Immuno-Mass Spectrometry Workflow for Quantification of Serum α-Fetoprotein Using Antibody-Immobilized Magnetic Beads and Modified Eluents.

Immuno-mass spectrometry (MS) is a powerful method for the quantitative analysis of low-abundance proteins in biological specimens. In these procedures, collecting specifically and efficiently the target protein antigens from the antigen-antibody complex generated on the surface of nanocarrier beads is crucial and can be performed by hydrolyzing the proteins directly on the beads or after elution. Herein, we optimized the conditions of the immunoaffinity purification via elution using serum α-fetoprotein (AFP) as a model and its specific antibody immobilized covalently on magnetic beads. Antibody-coated beads were incubated with human serum spiked with standard AFP for antigen-antibody reaction. AFP was then eluted from the beads using various eluents, including organic solvents, to optimize the elution conditions. After proteolytically hydrolyzing the eluted protein, stable isotope-labeled standard peptides were added to the hydrolysate to quantify the eluted AFP via liquid chromatography-tandem MS. Using an optimized workflow for quantitative analysis afforded a correlation between the amount of spiked AFP and heavy to light ratios calculated based on peptide ion peak areas, from which an endogenous AFP concentration of 2.3±0.6 ng/mL was determined in normal serum; this is consistent with previous reports using radioimmunoassay methods. The present immuno-MS workflow could apply to the detection and quantitation of other low-abundance biofluid biomarkers.

Phos-tag diagonal electrophoresis precisely detects the mobility change of phosphoproteins in Phos-tag SDS-PAGE.

Phos-tag diagonal electrophoresis was developed to identify precisely a change in electrophoretic mobility of phosphoproteins in Phos-tag SDS-PAGE. Previously, if a single protein band was detected, it was impossible to determine whether mobility of the protein altered by Mn2+ Phos-tag in Phos-tag SDS-PAGE gels because SDS-PAGE and Phos-tag SDS-PAGE were performed on different gels. Moreover, when multiple protein bands were detected, it was difficult to determine whether the band with the highest mobility was altered mobility by Mn2+ Phos-tag. However, these problems were resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns were provided on a single gel. Using this technique we identified phosphorylation states of various proteins such as α-lactalbumin, α- and ß-casein, ovalbumin, basic 7S globulin, and 26S proteasome subunits. In the analyses of 26S proteasome subunits from humans and yeast, we could confirm that all subunits are phosphorylated, and find that the number of major proteins with different phosphorylation states is a few in each of the subunits despite having many phosphorylation sites. SIGNIFICANCE: Previously, Phos-tag SDS-PAGE has been developed to identify a change in electrophoretic mobility of phosphoproteins. However, we had a problem in this technique; it was often difficult to recognize the mobility shift by Mn2+ Phos-tag when we used separately SDS-PAGE and Phos-tag SDS-PAGE. Such a problem was resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns are provided on a single gel. This technique was useful to identify phosphorylation states of various proteins. : Phos-tag diagonal electrophoresis, mass spectrometry, phosphoproteins, basic 7S globulin, proteasome.

Generation of a Nonhuman Primate Model of Severe Combined Immunodeficiency Using Highly Efficient Genome Editing.

Recent advances in genome editing have facilitated the generation of nonhuman primate (NHP) models, with potential to unmask the complex biology of human disease not revealed by rodent models. However, their broader use is hindered by the challenges associated with generation of adult NHP models as well as the cost of their production. Here, we describe the generation of a marmoset model of severe combined immunodeficiency (SCID). This study optimized zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) to target interleukin-2 receptor subunit gamma (IL2RG) in pronuclear stage marmoset embryos. Nine of 21 neonates exhibited mutations in the IL2RG gene, concomitant with immunodeficiency, and three neonates have currently survived from 240 days to 1.8 years. Our approach demonstrates highly efficient production of founder NHP with SCID phenotypes, with promises of multiple pre-clinical and translational applications.

Augmentation of multiple protein kinase activities associated with secondary imatinib resistance in gastrointestinal stromal tumors as revealed by quantitative phosphoproteome analysis.

