Immunoaffinity proteomics for kidney injury biomarkers in male beagle dogs.

Drug-induced kidney injury (DIKI) is a cause of drug development failure. Dogs represent a common non-rodent animal model in pre-clinical safety studies; however, biomarker assays for detecting nephrotoxicity in dogs are limited. To identify novel proteins and gain insight into the molecular mechanisms involved in DIKI, we developed an assay to evaluate proteomic changes associated with DIKI in male beagle dogs that received nephrotoxic doses of tobramycin for 10 consecutive days. Label-free quantitative discovery proteomics analysis on representative kidney cortex tissues collected on Day 11 showed that the tobramycin-induced kidney injury led to a significant differential regulation of 94 proteins mostly associated with mechanisms of nephrotoxicity such as oxidative stress and proteasome degradation. For verification of the proteomic results, we developed a multiplex peptide-centric immunoaffinity liquid chromatography tandem mass spectrometry assay (IA LC-MS/MS) to evaluate the association of eight DIKI protein biomarker candidates using kidney cortices collected on Day 11 and urine samples collected on Days -4, 1, 3, 7 and 10. The results showed that most biomarkers evaluated were detected in the kidney cortices and their expression profile in tissue aligned with the label-free data. Cystatin C was the most consistent marker regardless of the magnitude of the renal injury while fatty acid-binding protein-4 (FABP4) and kidney injury molecule-1 (KIM-1) were the most affected biomarkers in response to moderate proximal tubular injury in absence of changes in serum-based concentrations of blood urea nitrogen or creatinine. In the urine, clusterin is considered the most consistent biomarker regardless of the magnitude and time of the renal injury. To our knowledge, this is the most comprehensive multiplex assay for the quantitative analysis of mechanism-based proximal tubular injury biomarkers in dogs.

Okadaic Acid Activates JAK/STAT Signaling to Affect Xenobiotic Metabolism in HepaRG Cells.

Okadaic acid (OA) is a marine biotoxin that is produced by algae and accumulates in filter-feeding shellfish, through which it enters the human food chain, leading to diarrheic shellfish poisoning (DSP) after ingestion. Furthermore, additional effects of OA have been observed, such as cytotoxicity. Additionally, a strong downregulation of the expression of xenobiotic-metabolizing enzymes in the liver can be observed. The underlying mechanisms of this, however, remain to be examined. In this study, we investigated a possible underlying mechanism of the downregulation of cytochrome P450 (CYP) enzymes and the nuclear receptors pregnane X receptor (PXR) and retinoid-X-receptor alpha (RXRα) by OA through NF-κB and subsequent JAK/STAT activation in human HepaRG hepatocarcinoma cells. Our data suggest an activation of NF-κB signaling and subsequent expression and release of interleukins, which then activate JAK-dependent signaling and thus STAT3. Moreover, using the NF-κB inhibitors JSH-23 and Methysticin and the JAK inhibitors Decernotinib and Tofacitinib, we were also able to demonstrate a connection between OA-induced NF-κB and JAK signaling and the downregulation of CYP enzymes. Overall, we provide clear evidence that the effect of OA on the expression of CYP enzymes in HepaRG cells is regulated through NF-κB and subsequent JAK signaling.

Proteomic analysis of hepatic effects of phenobarbital in mice with humanized liver.

