Regulatory role of cysteines in (2R, 3R)-butanediol dehydrogenase BdhA of Bacillus velezensis strain GH1-13.

Bacillus velezensis strain GH1-13 contains a (2R,3R)-butanediol dehydrogenase (R-BDH) BdhA which converts acetoin to R-BD reversibly, however, little is known about its regulatory cysteine and biological significance. We performed site-directed mutation of three cysteines in BdhA. The C37S mutant had no enzyme activity and the C34S and C177S mutants differed from each other and wild type (WT). After zinc affinity chromatography, 1 mM ZnCl2 treatment resulted in a 3-fold enhancement of the WT activity, but reduced activity of the C34S mutant by more than 2 folds compared to the untreated ones. However, ZnCl2 treatment did not affect the activity of the C177S mutant. Most of the double and triple mutant proteins (C34S/C37S, C34S/C177S, C37S/C177S, and C34S/C37S/C177S) were aggregated in zinc resins, likely due to the decreased protein stability. All of the purified WT and single mutant proteins increased multiple intermolecular disulfide bonds in the presence of H2O2 as the buffer pH decreased from 7.5 to 5.5, whereas an intramolecular disulfide bond of cysteine 177 and another cysteine in the CGIC motif region was likely formed at pH higher than pKa of 7.5. When pH varied, WT and its C34S or C177S mutants reduced acetoin to R-BD at the optimum pH 5.5 and oxidized R-BD to acetoin at the optimum pH 10. This study demonstrated that cysteine residues in BdhA play a regulatory role for the production of acetoin and R-BD depending on pH as well as metal binding and oxidative stress.

Substrate-dependent effects of quaternary structure on RNase E activity.

RNase E is an essential, multifunctional ribonuclease encoded in E. coli by the rne gene. Structural analysis indicates that the ribonucleolytic activity of this enzyme is conferred by rne-encoded polypeptide chains that (1) dimerize to form a catalytic site at the protein-protein interface, and (2) multimerize further to generate a tetrameric quaternary structure consisting of two dimerized Rne-peptide chains. We identify here a mutation in the Rne protein's catalytic region (E429G), as well as a bacterial cell wall peptidoglycan hydrolase (Amidase C [AmiC]), that selectively affect the specific activity of the RNase E enzyme on long RNA substrates, but not on short synthetic oligonucleotides, by enhancing enzyme multimerization. Unlike the increase in specific activity that accompanies concentration-induced multimerization, enhanced multimerization associated with either the E429G mutation or interaction of the Rne protein with AmiC is independent of the substrate's 5' terminus phosphorylation state. Our findings reveal a previously unsuspected substrate length-dependent regulatory role for RNase E quaternary structure and identify cis-acting and trans-acting factors that mediate such regulation.

FOXL2 directs DNA double-strand break repair pathways by differentially interacting with Ku.

The balance between major DNA double-strand break (DSB) repair pathways is influenced by binding of the Ku complex, a XRCC5/6 heterodimer, to DSB ends, initiating non-homologous end joining (NHEJ) but preventing additional DSB end resection and homologous recombination (HR). However, the key molecular cue for Ku recruitment to DSB sites is unknown. Here, we report that FOXL2, a forkhead family transcriptional factor, directs DSB repair pathway choice by acetylation-dependent binding to Ku. Upon DSB induction, SIRT1 translocates to the nucleus and deacetylates FOXL2 at lysine 124, leading to liberation of XRCC5 and XRCC6 from FOXL2 and formation of the Ku complex. FOXL2 ablation enhances Ku recruitment to DSB sites, imbalances DSB repair kinetics by accelerating NHEJ and inhibiting HR, and thus leads to catastrophic genomic events. Our study unveils the SIRT1-(de)acetylated FOXL2-Ku axis that governs the balance of DSB repair pathways to maintain genome integrity.

A mitochondrial proteome profile indicative of type 2 diabetes mellitus in skeletal muscles.

