Optimal selection of epitopes for TXP-immunoaffinity mass spectrometry

<p>Abstract</p> <p>Background</p> <p>Mass spectrometry (MS) based protein profiling has become one of the key technologies in biomedical research and biomarker discovery. One bottleneck in MS-based protein analysis is sample preparation and an efficient fractionation step to reduce the complexity of the biological samples, which are too complex to be analyzed directly with MS. Sample preparation strategies that reduce the complexity of tryptic digests by using immunoaffinity based methods have shown to lead to a substantial increase in throughput and sensitivity in the proteomic mass spectrometry approach. The limitation of using such immunoaffinity-based approaches is the availability of the appropriate peptide specific capture antibodies. Recent developments in these approaches, where subsets of peptides with short identical terminal sequences can be enriched using antibodies directed against short terminal epitopes, promise a significant gain in efficiency.</p> <p>Results</p> <p>We show that the minimal set of terminal epitopes for the coverage of a target protein list can be found by the formulation as a set cover problem, preceded by a filtering pipeline for the exclusion of peptides and target epitopes with undesirable properties.</p> <p>Conclusions</p> <p>For small datasets (a few hundred proteins) it is possible to solve the problem to optimality with moderate computational effort using commercial or free solvers. Larger datasets, like full proteomes require the use of heuristics.</p

Combinatorial optimization for affinity proteomics

Biochemical test development can significantly benefit from combinatorial optimization. Multiplex assays do require complex planning decisions during implementation and subsequent validation. Due to the increasing complexity of setups and the limited resources, the need to work efficiently is a key element for the success of biochemical research and test development. The first approached problem was to systemically pool samples in order to create a multi-positive control sample. We could show that pooled samples exhibit a predictable serological profile and by using this prediction a pooled sample with the desired property. For serological assay validation it must be shown that the low, medium, and high levels can be reliably measured. It is shown how to optimally choose a few samples to achieve this requirements. Finally the latter methods were merged to validate multiplexed assays using a set of pooled samples. A novel algorithm combining fast enumeration and a set cover formulation has been introduced. The major part of the thesis deals with optimization and data analysis for Triple X Proteomics - immunoaffinity assays using antibodies binding short linear, terminal epitopes of peptides. It has been shown that the problem of choosing a minimal set of epitopes for TXP setups, which combine mass spectrometry with immunoaffinity enrichment, is equivalent to the well-known set cover problem. TXP Sandwich immunoassays capture and detect peptides by combining the C-terminal and N-terminal binders. A greedy heuristic and a meta-heuristic using local search is presented, which proves to be more efficient than pure ILP formulations. All models were implemented in the novel Java framework SCPSolver, which is applicable to many problems that can be formulated as integer programs. While the main design goal of the software was usability, it also provides a basic modelling language, easy deployment and platform independence. One question arising when analyzing TXP data was: How likely is it to observe multiple peptides sharing the same terminus? The algorithms TXP-TEA and MATERICS were able to identify binding characteristics of TXP antibodies from data obtained in immunoaffinity MS experiments, reducing the cost of such analyses. A multinomial statistical model explains the distributions of short sequences observed in protein databases. This allows deducing the average optimal length of the targeted epitope. Further a closed-from scoring function for epitope enrichment in sequence lists is derived

Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry

Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system

Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry

Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system

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.publishedVersio

Development of MS- based immunoassays for transporter and cytochrome P450 quantification in human, rat and mouse

