Plasma biomarkers TAP, CPA1, and CPA2 for the detection of pancreatic injury in rat: the development of a novel multiplex IA-LC-MS/MS assay and biomarker performance evaluation.

Drug-induced pancreatic injury (DIPI) is an issue seen in drug development both in nonclinical and clinical contexts. DIPI is typically monitored by measurement of lipase and/or amylase, however, both enzymes lack sensitivity and specificity. Although candidate protein biomarkers specific to pancreas exist, antibody-based assay development is difficult due to their small size or the rapid cleavage by proteolytic enzymes released during pancreatic injury. Here we report the development of a novel multiplexed immunoaffinity-based liquid chromatography mass spectrometric assay (IA-LC-MS/MS) for trypsinogen activation peptide (TAP) and carboxypeptidases A1 and A2 (CPA1, CPA2). This method is based on the enzymatic digestion of the target proteins, immunoprecipitation of the peptides with specific antibodies and LC-MS/MS analysis. This assay was used to detect TAP, CPA1, and CPA2 in 470 plasma samples collected from 9 in-vivo rat studies with pancreatic injury and 8 specificity studies with injury in other organs to assess their performance in monitoring exocrine pancreas injury. The TAP, CPA1, and CPA2 response was compared to histopathology, lipase, amylase and microRNA217. In summary, TAP, CPA1, and CPA2 proteins measured in rat plasma were sensitive and specific biomarkers for monitoring drug-induced pancreatic injury; outperforming lipase and amylase both by higher sensitivity of detection and by sustained increases in plasma observed over a longer time period. These protein-based assays and potentially others under development, are valuable tools for use in nonclinical drug development and as future translatable biomarkers for assessment in clinical settings to further improve patient safety.

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.

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.

Tris(hydroxymethyl)aminomethane Compatibility with N-Hydroxysuccinimide Ester Chemistry: Biotinylation of Peptides and Proteins in TRIS Buffer.

N-Hydroxysuccinimide esters of small molecules are widely used to modify biomolecules such as antibodies or proteins. Primary amine groups preferably react with the ester to form covalent amide bonds. Currently, protocols strongly recommend replacing the buffer reagent tris(hydroxymethyl)aminomethane, and it has even been proposed as a stop reagent. Here, we show that TRIS indeed does not interfere with biotinylation of biomolecules with NHS chemistry.

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.

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.

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.

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.

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.

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).

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.

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.

Variability in Mass Spectrometry-based Quantification of Clinically Relevant Drug Transporters and Drug Metabolizing Enzymes.

Many different methods are used for mass-spectrometry-based protein quantification in pharmacokinetics and systems pharmacology. It has not been established to what extent the results from these various methods are comparable. Here, we compared six different mass spectrometry-based proteomics methods by measuring the expression of clinically relevant drug transporters and metabolizing enzymes in human liver. Mean protein concentrations were in general quantified to similar levels by methods using whole tissue lysates. Methods using subcellular membrane fractionation gave incomplete enrichment of the proteins. When the enriched proteins were adjusted to levels in whole tissue lysates, they were on average 4-fold lower than those quantified directly in whole tissue lysates. The differences in protein levels were propagated into differences in predictions of hepatic clearance. In conclusion, caution is needed when comparing and applying quantitative proteomics data obtained with different methods, especially since membrane fractionation is common practice for protein quantification used in drug clearance predictions.

Monitoring interactions and dynamics of endogenous beta-catenin with intracellular nanobodies in living cells.

ß-catenin is the key component of the canonical Wnt pathway and plays a crucial role in a multitude of developmental and homeostatic processes. The different tasks of ß-catenin are orchestrated by its subcellular localization and participation in multiprotein complexes. To gain a better understanding of ß-catenin's role in living cells we have generated a new set of single domain antibodies, referred to as nanobodies, derived from heavy chain antibodies of camelids. We selected nanobodies recognizing the N-terminal, core or C-terminal domain of ß-catenin and applied these new high-affinity binders as capture molecules in sandwich immunoassays and co-immunoprecipitations of endogenous ß-catenin complexes. In addition, we engineered intracellularly functional anti-ß-catenin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. For the first time, we were able to visualize the subcellular localization and nuclear translocation of endogenous ß-catenin in living cells using these chromobodies. Moreover, the chromobody signal allowed us to trace the accumulation of diffusible, hypo-phosphorylated ß-catenin in response to compound treatment in real time using High Content Imaging. The anti-ß-catenin nanobodies and chromobodies characterized in this study are versatile tools that enable a novel and unique approach to monitor the dynamics of subcellular ß-catenin in biochemical and cell biological assays.

Evaluation of novel biomarkers of nephrotoxicity in Cynomolgus monkeys treated with gentamicin.