The pathogenesis of type 2 diabetes mellitus (T2DM) is closely associated with mitochondrial functions in insulin-responsive tissues. The mitochondrial proteome, compared with the mitochondrial genome, which only contains 37 genes in humans, can provide more comprehensive information for thousands of mitochondrial proteins regarding T2DM-associated mitochondrial functions. However, T2DM-associated protein signatures in insulin-responsive tissues are still unclear. Here, we performed extensive proteome profiling of mitochondria from skeletal muscles in nine T2DM patients and nine nondiabetic controls. A comparison of the mitochondrial proteomes identified 335 differentially expressed proteins (DEPs) between T2DM and nondiabetic samples. Functional and network analyses of the DEPs showed that mitochondrial metabolic processes were downregulated and mitochondria-associated ER membrane (MAM) processes were upregulated. Of the DEPs, we selected two (NDUFS3 and COX2) for downregulated oxidative phosphorylation and three (CALR, SORT, and RAB1A) for upregulated calcium and protein transport as representative mitochondrial and MAM processes, respectively, and then confirmed their differential expression in independent mouse and human samples. Therefore, we propose that these five proteins be used as a potential protein profile that is indicative of the dysregulation of mitochondrial functions in T2DM, representing downregulated oxidative phosphorylation and upregulated MAM functions.

Minimum InDel pattern analysis of the Zika virus.

BACKGROUND: The Zika virus (ZIKV) can cause microcephaly and congenital abnormalities in the foetus. Recent studies have provided insights into the evolution of ZIKV from the current and previous outbreaks, but the types have not been determined. RESULTS: We analysed the insertions and deletions (InDels) in 212 ZIKV polyproteins and 5 Dengue virus (DENV) reference sequences. Spearman correlation tests for the minimum InDel (minInDel) patterns were used to assess the type of polyprotein. Using the minInDel frequencies calculated from polyproteins with 11 elements, likelihood estimation was conducted to correct the evolutionary distance. The minInDel-corrected tree topology clearly distinguished between the ZIKV types (I and II) with a unique minInDel character in the E protein. From the 10-year average genetic distance, the African and Asian lineages of ZIKV-II were estimated to have occurred ~ 270 years ago, which is unlikely for ZIKV-I. CONCLUSIONS: The minInDel pattern analysis showed that the minInDel in the E protein is targetable for the rapid detection and determination of the virus types.

Determination of the binding site of 2-aminothiazole derivative with importin ß1 by UV-crosslinking experiment.

Importin ß1 (KPBN1) appears to be overexpressed in several cancer cells and siRNA-induced inhibition of KPNB1 shows significant inhibition of cancer cell proliferation, but do not affect normal cells. These results indicate that KPNB1 is a potential target and inhibition of KPNB1 can be used as a novel therapeutic approach for the treatment of cancer. Recently, we identified the aminothiazole derivative 1 as a KPNB1-targeted anticancer agent. Herein, we report that compound 1 binds strongly to KPNB1, in a pocket centered around serine-476, as shown by UV-crosslinking and tandem mass spectrometry experiments, and supported using a model derived from molecular docking.

Identification of KPNB1 as a Cellular Target of Aminothiazole Derivatives with Anticancer Activity.

We found that aminothiazole derivative (E)-N-(5-benzylthiazol-2-yl)-3-(furan-2-yl)acrylamide (1) has strong anticancer activity, and undertook proteomics approaches to identify the target protein of compound 1, importin ß1 (KPNB1). A competitive binding assay using fluorescein-labeled 1 showed that 1 has strong binding affinity for KPNB1 (Kd : ∼20 nm). Furthermore, through western blotting assays for KPNB1, KPNA2, EGFR, ErbB2, and STAT3, we confirmed that 1 has inhibitory effects on the importin pathway. KPBN1 appears to be overexpressed in several cancer cells, and siRNA-induced inhibition of KPNB1 shows significant inhibition of cancer cell proliferation, while leaving non-cancerous cells unaffected. Therefore, compound 1 is a promising new lead for the development of KPNB1-targeted anticancer agents. Fluorescein-labeled 1 could be a useful quantitative probe for the development of novel KPNB1 inhibitors.

MdsABC-Mediated Pathway for Pathogenicity in Salmonella enterica Serovar Typhimurium.

