identification of the latter molecule, beta-hydroxyisobutyrate (BHIB). A combination of dedicated 1D and 2D (1)H NMR experiments was employed for signal assignment. To our knowledge, BHIB has not previously been identified in (1)H NMR spectra of biofluids or biological tissues. Our assignment suggests new biochemical pathways involved in specific MS pathologies.     

New insights into the rat spermatogonial proteome: identification of 156 additional proteins

Despite the essential role played by spermatogonia in testicular function, little is known about these cells. To improve our understanding of their biology, our group recently identified a set of 53 spermatogonial proteins using two-dimensional (2-D) gel electrophoresis and mass spectrometry. To continue this work, we investigated a subset of the spermatogonial proteome using narrow range immobilized pH gradients to favor the detection of less abundant proteins. A 2-D reference map of spermatogonia in the pH range 4-9 was created, and protein entities fractionated in a pH 5-6 2-D gel were further processed for protein identification. A new set of 156 polypeptides was identified by peptide mass fingerprinting and tandem mass spectrometry. These polypeptides corresponded to 102 different proteins, which reflect the complexity of post-translational modifications. Seventy-nine of these proteins were identified for the first time in spermatogonia. All identified proteins were classified into functional groups. This work represents a first step toward the establishment of a systematic spermatogonia protein database.     

Improving sensitivity in proteome studies by analysis of false discovery rates for multiple search engines

LC‐MS experiments can generate large quantities of data, for which a variety of database search engines are available to make peptide and protein identifications. Decoy databases are becoming widely used to place statistical confidence in result sets, allowing the false discovery rate (FDR) to be estimated. Different search engines produce different identification sets so employing more than one search engine could result in an increased number of peptides (and proteins) being identified, if an appropriate mechanism for combining data can be defined. We have developed a search engine independent score, based on FDR, which allows peptide identifications from different search engines to be combined, called the FDR Score. The results demonstrate that the observed FDR is significantly different when analysing the set of identifications made by all three search engines, by each pair of search engines or by a single search engine. Our algorithm assigns identifications to groups according to the set of search engines that have made the identification, and re‐assigns the score (combined FDR Score). The combined FDR Score can differentiate between correct and incorrect peptide identifications with high accuracy, allowing on average 35% more peptide identifications to be made at a fixed FDR than using a single search engine.