Activation of the constitutive androstane receptor (CAR) may induce adaptive but also adverse effects in rodent liver, including the induction of drug-metabolizing enzymes, transient hepatocellular proliferation, and promotion of liver tumor growth. Human relevance of CAR-related adverse hepatic effects is controversially debated. Here, we used the chimeric FRG-KO mouse model with livers largely repopulated by human hepatocytes, in order to study human hepatocytes and their response to treatment with the model CAR activator phenobarbital (PB) in vivo. Mice received an intraperitoneal injection with 50 mg/kg body weight PB or saline, and were sacrificed after 72-144 h. Non-repopulated FRG-KO mice were used as additional control. Comprehensive proteomics datasets were generated by merging data obtained by targeted as well as non-targeted proteomics approaches. For the first time, a novel proteomics workflow was established to comparatively analyze the effects of PB on human and murine proteins within one sample. Analysis of merged proteome data sets and bioinformatics data mining revealed comparable responses in murine and human hepatocytes with respect to nuclear receptor activation and induction of xenobiotic metabolism. By contrast, activation of MYC, a key regulator of proliferation, was predicted only for mouse but not human hepatocytes. Analyses of 5-bromo-2'-deoxyuridine incorporation confirmed this finding. In summary, this study for the first time presents a comprehensive proteomic analysis of CAR-dependent effects in human and mouse hepatocytes from humanized FRG-KO mice. The data support the hypothesis that PB does induce adaptive metabolic responses, but not hepatocellular proliferation in human hepatocytes in vivo.

Matrix and Sampling Effects on Quantification of Protein Biomarkers of Drug-Induced Liver Injury.

Macrophage colony stimulating factor 1 receptor (MCSF1R), osteopontin (OPN), high-mobility group protein B1 (HMGB1), glutamate dehydrogenase (GLDH), keratin 18 (K18), and caspase-cleaved keratin 18 (ccK18) are considered promising mechanistic biomarkers for the diagnosis of drug-induced liver injury. Here, we aim to elucidate the impact of the sample matrix and handling on the quantification of these emerging protein biomarkers. We investigated effects such as time from collection to centrifugation during serum (± gel) or EDTA plasma preparation on two assay platforms: immunoaffinity liquid chromatography mass spectrometric assays and sandwich immunoassays. Furthermore, we measured GLDH activity with an enzymatic activity assay. Matrix effects were observed particularly for HMGB1 and MCSF1R. HMGB1 levels were higher in serum than in plasma, whereas higher concentrations of MCSF1R were observed in plasma than in serum. A comparison of sample collection to centrifugation time ranging from 15 to 60 min demonstrated increasing levels of HMGB1 in serum, while MCSF1R, OPN, GLDH, and ccK18 concentrations remained stable. Additionally, there was a poor correlation in HMGB1 and ccK18 levels between serum and plasma. Considering the observed matrix effects, we recommend plasma as a matrix of choice and cross-study comparison studies to be limited to those using the same matrix.

A targeted transcriptomics approach for the determination of mixture effects of pesticides.

While real-life exposure occurs to complex chemical mixtures, toxicological risk assessment mostly focuses on individual compounds. There is an increasing demand for in vitro tools and strategies for mixture toxicity analysis. Based on a previously established set of hepatotoxicity marker genes, we analyzed mixture effects of non-cytotoxic concentrations of different pesticides in exposure-relevant binary mixtures in human HepaRG hepatocarcinoma cells using targeted transcriptomics. An approach for mixture analysis at the level of a complex endpoint such as a transcript pattern is presented, including mixture design based on relative transcriptomic potencies and similarities. From a mechanistic point of view, goal of the study was to evaluate combinations of chemicals with varying degrees of similarity in order to determine whether differences in mechanisms of action lead to different mixtures effects. Using a model deviation ratio-based approach for assessing mixture effects, it was revealed that most data points are consistent with the assumption of dose addition. A tendency for synergistic effects was only observed at high concentrations of some combinations of the test compounds azoxystrobin, cyproconazole, difenoconazole, propiconazole and thiacloprid, which may not be representative of human real-life exposure. In summary, the findings of our study suggest that, for the pesticide mixtures investigated, risk assessment based on the general assumption of dose addition can be considered sufficiently protective for consumers. The way of data analysis presented in this paper can pave the way for a more comprehensive use of multi-gene expression data in experimental studies related to mixture toxicity.

Investigation of new biomarkers of kidney injury in renal transplant recipients undergoing graft biopsy.