MdsABC is a Salmonella-specific tripartite efflux pump that has been implicated in the virulence of Salmonella enterica serovar Typhimurium; however, little is known about the virulence factors associated with this pump. We observed MdsABC expression-dependent alterations in the degree of resistance to extracellular oxidative stress and macrophage-mediated killing. Thin-layer chromatography and tandem mass spectrometry analyses revealed that overexpression of MdsABC led to increased secretion of 1-palmitoyl-2-stearoyl-phosphatidylserine (PSPS), affecting the ability of the bacteria to invade and survive in host cells. Overexpression of MdsABC and external addition of PSPS similarly rendered the mdsABC deletion strain resistant to diamide. Diagonal gel analysis showed that PSPS treatment reduced the diamide-mediated formation of disulfide bonds, particularly in the membrane fraction of the bacteria. Salmonella infection of macrophages induced the upregulation of MdsABC expression and led to an increase of intracellular bacterial number and host cell death, similar to the effects of MdsABC overexpression and PSPS pretreatment on the mdsABC deletion strain. Our study shows that MdsABC mediates a previously uncharacterized pathway that involves PSPS as a key factor for the survival and virulence of S. Typhimurium in phagocytic cells.

Structural and Mechanistic Insights into the Pseudomonas fluorescens 2-Nitrobenzoate 2-Nitroreductase NbaA.

The bacterial 2-nitroreductase NbaA is the primary enzyme initiating the degradation of 2-nitrobenzoate (2-NBA), and its activity is controlled by posttranslational modifications. To date, the structure of NbaA remains to be elucidated. In this study, the crystal structure of a Cys194Ala NbaA mutant was determined to a 1.7-Å resolution. The substrate analog 2-NBA methyl ester was used to decipher the substrate binding site by inhibition of the wild-type NbaA protein. Tandem mass spectrometry showed that 2-NBA methyl ester produced a 2-NBA ester bond at the Tyr193 residue in the wild-type NbaA but not residues in the Tyr193Phe mutant. Moreover, covalent binding of the 2-NBA methyl ester to Tyr193 reduced the reactivity of the Cys194 residue on the peptide link. The Tyr193 hydroxyl group was shown to be essential for enzyme catalysis, as a Tyr193Phe mutant resulted in fast dissociation of flavin mononucleotide (FMN) from the protein with the reduced reactivity of Cys194. FMN binding to NbaA varied with solution NaCl concentration, which was related to the catalytic activity but not to cysteine reactivity. These observations suggest that the Cys194 reactivity is negatively affected by a posttranslational modification of the adjacent Tyr193 residue, which interacts with FMN and the substrate in the NbaA catalytic site.

Identification and Validation of Differential Phosphorylation Sites of the Nuclear FOXL2 Protein as Potential Novel Biomarkers for Adult-Type Granulosa Cell Tumors.

Granulosa cell tumor (GCT) is a rare form of ovarian cancer classified as a sex cord-stromal tumor. The c.402C→G missense mutation in the FOXL2 gene that changes cysteine 134 to tryptophan (C134W) is found in more than 97% of adult-type GCTs, and the C134W FOXL2 mutant is hyperphosphorylated. We identified three differential phosphorylation sites, at serine 33 (S33), tyrosine 186 (Y186), and serine 238 (S238), of the C134W mutant by tandem mass spectrometry. Among these sites, antibodies were raised against the pS33 and pY186 epitopes using specific peptides, and they were tested by immunostaining tissue microarrays of archival adult-type GCT specimens, other tumors, and normal tissues. The pS33 antibody showed greater sensitivity and specificity for the detection of adult-type GCTs than that of the other phospho and nonphospho antibodies. The specificity of the pS33 antibody to the pS33 epitope was further confirmed by enriching the pS33 peptide by affinity chromatography using the immobilized antibody, followed by mass spectrometric and western blot analyses from whole cell lysates of the adult-type GCT cell line, KGN. pS33 FOXL2 immunostaining levels were significantly higher in adult-type GCTs than those in other tumors and tissues. The receiver operating characteristic curve analysis of pS33 FOXL2 showed high sensitivity (1.0) and specificity (0.76) to adult-type GCTs with a cutoff score of >30% positive cells, and the area under the curve was 0.96. This suggests the potential of pS33 FOXL2 to serve as a new biomarker for the diagnosis of adult-type GCT.