Most studies to evaluate kidney safety biomarkers have been performed in rats. This study was conducted in Cynomolgus monkeys in order to evaluate the potential usefulness of novel biomarkers of nephrotoxicity in this species. Groups of 3 males were given daily intramuscular injections of gentamicin, a nephrotoxic agent known to produce lesions in proximal tubules, at dose-levels of 10, 25, or 50mg/kg/day for 10days. Blood and 16-h urine samples were collected on Days -7, -3, 2, 4, 7, and at the end of the dosing period. Several novel kidney safety biomarkers were evaluated, with single- and multiplex immunoassays and in immunoprecipitation-LC/MS assays, in parallel to histopathology and conventional clinical pathology parameters. Treatment with gentamicin induced a dose-dependent increase in kidney tubular cell degeneration/necrosis, ranging from minimal to mild severity at 10mg/kg/day, moderate at 25mg/kg/day, and to severe at 50mg/kg/day. The results showed that the novel urinary biomarkers, microalbumin, α1-microglobulin, clusterin, and osteopontin, together with the more traditional clinical pathology parameters, urinary total protein and N-acetyl-ß-D-glucosaminidase (NAG), were more sensitive than blood urea nitrogen (BUN) and serum creatinine (sCr) to detect kidney injury in the monkeys given 10mg/kg/day gentamicin for 10days, a dose leading to an exposure which is slightly higher than the desired therapeutic exposure in clinics. Therefore, these urinary biomarkers represent non-invasive biomarkers of proximal tubule injury in Cynomolgus monkeys which may be potentially useful in humans.

Cited1 deficiency suppresses intestinal tumorigenesis.

Conditional deletion of Apc in the murine intestine alters crypt-villus architecture and function. This process is accompanied by multiple changes in gene expression, including upregulation of Cited1, whose role in colorectal carcinogenesis is unknown. Here we explore the relevance of Cited1 to intestinal tumorigenesis. We crossed Cited1 null mice with Apc(Min/+) and AhCre(+)Apc(fl/fl) mice and determined the impact of Cited1 deficiency on tumour growth/initiation including tumour multiplicity, cell proliferation, apoptosis and the transcriptome. We show that Cited1 is up-regulated in both human and murine tumours, and that constitutive deficiency of Cited1 increases survival in Apc(Min/+) mice from 230.5 to 515 days. However, paradoxically, Cited1 deficiency accentuated nearly all aspects of the immediate phenotype 4 days after conditional deletion of Apc, including an increase in cell death and enhanced perturbation of differentiation, including of the stem cell compartment. Transcriptome analysis revealed multiple pathway changes, including p53, PI3K and Wnt. The activation of Wnt through Cited1 deficiency correlated with increased transcription of ß-catenin and increased levels of dephosphorylated ß-catenin. Hence, immediately following deletion of Apc, Cited1 normally restrains the Wnt pathway at the level of ß-catenin. Thus deficiency of Cited1 leads to hyper-activation of Wnt signaling and an exaggerated Wnt phenotype including elevated cell death. Cited1 deficiency decreases intestinal tumourigenesis in Apc(Min/+) mice and impacts upon a number of oncogenic signaling pathways, including Wnt. This restraint imposed by Cited1 is consistent with a requirement for Cited1 to constrain Wnt activity to a level commensurate with optimal adenoma formation and maintenance, and provides one mechanism for tumour repression in the absence of Cited1.

Catch and measure-mass spectrometry-based immunoassays in biomarker research.

Mass spectrometry-based (MS) methods are effective tools for discovering protein biomarker candidates that can differentiate between physiological and pathophysiological states. Promising candidates are validated in studies comprising large patient cohorts. Here, targeted protein analytics are used to increase sample throughput. Methods involving antibodies, such as sandwich immunoassays or Western blots, are commonly applied at this stage. Highly-specific and sensitive mass spectrometry-based immunoassays that have been established in recent years offer a suitable alternative to sandwich immunoassays for quantifying proteins. Mass Spectrometric ImmunoAssays (MSIA) and Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA/iMALDI) are two prominent types of MS-based immunoassays in which the capture is done either at the protein or the peptide level. We present an overview of these emerging types of immunoassays and discuss their suitability for the discovery and validation of protein biomarkers. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Identification of short terminal motifs enriched by antibodies using peptide mass fingerprinting.

MOTIVATION: Mass spectrometry-based protein profiling has become a key technology in biomedical research and biomarker discovery. Sample preparation strategies that reduce the complexity of tryptic digests by immunoaffinity substantially increase throughput and sensitivity in proteomic mass spectrometry. The scarce availability of peptide-specific capture antibodies limits these approaches. Recently antibodies directed against short terminal motifs were found to enrich subsets of peptides with identical terminal sequences. This approach holds the promise of a significant gain in efficiency. TXP (Triple X Proteomics) and context-independent motif specific/global proteome survey binders are variants of this concept. Principally the binding motifs of such antibodies have to be elucidated after generating these antibodies. This entails a substantial effort in the lab, as it requires synthetic peptide libraries and numerous mass spectrometry experiments. RESULTS: We present an algorithm for predicting the antibody-binding motif in a mass spectrum obtained from a tryptic digest of a common cell line after immunoprecipitation. The epitope prediction, based on peptide mass fingerprinting, reveals the most enriched terminal epitopes. The tool provides a P-value for each potential epitope, estimated by sampling random spectra from a peptide database. The second algorithm combines the predicted sequences to more complex binding motifs. A comparison with library screenings shows that the predictions made by the novel methods are reliable and reproducible indicators of the binding properties of an antibody.