AIM: Urinary and blood kidney biomarkers (BM) remain insufficient for early kidney injury detection. We aimed to compare new kidney BM with histopathological data in kidney allograft recipients. METHODS: Blood and urine samples were collected from consecutive adult patients just before graft biopsy. All kidney samples were classified according to the Banff 2007 classification. The diagnostic performance of 16 new BM was compared to those of urinary proteins, blood urea nitrogen, eGFR, and serum creatinine to identify histopathological groups. RESULTS: Two hundred and twenty-three patients were analyzed. Microalbuminuria and urinary proteins performed well to discriminate glomerular injury from slightly modified renal parenchyma (SMRP). Urinary neutrophil gelatinase-associated lipocalin (NGAL) had the best performance relative to SMRP (AUROC .93) for acute tubular necrosis (ATN) diagnosis. Other BM had a slightly lower AUROC (.89). For the comparison of ATN to acute rejection, several new urinary BM (NGAL, cystatin C, MCP1) and classical BM (eGFR, serum creatinine) gave similar AUROC values (from .80 to .85). Urinary NGAL values in patients with ATN were 10-time higher than those with acute rejection (P=.0004). CONCLUSION: The new BM did not outperform classical BM in the context of renal transplantation. Urinary NGAL may be useful for distinguishing between ATN and acute rejection.

Precise Quantitation of PTEN by Immuno-MRM: A Tool To Resolve the Breast Cancer Biomarker Controversy.

The tumor suppressor PTEN is the main negative regulator of PI3K/AKT/mTOR signaling and is commonly found downregulated in breast cancer (BC). Conflicting data from conventional immunoassays such as immunohistochemistry (IHC) has sparked controversy about PTEN's role as a prognostic and predictive biomarker in BC, which can be largely attributed to the lack of specificity, sensitivity, and interlaboratory standardization. Here, we present a fully standardized, highly sensitive, robust microflow immuno-MRM (iMRM) assay that enables precise quantitation of PTEN concentrations in cells and fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissues, down to 0.1 fmol/10 µg of extracted protein, with high interday and intraday precision (CV 6.3%). PTEN protein levels in BC PDX samples that were determined by iMRM correlate well with semiquantitative IHC and WB data. iMRM, however, allowed the precise quantitation of PTEN-even in samples that were deemed to be PTEN negative by IHC or western blot (WB)-while requiring substantially less tumor tissue than WB. This is particularly relevant because the extent of PTEN downregulation in tumors has been shown to correlate with severity. Our standardized and robust workflow includes an 11 min microflow LC-MRM analysis on a triple-quadrupole MS and thus provides a much needed tool for the study of PTEN as a potential biomarker for BC.

Using Two Peptide Isotopologues as Internal Standards for the Streamlined Quantification of Low-Abundance Proteins by Immuno-MRM and Immuno-MALDI.

Mass spectrometry (MS), particularly targeted proteomics, is increasingly being used for quantifying specific proteins and peptides in clinical specimens. The coupling of immuno-enrichment of proteotypic peptides with MS [e.g., immuno-multiple reaction monitoring (MRM) and immuno-matrix-assisted laser desorption ionization (MALDI)] enables the development of highly sensitive and specific assays for low-abundance signaling proteins. By incorporating stable isotope-labeled standards, these workflows allow the determination of endogenous protein concentrations. This is typically achieved through external calibration, often using surrogate matrices, which has inherent limitations for the analysis of clinical specimens as there are often substantial variations in the sample matrix, and sample amounts are typically limited. We have previously introduced the use of two peptide isotopologues for generating external calibration curves in plasma. Here, we present a two-point internal calibration (2-PIC) strategy using two isotopologues for immuno-MS assays and demonstrate its flexibility and robustness. Quantification of the tumor suppressor PTEN in Colo-205 cells by immuno-MRM and immuno-MALDI using 2-PIC and external calibration yielded very similar results (relative standard deviation between 2-PIC and external calibration: 4.9% for immuno-MRM; 1.1% for immuno-MALDI), without the need for a surrogate matrix or additional patient material for calibration, while concurrently reducing the instrument time and cost. Although our PTEN immuno-MRM and immuno-MALDI assays can be considered to be orthogonal as they utilized entirely different sample preparation and MS analysis workflows, targeted different PTEN peptides, and were performed in different laboratories, the endogenous Colo-205 PTEN levels determined with 2-PIC showed a good correlation (r2 = 0.9966) and good agreement (0.48 ± 0.01 and 0.29 ± 0.02 fmol/µg of total protein) between immuno-MRM and immuno-MALDI.