Periplasmic disulfide isomerase DsbC is involved in the reduction of copper binding protein CueP from Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen with the ability to survive and replicate in macrophages. Periplasmic copper binding protein CueP is known to confer copper resistance to S. Typhimurium, and has been implicated in ROS scavenge activity by transferring the copper ion to a periplasmic superoxide dismutase or by directly reducing the copper ion. Structural and biochemical studies on CueP showed that its copper binding site is surrounded by conserved cysteine residues. Here, we present evidence that periplasmic disulfide isomerase DsbC plays a key role in maintaining CueP protein in the reduced state. We observed purified DsbC protein efficiently reduced the oxidized form of CueP, and that it acted on two (Cys104 and Cys172) of the three conserved cysteine residues. Furthermore, we found that a surface-exposed conserved phenylalanine residue in CueP was important for this process, which suggests that DsbC specifically recognizes the residue of CueP. An experiment using an Escherichia coli system confirmed the critical role played by DsbC in the ROS scavenge activity of CueP. Taken together, we propose a molecular insight into how CueP collaborates with the periplasmic disulfide reduction system in the pathogenesis of the bacteria.

Direct ROS scavenging activity of CueP from Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that has evolved to survive in the phagosome of macrophages. The periplasmic copper-binding protein CueP was initially known to confer copper resistance to S. Typhimurium. Crystal structure and biochemical studies on CueP revealed a putative copper binding site surrounded by the conserved cysteine and histidine residues. A recent study reported that CueP supplies copper ions to periplasmic Cu, Zn-superoxide dismutase (SodCII) at a low copper concentration and thus enables the sustained SodCII activity in the periplasm. In this study, we investigated the role of CueP in copper resistance at a high copper concentration. We observed that the survival of a cueP-deleted strain of Salmonella in macrophage phagosome was significantly reduced. Subsequent biochemical experiments revealed that CueP specifically mediates the reduction of copper ion using electrons released during the formation of the disulfide bond. We observed that the copper ion-mediated Fenton reaction in the presence of hydrogen peroxide was blocked by CueP. This study provides insight into how CueP confers copper resistance to S. Typhimurium in copper-rich environments such as the phagosome of macrophages.

FOXL2 posttranslational modifications mediated by GSK3ß determine the growth of granulosa cell tumours.

Approximately 97% of patients with ovarian granulosa cell tumours (GCTs) bear the C134W mutation in FOXL2; however, the pathophysiological mechanism of this mutation is unknown. Here we report how this mutation affects GCT development. Sequential posttranslational modifications of the C134W mutant occur where hyperphosphorylation at serine 33 (S33) by GSK3ß induces MDM2-mediated ubiquitination and proteasomal degradation. In contrast, S33 of wild-type FOXL2 is underphosphorylated, leading to its SUMOylation and stabilization. This prominent hyperphosphorylation is also observed at S33 of FOXL2 in GCT patients bearing the C134W mutation. In xenograft mice, the S33 phosphorylation status correlates with the oncogenicity of FOXL2, and the inhibition of GSK3ß efficiently represses GCT growth. These findings reveal a previously unidentified regulatory mechanism that determines the oncogenic attributes of the C134W mutation via differential posttranslational modifications of FOXL2 in GCT development.

Structure of the periplasmic copper-binding protein CueP from Salmonella enterica serovar Typhimurium.

CueP was initially identified as a copper-resistance gene in Salmonella enterica serovar Typhimurium, which has evolved to survive in the phagosomes of macrophages. Recently, CueP was determined to be a periplasmic copper-binding protein and has been implicated in the transfer of copper ions to SodCII in the periplasm. In this study, the crystal structure of CueP has been determined, revealing a V-shaped dimeric structure. The conserved cysteine and histidine residues are clustered on the surface of one side of the C-terminal domain, suggesting that this cysteine- and histidine-rich region is related to the function of CueP. LC-MS/MS analysis established the presence of a disulfide bond between Cys96 and Cys176 under aerobic conditions. Subsequent biophysical analyses showed that the CueP protein binds copper and zinc, and the mutation of Cys104 to serine (C104S) dramatically reduced the binding affinity for copper and zinc, suggesting that the cysteine- and histidine-rich cluster is responsible for copper binding. This study provides a structural basis for the participation of CueP in the resistance of the intracellular pathogen Salmonella to copper.

2-nitrobenzoate 2-nitroreductase (NbaA) switches its substrate specificity from 2-nitrobenzoic acid to 2,4-dinitrobenzoic acid under oxidizing conditions.