CYPs and transporters are important for the turnover of xenobiotic compounds. Their expression levels and activity influence bioavailability and convey drug-drug interactions. Moreover, transporters mediate barrier maintenance of several organs such as the blood – brain - barrier and the placenta-barrier. Overexpression of export transporters in tumors can lead to multiple drug resistance. Therefore, they are investigated thoroughly during drug development. However, it is still difficult to establish conventional assays such as sandwich immunoassays to quantify these proteins because CYP and transporter families are homologous and additionally transporters are very hydrophobic. The objective of this thesis was to develop MS – based immunoassays for ABC and SLC transporters as well as CYPs in up to three species which are relevant for research and drug development. During this thesis, nineteen TXP – multiplex assays were successfully developed which allow the quantification of up to 14 transporters in human, rat and mouse as well as up to 18 CYPs in rat and mouse. In total this corresponds to 61 different proteins. Additionally, it was shown that the TXP methodology is sensitive enough to quantify the low abundant target proteins from whole cell lysates without further enrichment such as membrane enrichment. The developed TXP -assays were used to analyze several independent studies: The amounts of transporters quantified in healthy human liver tissue was well line with published data. The protein expression profile of pericentral and periportal murine hepatocytes was analyzed and correlated with published immunohistochemistry as well as RNA expression data. Furthermore, a cross species study investigating the effects of azole fungicides in human cell culture, rat and wildtype as well as humanized mouse models was analyzed. It could be shown that high dosed fungicide treatment induced CAR, PXR and AhR target proteins as well as that the extent of the response differed between the models.CYPs und Transporter sind wichtige Bestandteile des Fremdstoffmetabolismus, deren Expression und Aktivität durch Nahrungsbestandteile und Medikamente moduliert werden. Sie beeinflussen die Bioverfügbarkeit von Medikamenten und vermitteln Wechselwirkungen zwischen verschiedenen Medikamenten oder Nahrungs-bestandteilen. Des Weiteren sind Transporter an der Aufrechterhaltung von Blutschranken verschiedener Organe beteiligt wie zum Beispiel der Blut – Hirn – Schranke. Das Phänomen der multiple drug resistance wird unter anderem durch die Überexpression von Exportpumpen vermittelt. Da CYPs und Transporter an so vielen Prozessen beteiligt sind, werden sie während der Medikamentenentwicklung ausführlich untersucht. Allerdings ist es immer noch schwierig sie mit konventionellen Methoden wie zum Beispiel Sandwich - Assays zu quantifizieren, weil sie zu sehr homologen Proteinfamilien gehören und Transporter des Weiteren auch noch sehr hydrophob sind. Aus diesem Grund, war das Ziel dieser Arbeit MS-basierte Immunoassays zu entwickeln um ABC und SLC Transporter sowie CYPs in bis zu drei für die Forschung und Medikamentenentwicklung wichtigen Spezies zu quantifizieren. Es wurden neunzehn TXP-Assays entwickelt mit denen bis zu 14 Transporter in Mensch, Ratte und Maus sowie bis zu 18 verschiedene CYPs in Ratte und Maus gemessen werden können. Das entspricht insgesamt 61 verschiedenen Proteinen. Zusätzlich konnte gezeigt werden, dass die TXP – Methode sensitiv genug ist, sodass auf zusätzlich Anreicherungsverfahren wie verzichtet werden kann. Die entwickelten TXP – Assays wurden verwendet um bei vier verschiedenen Studien CYPs und Transporter zu quantifizieren. Die in humanem Normalgewebe aus der Leber bestimmten Mengen sowie die Zonierung in Mausleber, stimmten gut mit den Literaturwerten überein. Des Weiteren wurde eine speziesübergreifende Studie zu den Effekten von Pyrrolfungiziden analysiert. Humane Zellkultur, Ratten und Wildtyp - sowie humanisierte Mäuse wurden als Modelle verwendet. Es konnte gezeigt werden, dass die Fungizide in hohen Dosen die Expression von CAR, PXR und AhR Zielproteinen in den vier Modellen beeinflussen. Das Ausmaß der Antwort hängt jedoch von dem verwendeten Modell ab

Immunoaffinity-Based Mass Spectrometry for the Species Identification and Quantification of Processed Animal Proteins in Feed

The present work introduced immunoaffinity-based mass spectrometry to feed analysis and improved the detection of banned processed animal proteins (PAPs) in animal feed. Current analytical methods show deficiencies in either sensitivity, species and tissue specificity or quantification ability. To address this issue, a peptide-centric workflow that comprises a more efficient sample preparation, an immunoaffinity enrichment of species- and tissue-specific peptides, and a LC-MS/MS analysis for identification and quantification using stable isotope labeled standard peptides, was established. The release of peptides from poorly soluble PAPs and blood products was improved by a direct digestion in suspension. Further time-consuming clean-up steps are not necessary since reagents and salts are removed during the immunoenrichment. The enrichment also allows a fast peptide separation using short gradients with a 10 min cycle time and therefore an increased sample throughput. The species differentiation of the 8 livestock species cattle, sheep/goat, pig, horse, turkey chicken, duck and goose, was addressed in a multispecies approach. Therefore, a cross-species polyclonal antibody was generated, which is able to enrich 8 homologous peptides from processed meat and bone meal, blood meal and spray-dried plasma, hence allowing a comprehensive analysis of common feed additives. A second multiplex assay was developed to differentiate ruminant tissues by targeting 7 peptides of meat, bone, cartilage, blood and milk proteins. This allows a differentiation of legal and illegal ruminant protein additives. The assays’ basic analytical parameters were validated. Both assays showed a detection limit in the picomolar concentration range allowing a qualitative detection over 4 to 5 orders of magnitude and a quantification over 3 to 4 orders of magnitude. Depending on the tissue type, 0.05%-0.75% PAP was specifically and quantitatively determined in an animal feed background. The multiplex assays were finally applied to official proficiency test samples from the European Reference Laboratory for Animal Proteins (EURL-AP, Gembloux, Belgium). The developed assays showed an unambiguous differentiation and quantification of species and tissues on a contamination level of 0.1% PAP in feed. As a final conclusion, immunoaffinity-based mass spectrometry was shown to overcome the current limitations in PAP detection and meets the requirements for future feed authentication methods

Etablierung eines Nachweissystems zur Quantifizierung von Cytochrom P450 Enzymen in der menschlichen Leber

Den Abbauprozessen von Fremdstoffen kommt vor allem in der pharmazeutischen Industrie großes Interesse zu. Die größtenteils auf mRNA-Expressionsstudien und Enzymaktivitätstest beruhende Erforschung des Fremdstoffmetabolismus kann durch die in dieser Arbeit entwickelte Quantifizierungsstrategie um die Information des Proteingehalts ergänzt werden. Der Konzentrationen der Cytochrom P450 Enzyme stellen einen wichtigen Baustein für die Untersuchung pharmakokinetischer Prozesse dar. Die entwickelten CYP-TXP-Tests quantifizieren die im menschlichen Fremdstoffmetabolismus beteiligten Cytochrom P450 Enzyme spezifisch und sensitiv. Die Tests erlauben es, simultan in einem einzelnen LC-MS-Lauf mehrere Analyte zu vermessen. Die zwei entwickelten multiplexen Quantifizierungsaufbauten sind in der Lage, nach Immunopräzipitation die Quantifizierung aller 15 im Fremdstoffmetabolismus relevanten Cytochrom P450 Enzyme, sowie des ABC-Transporters MDR1 und der NADPH-Cytochrom-P450-Oxidoreduktase zu ermöglichen. Die verwendeten TXP-Antikörper wurden eingehend charakterisiert und validiert. Neben der Bestimmung der Dissoziationskonstanten und Bindekapazitäten wurde der lineare Messbereich der TXP-Tests bestimmt. Ein breiter linearer Messbereich ermöglicht es, stark variierende Konzentrationsspiegel ohne Anpassung des Probevolumens zu vermessen. Somit sind die TXP-Tests besonders für Induktionsstudien geeignet und werden gleichzeitig den in der Bevölkerung stark schwankenden Expressionsspiegeln der Cytochrom P450 Enzyme gerecht. In Induktionsexperimenten konnten hierbei in der Literatur beschriebene Induktionsprozesse bestätigt werden. In Adriamycin-resistenten Zellen wurde zudem eine gesteigerte Expression des MDR1-Transporters nachgewiesen. Die hohen Sensitivitäten der TXP-Tests erlauben es, Cytochrom P450 Enzyme aus Lebergewebe ohne vorhergehende Mikrosomenpräparation zu quantifizieren. Die Quantifizierung der Cytochrom P450-Enzyme kann hierbei sowohl aus mikrosomalen Präparationen, als auch aus Gewebe erfolgen. Kleinste Gewebe- oder Zelllysatmengen von weniger als 10 μg Gewebe reichen für eine robuste Quantifizierung aus. Die entwickelten CYP-TXP-Tests eignen sich somit für die Quantifizierung in miniaturisierten Zellkulturexperimenten im 96-Well-Format.The degradation processes of xenobiotics are primarily of interest to the pharmaceutical industry. The quantification strategy developed in this thesis completes the research of drug metabolism mostly based on mRNA expression studies and enzyme activity tests with the knowledge of the protein amount. The concentration levels of cytochrome P450 enzymes build an important component for the investigation of pharmacokinetic processes. The developed CYP-TXP-tests are able to quantify the cytochrome P450 enzymes in a specific and sensitive way. The tests permit the simultaneous quantification of several analytes in only one LC-MS run. Two quantification setups were developed which have the ability to quantify 15 cytochrome P450 enzymes as well as the abc-tansporter MDR1 and the NADPH-cytochrome-p450-oxidoreductase after immunoprecipitation. Employed TXP-antibodies were characterized and validated in-depth. In addition to dissociation constants and binding capacities the linear ranges of the TXP-tests were determined. A broad linear range affords to measure highly varying concentration levels without adapting sample volumes. Thus the TXP tests are particularly suitable to conduct induction studies and meet very fluctuating expression levels of cytochrome P450 enzymes in population. Induction experiments confirmed induction processes described in literature. Furthermore an increased expression of the transporter protein MDR1 was demonstrated in Adriamycin-resistant cells. The high sensitivities of the TXP-tests enable the quantification of cytochrome P450 enzymes in liver tissue without preparing microsomes. It is possible to quantify cytochrome P450 enzymes out of both microsomal preparation and tissue. Very small amounts of tissue or cell lysate are sufficient to quantify out of 10 µg tissue in a robust way. Therefore the developed CYP-TXP-tests are suited for the quantification in miniaturised cell culture experiments using the 96-well-format