Systematic Optimization of the iMALDI Workflow for the Robust and Straightforward Quantification of Signaling Proteins in Cancer Cells.

PURPOSE: Immuno-MALDI (iMALDI) combines immuno-enrichment of biomarkers with MALDI-MS for fast, precise, and specific quantitation, making it a valuable tool for developing clinical assays. iMALDI assays are optimized for the PI3-kinase signaling pathway members phosphatase and tensin homolog (PTEN) and PI3-kinase catalytic subunit alpha (p110α), with regard to sensitivity, robustness, and throughput. A standardized template for developing future iMALDI assays, including automation protocols to streamline assay development and translation, is provided. EXPERIMENTAL DESIGN: Conditions for tryptic digestion and immuno-enrichment (beads, bead:antibody ratios, incubation times, direct vs. indirect immuno-enrichment) are rigorously tested. Different strategies for calibration and data readout are compared. RESULTS: Digestion using 1:2 protein:trypsin (wt:wt) for 1 h yielded high and consistent peptide recoveries. Direct immuno-enrichment (antibody-bead coupling prior to antigen-enrichment) yielded 30% higher peptide recovery with a 1 h shorter incubation time than indirect enrichment. Immuno-enrichment incubation overnight yielded 1.5-fold higher sensitivities than 1 h incubation. Quantitation of the endogenous target proteins is not affected by the complexity of the calibration matrix, further simplifying the workflow. CONCLUSIONS AND CLINICAL RELEVANCE: This optimized and automated workflow will facilitate the clinical translation of high-throughput sensitive iMALDI assays for quantifying cell-signaling proteins in individual tumor samples, thereby improving patient stratification for targeted treatment.

P-Glycoprotein Inhibition Exacerbates Paclitaxel Neurotoxicity in Neurons and Patients With Cancer.

Paclitaxel-induced peripheral neuropathy (PIPN) is a common and dose-limiting adverse event. The role of P-glycoprotein (P-gp) in the neuronal efflux of paclitaxel was assessed using a translational approach. SH-SY5Y cells were differentiated to neurons and paclitaxel toxicity in the absence and presence of a P-gp inhibitor was determined. Paclitaxel caused marked dose-dependent toxicity in SH-SY5Y-derived neurons. Paclitaxel neurotoxicity was exacerbated with concomitant P-gp inhibition by valspodar and verapamil, consistent with increased intracellular accumulation of paclitaxel. Patients with cancer treated with paclitaxel and P-gp inhibitors had a 2.4-fold (95% confidence interval (CI) 1.3-4.3) increased risk of peripheral neuropathy-induced dose modification while a 4.7-fold (95% CI 1.9-11.9) increased risk for patients treated with strong P-gp inhibitors was observed, and a 7.0-fold (95% CI 2.3-21.5) increased risk in patients treated with atorvastatin. Atorvastatin also increased neurotoxicity by paclitaxel in SH-SY5Y-derived neurons. Clinicians should be aware that comedication with P-gp inhibitors may lead to increased risk of PIPN.

RNA-protein correlation of liver toxicity markers in HepaRG cells.

The liver is a main target organ for the toxicity of many different compounds. While in general, in vivo testing is still routinely used for assessing the hepatotoxic potential of test chemicals, the use of in vitro models offers advantages with regard to throughput, consumption of resources, and animal welfare aspects. Using the human hepatoma cell line HepaRG, we performed a comparative evaluation of a panel of hepatotoxicity marker mRNAs and proteins after exposure of the cells to 30 different pesticidal active compounds comprising herbizides, fungicides, insecticides, and others. The panel of hepatotoxicity markers included nuclear receptor target genes, key players of fatty acid and bile acid metabolism-related pathways, as well as recently identified biomarkers of drug-induced liver injury. Moreover, marker genes and proteins were identified, for example, S100P, ANXA10, CYP1A1, and CYP7A1. These markers respond with high sensitivity to stimulation with chemically diverse test compounds already at non-cytotoxic concentrations. The potency of the test compounds, determined as an overall parameter of their ability to deregulate marker expression in vitro, was very similar between the mRNA and protein levels. Thus, this study does not only characterize the response of human liver cells to 30 different pesticides but also demonstrates that hepatotoxicity testing in human HepaRG cells yields well comparable results at the mRNA and protein levels. Furthermore, robust hepatotoxicity marker genes and proteins were identified in HepaRG cells.