2-Nitrobenzoate 2-nitroreductase (NbaA) of Pseudomonas fluorescens strain KU-7 is a unique enzyme, transforming 2-nitrobenzoic acid (2-NBA) and 2,4-dinitrobenzoic acid (2,4-DNBA) to the 2-hydroxylamine compounds. Sequence comparison reveals that NbaA contains a conserved cysteine residue at position 141 and two variable regions at amino acids 65 to 74 and 193 to 216. The truncated mutant Δ65-74 exhibited markedly reduced activity toward 2,4-DNBA, but its 2-NBA reduction activity was unaffected; however, both activities were abolished in the Δ193-216 mutant, suggesting that these regions are necessary for the catalysis and specificity of NbaA. NbaA showed different lag times for the reduction of 2-NBA and 2,4-DNBA with NADPH, and the reduction of 2,4-DNBA, but not 2-NBA, failed in the presence of 1 mM dithiothreitol or under anaerobic conditions, indicating oxidative modification of the enzyme for 2,4-DNBA. The enzyme was irreversibly inhibited by 5,5'-dithio-bis-(2-nitrobenzoic acid) and ZnCl(2), which bind to reactive thiol/thiolate groups, and was eventually inactivated during the formation of higher-order oligomers at high pH, high temperature, or in the presence of H(2)O(2). SDS-PAGE and mass spectrometry revealed the formation of intermolecular disulfide bonds by involvement of the two cysteines at positions 141 and 194. Site-directed mutagenesis indicated that the cysteines at positions 39, 103, 141, and 194 played a role in changing the enzyme activity and specificity toward 2-NBA and 2,4-DNBA. This study suggests that oxidative modifications of NbaA are responsible for the differential specificity for the two substrates and further enzyme inactivation through the formation of disulfide bonds under oxidizing conditions.

Overexpression of reactive cysteine-containing 2-nitrobenzoate nitroreductase (NbaA) and its mutants alters the sensitivity of Escherichia coli to reactive oxygen species by reprogramming a regulatory network of disulfide-bonded proteins.

The effects of redox-sensitive proteins on Escherichia coli were investigated by overexpressing Pseudomonas 2-nitrobenzoate nitroreductase (NbaA) and its mutants. Overexpression of wild-type and mutant NbaA proteins significantly altered the sensitivity of E. coli to antibiotics and reactive oxygen species regardless of the enzyme activity for reduction of 2-nitrobenzoic acid. The overexpressed proteins rendered cells 100-10000-fold more sensitive to superoxide anion (O2(•-))-generating paraquat and 10-100-fold more resistant to H2O2. A significant increase in intracellular levels of O2(•-), but not H2O2, was observed during expression of wild-type and truncated (Δ65-74, Δ193-216, and Δ65-74Δ193-216) NbaA. From two-dimensional nonreducing/reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry analyses, 29 abundant proteins in the cytoplasm were identified to form interchain disulfide bonds, when cells were exposed to polymyxin B. Of them, down-regulation and modifications of SodB, KatE, and KatG were strongly associated with elevated cellular O2(•-) levels. Western blotting showed up-regulation of cell death signal sensor, CpxA, and down-regulation of cytoplasmic superoxide dismutase, SodB, with ∼2-fold up-regulation of heterodimeric integration host factor, Ihf. Activity gel assays revealed significant reduction of glyceraldehyde-3-phosphate dehydrogenase with constant levels of 6-phosphogluconate dehydrogenase. These changes would support a high level of NADPH to reduce H2O2-induced disulfide bonds by forced expression of thioredoxin A via thioredoxin reductase. Thus, overexpression of wild-type and truncated NbaA partially compensates for the lack of KatE and KatG to degrade H2O2, thereby enhancing disulfide bond formation in the cytoplasm, and modifies a regulatory network of disulfide-bonded proteins to increase intracellular O2(•-) levels.

Reprogramming of EBV-immortalized B-lymphocyte cell lines into induced pluripotent stem cells.