Mutations in the Kit receptor tyrosine kinase gene (KIT), which result in constitutive activation of the protein (KIT), are causally related to the development of gastrointestinal stromal tumors (GISTs). Imatinib, a targeted anticancer drug, exerts a therapeutic effect against GISTs by repressing the kinase activity of KIT. Long-term administration of this drug, however, causes the emergence of imatinib-resistant GISTs. We performed quantitative phosphoproteome analysis using a cell-based GIST model system comprising an imatinib-sensitive GIST cell line (GIST882), GIST882 under treatment with imatinib (GIST882-IM), and secondary imatinib-resistant GIST882 (GIST882-R). Phosphorylated peptides were purified from each cell line using titania-based affinity chromatography or anti-phosphotyrosine immunoprecipitation, and then subjected to LC-MS/MS based quantitative phosphoproteome analysis. Using this method we identified augmentation of the kinase activities of multiple elements of the signal transduction pathway, especially KIT and EGFR. Although, these elements were up-regulated in GIST882-R, no additionally mutated KIT mRNA was found in secondary imatinib-resistant GIST cells. Treatment of GIST882-R with imatinib in combination with gefitinib, an EGFR inhibitor, partially prevented cell growth, implying that EGFR may be involved in acquisition of secondary imatinib resistance in GIST. BIOLOGICAL SIGNIFICANCE: In this study, we performed a quantitative phosphoproteome analysis using a cell culture-based GIST model system. The goal of the study was to investigate the mechanism of acquired resistance in GISTs against imatinib, a molecularly targeted drug that inhibits kinase activity of the KIT protein and that has been approved for the treatment of GISTs. In imatinib-resistant GIST cells, we observed elevated expression of KIT and restoration of its kinase activity, as well as activation of multiple proliferative signaling pathways. Our results indicate that the effects of even so-called 'molecularly targeted' drugs, are broad rather than convergent, and that the mechanisms of action of such drugs during continuous administration are extremely complex.

A neuronal transmembrane protein LRFN4 complexes with 14-3-3s and NCK1 to induce morphological change in monocytic cells via Rac1-mediated actin cytoskeleton reorganization.

We previously reported that leucine-rich repeat and fibronectin type III domain-containing 4 (LRFN4) functioned in migration and morphological change (i.e. cell elongation) of monocytic cells. Here, we examined a molecular mechanism regulating LRFN4-mediated cell elongation. We found that 14-3-3 and NCK proteins complexed with LRFN4, and they were involved in LRFN4-mediated cell elongation. We also identified the regions of LRFN4 interacting with NCK1 and 14-3-3s. Finally, we demonstrated that a Rac1 small GTPase was involved in LRFN4-mediated cell elongation. These results indicated that LRFN4 complexed with 14-3-3s and NCK1 to mediate elongation in monocytic cells via Rac-1-mediated actin cytoskeleton reorganization.

The ASB2ß Ubiquitin-interacting motif is involved in its monoubiquitination.

ASB2 proteins are E3 ubiquitin (Ub) ligases that ubiquitinate filamins. There are two ASB2 splice variants, ASB2α and ASB2ß. ASB2ß has a ubiquitin-binding motif (UIM) at the N-terminal region but ASB2α does not. Here, we provide the first evidence that ASB2ß but not ASB2α is monoubiquitinated and that this monoubiquitination involves the UIM. Myc-tagged ASB2ß and hemagglutinin (HA)-tagged Ub were co-expressed in HEK293 cells using the pCMV expression vector. Immunoprecipitation with an anti-Myc antibody followed by immunoblotting with anti-Myc and anti-HA antibodies showed an additional ASB2ß protein band that had both a Myc and a HA tag. The molecular weight of this protein was larger than that of ASB2ß, and the difference in molecular weight between these two proteins corresponded to the molecular weight of monoubiquitin, strongly implying that monoubiquitinated ASB2ß is produced in cells. ASB2ß with mutations in the UIM motif; either Glu·Asp·Glu27-29Ala·Ala·Ala mutations (ASB2ß M1) or a Ser38Ala mutation, (ASB2ß M2) were not monoubiquitinated, suggesting the importance of the UIM for ASB2ß monoubiquitination. Furthermore, an ASB2ß mutant that lacked a SOCS box (ASB2ß ΔC) and did not show E3 Ub ligase activity was monoubiquitinated to the same extent as the wild-type ASB2ß. In contrast, an ASB2ß mutant that lacked the UIM-containing domain (ASB2ß ΔN) was not monoubiquitinated. These results suggest that ASB2ß but not ASB2α might be monoubiquitinated and that the ASB2ß UIM motif, but not its E3 Ub ligase activity, plays a pivotal role in this monoubiquitination.

Sec14l3 is specifically expressed in mouse airway ciliated cells.

Airway epithelium is a key component for airway integrity. Previously, we found that expression of the Sec14l3 gene that encodes a 45-kDa secretory protein is inversely associated with the progression of experimentally induced airway inflammation and degeneration/necrosis of alveolar epithelium. In this report, using in situ hybridization we demonstrated that the ciliated cells in mouse lung selectively express Sec14l3 mRNA. In a three-dimensional culture of mouse tracheal epithelial cells, levels of the Sec14l3 mRNA correlated with the differentiation of ciliated cells. Intranasal infection of adult mice with influenza virus resulted in a 20-fold, progressive decrease in Sec14l3 mRNA expression over 10 days post infection. These results enhance the potential value of Sec14l3 as a ciliated epithelial cell-specific biomarker for the progression of airway inflammations such as airway viral infection and asthma.

Comprehensive trapping of polyubiquitinated proteins using the UIM peptide of ASB2a.

An alternative splicing variant of E3 ubiquitin ligase ASB2, termed ASB2a, has a distinct N-terminal sequence containing a ubiquitin-interacting motif (UIM) consensus sequence. Examination of the minimal essential region for binding to polyubiquitinated proteins indicated that the UIM consensus sequence (residues 26-41) alone is not enough, and that amino acids 12-41 from the N-terminus of ASB2a is essential for binding. ASB2a(12-41) peptide was chemically synthesized and coupled to Sepharose 4B via disulfide bonds. This ASB2a(12-41) peptide-coupled affinity resin bound both K48- and K63-linked polyubiquitinated proteins in cell lysates and comprehensively captured polyubiquitinated proteins, including polyubiquitinated ß-catenin, I-κB, and EGF receptor, which were eluted with 2-mercaptoethanol under non-denaturing conditions. These results indicate that this UIM affinity purification (designated as ubiquitin-trapping) is a useful method to discover polyubiquitinated proteins and their associated proteins.

Identification by differential tissue proteome analysis of talin-1 as a novel molecular marker of progression of hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is characterized by a multistage process of tumor progression. This study addressed its molecular features to identify novel protein candidates involved in HCC progression. METHODS: Using liquid chromatography-tandem mass spectrometry, proteomes of 4 early HCCs and 4 non-HCC tissues derived from 2 cases of liver transplant surgery were compared with respect to the separation profiles of their tryptic peptides. Immunohistochemistry was performed on 106 HCC nodules to confirm the results of the proteomic analysis. RESULTS: Statistical analysis of the profiles selected the peptide peaks differentiating HCC from non-HCC. A database search of the tandem mass spectrometry data from those peptide peaks identified 61 proteins, including a cytoskeletal protein, talin-1, as upregulated in HCC. Talin-1 expression levels in HCC nodules were significantly associated with the dedifferentiation of HCC (p = 0.001). A follow-up survey of the examined clinical cases revealed a correlation between talin-1 upregulation and a shorter time to recurrence after resection (p = 0.039), which may be related to the higher rate of portal vein invasion in HCCs with talin-1 up-regulation (p = 0.029). CONCLUSIONS: Proteomic analysis led to identification of talin-1 as a promising HCC marker. Talin-1 upregulation is associated with HCC progression and may serve as a prognostic marker.

Proteomic biomarkers for acute interstitial lung disease in gefitinib-treated Japanese lung cancer patients.

Interstitial lung disease (ILD) events have been reported in Japanese non-small-cell lung cancer (NSCLC) patients receiving EGFR tyrosine kinase inhibitors. We investigated proteomic biomarkers for mechanistic insights and improved prediction of ILD. Blood plasma was collected from 43 gefitinib-treated NSCLC patients developing acute ILD (confirmed by blinded diagnostic review) and 123 randomly selected controls in a nested case-control study within a pharmacoepidemiological cohort study in Japan. We generated ∼7 million tandem mass spectrometry (MS/MS) measurements with extensive quality control and validation, producing one of the largest proteomic lung cancer datasets to date, incorporating rigorous study design, phenotype definition, and evaluation of sample processing. After alignment, scaling, and measurement batch adjustment, we identified 41 peptide peaks representing 29 proteins best predicting ILD. Multivariate peptide, protein, and pathway modeling achieved ILD prediction comparable to previously identified clinical variables; combining the two provided some improvement. The acute phase response pathway was strongly represented (17 of 29 proteins, p = 1.0×10(-25)), suggesting a key role with potential utility as a marker for increased risk of acute ILD events. Validation by Western blotting showed correlation for identified proteins, confirming that robust results can be generated from an MS/MS platform implementing strict quality control.

A neuronal transmembrane protein LRFN4 induces monocyte/macrophage migration via actin cytoskeleton reorganization.

Leucine-rich repeat and fibronectin type III domain-containing (LRFN) family proteins are thought to be neuronal-specific proteins that play essential roles in neurite outgrowth and synapse formation. Here, we focused on expression and function of LRFN4, the fourth member of the LRFN family, in non-neural tissues. We found that LRFN4 was expressed in a wide variety of cancer and leukemia cell lines. We also found that expression of LRFN4 in the monocytic cell line THP-1 and in primary monocytes was upregulated following macrophage differentiation. Furthermore, we demonstrated that LRFN4 signaling regulated both the transendothelial migration of THP-1 cells and the elongation of THP-1 cells via actin cytoskeleton reorganization. Our data indicate that LRFN4 signaling plays an important role in the migration of monocytes/macrophages.

Inverse relationship between Sec14l3 mRNA/protein expression and allergic airway inflammation.

Bronchial asthma is an inflammatory disease of the airways. The Sec14l3 gene, encoding a 45-kDa secretory protein, is specifically expressed in airway epithelium. Here, we report on the kinetics of Sec14l3 expression following allergic inflammation of the lung. Brown Norway rats were sensitized by intraperitoneal injection of ovalbumin, followed by challenge with aerosolized ovalbumin after a 3-week interval. This animal model showed many features similar to human allergic asthma: an increase in inflammatory cells such as eosinophils, lymphocytes and neutrophils in bronchoalveolar lavage (BAL) fluid and histopathological alteration of lung tissue, exhibiting infiltration of these inflammatory cells and degeneration and necrosis of alveolar epithelium. These parameters reached their maximal level 24h after allergen challenge. In contrast, quantitative polymerase chain reaction analyses demonstrated a rapid and significant reduction of Sec14l3 mRNA in lung tissue and maximum reduction (to 1.4% of the control) was observed at 24h. Pretreatment with dexamethasone significantly suppressed both the Sec14I3 mRNA reduction and all of the inflammatory changes. The 45-kDa secretory protein was identified in the supernatant of BAL fluids. Two-dimensional gel images of the supernatant proteome also revealed down-regulation of the protein following inflammation (to approximately 30% of the control at 24h). Thus, Sec14l3 expression is highly and inversely associated with the progression of airway inflammation. Sec14l3 mRNA and protein may function in the homeostasis of airway epithelial cells under normal conditions.

Synuclein-gamma is closely involved in perineural invasion and distant metastasis in mouse models and is a novel prognostic factor in pancreatic cancer.

PURPOSE: Perineural invasion is associated with the high incidence of local recurrence and a dismal prognosis in pancreatic cancer. We previously reported a novel perineural invasion model and distinguished high- and low-perineural invasion groups in pancreatic cancer cell lines. This study aimed to elucidate the molecular mechanism of perineural invasion. EXPERIMENTAL DESIGN: To identify key biological markers involved in perineural invasion, differentially expressed molecules were investigated by proteomics and transcriptomics. Synuclein-gamma emerged as the only up-regulated molecule in high-perineural invasion group by both analyses. The clinical significance and the biological property of synuclein-gamma were examined in 62 resected cases of pancreatic cancer and mouse models. RESULTS: Synuclein-gamma overexpression was observed in 38 (61%) cases and correlated with major invasive parameters, including perineural invasion and lymph node metastasis (P < 0.05). Multivariate analyses revealed synuclein-gamma overexpression as the only independent predictor of diminished overall survival [hazard ratio, 3.4 (95% confidence interval, 1.51-7.51)] and the strongest negative indicator of disease-free survival [2.8 (1.26-6.02)]. In mouse perineural invasion and orthotopic transplantation models, stable synuclein-gamma suppression by short hairpin RNA significantly reduced the incidence of perineural invasion (P = 0.009) and liver/lymph node metastasis (P = 0.019 and P = 0.020, respectively) compared with the control. CONCLUSIONS: This is the first study to provide in vivo evidence that synuclein-gamma is closely involved in perineural invasion/distant metastasis and is a significant prognostic factor in pancreatic cancer. Synuclein-gamma may serve as a promising molecular target of early diagnosis and anticancer therapy.

Personalized medicine and proteomics: lessons from non-small cell lung cancer.

Personalized medicine allows the selection of treatments best suited to an individual patient and disease phenotype. To implement personalized medicine, effective tests predictive of response to treatment or susceptibility to adverse events are needed, and to develop a personalized medicine test, both high quality samples and reliable data are required. We review key features of state-of-the-art proteomic profiling and introduce further analytic developments to build a proteomic toolkit for use in personalized medicine approaches. The combination of novel analytical approaches in proteomic data generation, alignment and comparison permit translation of identified biomarkers into practical assays. We further propose an expanded statistical analysis to understand the sources of variability between individuals in terms of both protein expression and clinical variables and utilize this understanding in a predictive test.

Disease proteomics toward bedside reality.

The human genome has been sequenced, and investigation of its products has become possible in a sequence-based framework. More than 200,000 protein species are expressed in the body from approximately 30000 human genes. The term proteome, coined as a linguistic equivalent to the concept of genome, is used to describe the complete set of proteins that is expressed, and modified following expression, by the entire genome in a cell at any one time. Protein types and amounts expressed in a body vary greatly depending upon whether it is healthy or ill. Therefore, proteomics is attracting an increasing interest in its application to better understanding of disease processes, to development of new biomarkers for diagnosis and early detection of disease, and to accelerate drug development. There are numerous opportunities for medicine, although it is quite challenging to meet the needs for high sensitivity and high throughput required for disease-related investigations.

Protein identification from product ion spectra of peptides validated by correlation between measured and predicted elution times in liquid chromatography/mass spectrometry.

Reversed-phase liquid chromatography (LC) directly coupled with electrospray-tandem mass spectrometry (MS/MS) is a successful choice to obtain a large number of product ion spectra from a complex peptide mixture. We describe a search validation program, ScoreRidge, developed for analysis of LC-MS/MS data. The program validates peptide assignments to product ion spectra resulting from usual probability-based searches against primary structure databases. The validation is based only on correlation between the measured LC elution time of each peptide and the deduced elution time from the amino acid sequence assigned to product ion spectra obtained from the MS/MS analysis of the peptide. Sufficient numbers of probable assignments gave a highly correlative curve. Any peptide assignments within a certain tolerance from the correlation curve were accepted for the following arrangement step to list identified proteins. Using this data validation program, host protein candidates responsible for interaction with human hepatitis B virus core protein were identified from a partially purified protein mixture. The present simple and practical program complements protein identification from usual product ion search algorithms and reduces manual interpretation of the search result data. It will lead to more explicit protein identification from complex peptide mixtures such as whole proteome digests from tissue samples.

Interaction of the hepatitis B virus X protein (HBx) with heat shock protein 60 enhances HBx-mediated apoptosis.

Understanding the function of the hepatitis B virus X protein (HBx) is fundamental to elucidating the underlying mechanisms of hepatitis and hepatocarcinogenesis caused by hepatitis B virus (HBV) infection. We identified heat shock protein 60 (Hsp60) as a novel cellular target of HBx by the combination of affinity purification and mass spectrometry. Physical interaction between HBx and Hsp60 was confirmed by standard immunoprecipitation and immunoblot methods. Analysis of HBx deletion constructs showed that amino acids 88-117 of HBx were responsible for the binding to Hsp60. Confocal laser microscopy demonstrated that HBx and Hsp60 colocalized in mitochondria. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL) revealed that the introduction of Hsp60 into cells facilitated HBx-induced apoptosis. These findings suggest the importance of the molecular chaperon protein Hsp60 to the function of HBV viral proteins.

Proteomic approach to apoptotic thymus maturation.

Apoptosis is an essential process for selection of T lymphocytes specific for foreign antigen in the process of mammalian thymus maturation. Proteomics, a comprehensive study of proteins expressed in a cell, will facilitate the systematic analysis of protein molecules related to such a complicated biological system. Protein expression profiles including information about protein signatures, localization and their quantitative changes with extracellular stimulations are extremely useful to construct intracellular pathway models resulting in the apoptotic cell death.