Polycyclic Aromatic Hydrocarbons Activate the Aryl Hydrocarbon Receptor and the Constitutive Androstane Receptor to Regulate Xenobiotic Metabolism in Human Liver Cells.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants produced by incomplete combustion of organic matter. They induce their own metabolism by upregulating xenobiotic-metabolizing enzymes such as cytochrome P450 monooxygenase 1A1 (CYP1A1) by activating the aryl hydrocarbon receptor (AHR). However, previous studies showed that individual PAHs may also interact with the constitutive androstane receptor (CAR). Here, we studied ten PAHs, different in carcinogenicity classification, for their potential to activate AHR- and CAR-dependent luciferase reporter genes in human liver cells. The majority of investigated PAHs activated AHR, while non-carcinogenic PAHs tended to activate CAR. We further characterized gene expression, protein abundancies and activities of the AHR targets CYP1A1 and 1A2, and the CAR target CYP2B6 in human HepaRG hepatoma cells. Enzyme induction patterns strongly resembled the profiles obtained at the receptor level, with AHR-activating PAHs inducing CYP1A1/1A2 and CAR-activating PAHs inducing CYP2B6. In summary, this study provides evidence that beside well-known activation of AHR, some PAHs also activate CAR, followed by subsequent expression of respective target genes. Furthermore, we found that an increased PAH ring number is associated with AHR activation as well as the induction of DNA double-strand breaks, whereas smaller PAHs activated CAR but showed no DNA-damaging potential.

Okadaic acid activates Wnt/ß-catenin-signaling in human HepaRG cells.

The lipophilic phycotoxin okadaic acid (OA) occurs in the fatty tissue and hepatopancreas of filter-feeding shellfish. The compound provokes the diarrhetic shellfish poisoning (DSP) syndrome after intake of seafood contaminated with high levels of the DSP toxin. In animal experiments, long-term exposure to OA is associated with an elevated risk for tumor formation in different organs including the liver. Although OA is a known inhibitor of the serine/threonine protein phosphatase 2A, the mechanisms behind OA-induced carcinogenesis are not fully understood. Here, we investigated the influence of OA on the ß-catenin-dependent Wnt-signaling pathway, addressing a major oncogenic pathway relevant for tumor development. We analyzed OA-mediated effects on ß-catenin and its biological function, cellular localization, post-translational modifications, and target gene expression in human HepaRG hepatocarcinoma cells treated with non-cytotoxic concentrations up to 50 nM. We detected concentration- and time-dependent effects of OA on the phosphorylation state, cellular redistribution as well as on the amount of transcriptionally active ß-catenin. These findings were confirmed by quantitative live-cell imaging of U2OS cells stably expressing a green fluorescent chromobody which specifically recognize hypophosphorylated ß-catenin. Finally, we demonstrated that nuclear translocation of ß-catenin mediated by non-cytotoxic OA concentrations results in an upregulation of Wnt-target genes. In conclusion, our results show a significant induction of the canonical Wnt/ß-catenin-signaling pathway by OA in human liver cells. Our data contribute to a better understanding of the molecular mechanisms underlying OA-induced carcinogenesis.

Application of Mass Spectrometry-Based Immunoassays for the Species- and Tissue-Specific Quantification of Banned Processed Animal Proteins in Feeds.