EBV-immortalized B lymphocyte cell lines have been widely banked for studying a variety of diseases, including rare genetic disorders. These cell lines represent an important resource for disease modeling with the induced pluripotent stem cell (iPSC) technology. Here we report the generation of iPSCs from EBV-immortalized B-cell lines derived from multiple inherited disease patients via a nonviral method. The reprogramming method for the EBV cell lines involves a distinct protocol compared with that of patient fibroblasts. The B-cell line-derived iPSCs expressed pluripotency markers, retained the inherited mutation and the parental V(D)J rearrangement profile, and differentiated into all 3 germ layer cell types. There was no integration of the reprogramming-related transgenes or the EBV-associated genes in these iPSCs. The ability to reprogram the widely banked patient B-cell lines will offer an unprecedented opportunity to generate human disease models and provide novel drug therapies.

The WNT antagonist Dickkopf2 promotes angiogenesis in rodent and human endothelial cells.

Neovessel formation is a complex process governed by the orchestrated action of multiple factors that regulate EC specification and dynamics within a growing vascular tree. These factors have been widely exploited to develop therapies for angiogenesis-related diseases such as diabetic retinopathy and tumor growth and metastasis. WNT signaling has been implicated in the regulation and development of the vascular system, but the detailed mechanism of this process remains unclear. Here, we report that Dickkopf1 (DKK1) and Dickkopf2 (DKK2), originally known as WNT antagonists, play opposite functional roles in regulating angiogenesis. DKK2 induced during EC morphogenesis promoted angiogenesis in cultured human endothelial cells and in in vivo assays using mice. Its structural homolog, DKK1, suppressed angiogenesis and was repressed upon induction of morphogenesis. Importantly, local injection of DKK2 protein significantly improved tissue repair, with enhanced neovascularization in animal models of both hind limb ischemia and myocardial infarction. We further showed that DKK2 stimulated filopodial dynamics and angiogenic sprouting of ECs via a signaling cascade involving LRP6-mediated APC/Asef2/Cdc42 activation. Thus, our findings demonstrate the distinct functions of DKK1 and DKK2 in controlling angiogenesis and suggest that DKK2 may be a viable therapeutic target in the treatment of ischemic vascular diseases.

In vivo liver regeneration potential of human induced pluripotent stem cells from diverse origins.

Human induced pluripotent stem cells (iPSCs) are a potential source of hepatocytes for liver transplantation to treat end-stage liver disease. In vitro differentiation of human iPSCs into hepatic cells has been achieved using a multistage differentiation protocol, but whether these cells are functional and capable of engrafting and regenerating diseased liver tissue is not clear. We show that human iPSC-derived hepatic cells at various differentiation stages can engraft the liver in a mouse transplantation model. Using the same differentiation and transplantation protocols, we also assessed the ability of human iPSCs derived from each of the three developmental germ layer tissues (that is, ectoderm, mesoderm, and endoderm) to regenerate mouse liver. These iPSC lines, with similar but distinct global DNA methylation patterns, differentiated into multistage hepatic cells with an efficiency similar to that of human embryonic stem cells. Human hepatic cells at various differentiation stages derived from iPSC lines of different origins successfully repopulated the liver tissue of mice with liver cirrhosis. They also secreted human-specific liver proteins into mouse blood at concentrations comparable to that of proteins secreted by human primary hepatocytes. Our results demonstrate the engraftment and liver regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo and suggest that human iPSCs of distinct origins and regardless of their parental epigenetic memory can efficiently differentiate along the hepatic lineage.

A proteomic approach based on multiple parallel separation for the unambiguous identification of an antibody cognate antigen.

Autoantibodies obtained from cancer patients have been identified as useful tools for cancer diagnostics, prognostics, and as potential targets for immunotherapy. Serological proteome analysis in combination with 2-DE is a classic strategy for identification of tumor-associated antigens in the serum of cancer patients. However, serological proteome analysis cannot always indicate the true antigen out of a complex proteome identified from a single protein spot because the most abundant protein is not always the most antigenic. To address this problem, we utilized multiple parallel separation (MPS) for proteome separation. The common identities present in the fractions obtained using different separation methods were regarded as the true antigens. The merit of our MPS technique was validated using anti-ARPC2 and anti-PTEN antibodies. Next, we applied the MPS technique for the identification of glycyl-tRNA synthetase as the cognate antigen for an autoantibody that was overexpressed in the plasma of breast cancer patients. These results reveal that MPS can unambiguously identify an antibody cognate antigen by reducing false-positives. Therefore, MPS could be used for the characterization of diagnostic antibodies raised in laboratory animals as well as autoantibodies isolated from diseased patients.