Processed Animal Proteins (PAPs) are considered as a sustainable protein source to improve the nutritional profile of feed for livestock and aquaculture. However, the use of these proteins is strongly regulated since the bovine spongiform encephalopathy (BSE) crisis. The reintroduction of nonruminant PAPs for use in aquaculture in 2013 has driven the need for alternative analytical methods to determine the species origin as well as the tissue source (legal or not). The current official methods, light microscopy and polymerase chain reaction, do not fulfill these requirements. Furthermore, future methods need to be quantitative, because the pending zero-tolerance-concept is planned to be replaced by accurate thresholds. Here, we developed a 7-plex mass spectrometry-based immunoassay that is capable of quantifying 0.1% (w/w) ruminant PAP in feed in a tissue- and species-specific way. The workflow comprises a 2 h tryptic digestion of PAPs in suspension, an immunoaffinity enrichment of peptides, and LC-MS/MS-based quantification. In combination with a previously published assay for species identification, we were able to confirm the species and tissue origin of six ring trial samples obtained in former PCR and microscopy proficiency tests. The sensitive, quantitative, species- and tissue-specific character of the developed assays meets the requirements for new methods for PAP detection and can be used in future feed authentication studies.

Peptide-Based Sandwich Immunoassay for the Quantification of the Membrane Transporter Multidrug Resistance Protein 1.

Multitransmembrane proteins are notoriously difficult to analyze. To date, rapid, and cost-efficient detection methods are lacking and only mass spectrometry-based systems allow reliable quantification of these proteins. Here, we present a novel type of sandwich immunoassay that is capable of sensitively detecting multidrug resistance protein 1 (MDR1), a prototypic 12-transmembrane-domains transporter. In a first assay step, complex samples are enzymatically fragmented into peptides as routinely done for mass spectrometry. A proteotypic peptide derived from MDR1 was chosen and antibodies targeting this peptide were used to build a sandwich immunoassay. Validation of the optimized assay showed good sensitivity, reproducibility and it allowed reliable quantification of MDR1; cross-validation by mass spectrometry demonstrated the applicability for routine analyses in clinical and pharmaceutical research. MDR1 was quantified in primary human renal cell carcinoma and corresponding normal tissue and down-regulation or expression loss was found in tumor tissue corroborating its importance in drug resistance and efficacy.

Mass Spectrometry-Based Immunoassay for the Quantification of Banned Ruminant Processed Animal Proteins in Vegetal Feeds.

The ban of processed animal proteins (PAPs) in feed for farmed animals introduced in 2001 was one of the main EU measures to control the bovine spongiform encephalopathy (BSE) crisis. Currently, microscopy and polymerase chain reaction (PCR) are the official methods for the detection of illegal PAPs in feed. However, the progressive release of the feed ban, recently with the legalization of nonruminant PAPs for the use in aquaculture, requires the development of alternative methods to determine the species origin and the source (legal or not). Additionally, discussions about the need for quantitative tests came up, particularly if the zero-tolerance-concept is replaced by introducing PAP thresholds. To address this issue, we developed and partially validated a multiplex mass spectrometry-based immunoassay to quantify ruminant specific peptides in vegetal cattle feed. The workflow comprises a new sample preparation procedure based on a tryptic digestion of PAPs in suspension, a subsequent immunoaffinity enrichment of the released peptides, and a LC-MS/MS-based analysis for peptide quantification using isotope labeled standard peptides. For the very first time, a mass spectrometry-based method is capable of detecting and quantifying illegal PAPs in animal feed over a concentration range of 4 orders of magnitude with a detection limit in the range of 0.1% to 1% (w/w).

Direct Quantification of Cytochromes P450 and Drug Transporters-A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates.

The quantification of drug metabolizing enzymes and transporters has recently been revolutionized on the basis of targeted proteomic approaches. Isotope-labeled peptides are used as standards for the quantification of the corresponding proteins in enzymatically fragmented samples. However, hurdles in these approaches are low throughput and tedious sample prefractionation steps prior to mass spectrometry (MS) readout. We have developed an assay platform using sensitive and selective immunoprecipitation coupled with mass spectrometric readout allowing the quantification of proteins directly from whole cell lysates using less than 20,000 cells per analysis. Peptide group-specific antibodies (triple X proteomics antibodies) enable the enrichment of proteotypic peptides sharing a common terminus. These antibodies were employed to establish a MS-based immunoassay panel for the quantification of 14 cytochrome P450 (P450) enzymes and nine transporters. We analyzed the P450 enzyme and transporter levels in genotyped liver tissue homogenates and microsomes, and in samples from a time course induction experiment in human hepatocytes addressing different induction pathways. For the analysis of P450 enzymes and transporters only a minute amount of sample is required and no prefractionation is necessary, thus the assay platform bears the potential to bridge cell culture model experiments and results from whole organ tissue studies.

Immuno-Matrix-Assisted Laser Desorption/Ionization Assays for Quantifying AKT1 and AKT2 in Breast and Colorectal Cancer Cell Lines and Tumors.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway is one of the most commonly dysregulated signaling pathways that is linked to cancer development and progression, and its quantitative protein analysis holds the promise to facilitate patient stratification for targeted therapies. Whereas immunohistochemistry (IHC) and immunoassays are routinely used for clinical analysis of signaling pathways, mass spectrometry-based approaches such as liquid chromatography/electrospray ionization multiple reaction monitoring mass spectrometry (LC/ESI-MRM-MS) are more commonly used in clinical research. Both technologies have certain disadvantages, namely, the nonspecificity of IHC and immunoassays, and potentially long analysis times per sample of LC/ESI-MRM-MS. To create a robust, fast, and sensitive protein quantification tool, we developed immuno-matrix-assisted laser desorption/ionization (iMALDI) assays with automated liquid handling. The assays are able to quantify AKT1 and AKT2 from breast cancer and colon cancer cell lines and flash-frozen tumor lysates with a linear range of 0.05-2.0 fmol/µg of total lysate protein and with coefficients of variation < 15%. Compared to other mass spectrometric methods, the developed assays require less sample per analysis-only 25 µg of total protein-and are therefore suitable for analysis of needle biopsies. Furthermore, the presented iMALDI technique is the first MS-based method for absolute quantitation of AKT peptides from cancer tissues. This study demonstrates the suitability of iMALDI for low limit-of-detection and reproducible quantitation of signaling pathway members using a benchtop MALDI mass spectrometer within approximately 6-7 h.

Inhibition of ß-catenin signaling by phenobarbital in hepatoma cells in vitro.

The antiepileptic drug phenobarbital (PB) exerts hepatic effect based on indirect activation of the constitutive androstane receptor (CAR) via inhibition of the epidermal growth factor receptor (EGFR) and the kinase Src. It has furthermore been observed that in mice PB suppresses the growth of hepatocellular carcinoma with overactive signaling through the oncogenic Wnt/ß-catenin pathway, thus suggesting an interference of PB with ß-catenin signaling. The present work was aimed to characterize effects of PB on ß-catenin signaling at different cellular levels and to elucidate molecular details of the interaction of PB and ß-catenin in an in vitro system of mouse hepatoma cells. PB efficiently inhibited signaling through ß-catenin. This phenomenon was in-depth characterized at the levels of ß-catenin protein accumulation and transcriptional activity. Mechanistic analyses revealed that the effect of PB on ß-catenin signaling was independent of the activation of CAR and also independent of the cytosolic multi-protein complex responsible for physiological post-translation control of the ß-catenin pathway via initiation of ß-catenin degradation. Instead, evidence is provided that PB diminishes ß-catenin protein production by inhibition of protein synthesis via signal transduction through EGFR and Src. The proposed mechanism is well in agreement with previously published activities of PB at the EGFR and Src-mediated regulation of ß-catenin mRNA translation. Inhibition of ß-catenin signaling by PB through the proposed mechanism might explain the inhibitory effect of PB on the growth of specific sub-populations of mouse liver tumors. In conclusion, the present data comprehensively characterize the effect of PB on ß-catenin signaling in mouse hepatoma cells in vitro and provides mechanistic insight into the molecular processes underlying the observed effect.

Peptides in headlock--a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy.

Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a ß-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies.