Nature as blueprint: Global phenotype engineering of CHO production cells based on a multi-omics comparison with plasma cells.

Especially for the production of artificial, difficult to express molecules a further development of the CHO production cell line is required to keep pace with the continuously increasing demands. However, the identification of novel targets for cell line engineering to improve CHO cells is a time and cost intensive process. Since plasma cells are evolutionary optimized for a high antibody expression in mammals, we performed a comprehensive multi-omics comparison between CHO and plasma cells to exploit optimized cellular production traits. Comparing the transcriptome, proteome, miRNome, surfaceome and secretome of both cell lines identified key differences including 392 potential overexpression targets for CHO cell engineering categorized in 15 functional classes like transcription factors, protein processing or secretory pathway. In addition, 3 protein classes including 209 potential knock-down/out targets for CHO engineering were determined likely to affect aggregation or proteolysis. For production phenotype engineering, several of these novel targets were successfully applied to transient and transposase mediated overexpression or knock-down strategies to efficiently improve productivity of CHO cells. Thus, substantial improvement of CHO productivity was achieved by taking nature as a blueprint for cell line engineering.

Spatial Proteomics Reveals Differences in the Cellular Architecture of Antibody-Producing CHO and Plasma Cell-Derived Cells.

Most of the recombinant biotherapeutics employed today to combat severe illnesses, for example, various types of cancer or autoimmune diseases, are produced by Chinese hamster ovary (CHO) cells. To meet the growing demand of these pharmaceuticals, CHO cells are under constant development in order to enhance their stability and productivity. The last decades saw a shift from empirical cell line optimization toward rational cell engineering using a growing number of large omics datasets to alter cell physiology on various levels. Especially proteomics workflows reached new levels in proteome coverage and data quality because of advances in high-resolution mass spectrometry instrumentation. One type of workflow concentrates on spatial proteomics by usage of subcellular fractionation of organelles with subsequent shotgun mass spectrometry proteomics and machine learning algorithms to determine the subcellular localization of large portions of the cellular proteome at a certain time point. Here, we present the first subcellular spatial proteome of a CHO-K1 cell line producing high titers of recombinant antibody in comparison to the spatial proteome of an antibody-producing plasma cell-derived myeloma cell line. Both cell lines show colocalization of immunoglobulin G chains with chaperones and proteins associated in protein glycosylation within the endoplasmic reticulum compartment. However, we report differences in the localization of proteins associated to vesicle-mediated transport, transcription, and translation, which may affect antibody production in both cell lines. Furthermore, pairing subcellular localization data with protein expression data revealed elevated protein masses for organelles in the secretory pathway in plasma cell-derived MPC-11 (Merwin plasma cell tumor-11) cells. Our study highlights the potential of subcellular spatial proteomics combined with protein expression as potent workflow to identify characteristics of highly efficient recombinant protein-expressing cell lines. Data are available via ProteomeXchange with identifier PXD029115.

A Novel PNGase Rc for Improved Protein N-Deglycosylation in Bioanalytics and Hydrogen-Deuterium Exchange Coupled With Mass Spectrometry Epitope Mapping under Challenging Conditions.

N-linked glycosylation is a ubiquitous posttranslational modification of proteins. While it plays an important role in the biological function of proteins, it often poses a major challenge for their analytical characterization. Currently available peptide N-glycanases (PNGases) are often inefficient at deglycosylating proteins due to sterically inaccessible N-glycosylation sites. This usually leads to poor sequence coverage in bottom-up analysis using liquid chromatography with tandem mass spectrometry and makes it impossible to obtain an intact mass signal in top-down MS analysis. In addition, most PNGases operate optimally only in the neutral to slightly acidic pH range and are severely compromised in the presence of reducing and denaturing substances, which limits their use for advanced bioanalysis based on hydrogen-deuterium exchange in combination with mass spectrometry (HDX-MS). Here, we present a novel peptide N-glycanase from Rudaea cellulosilytica (PNGase Rc) for which we demonstrate broad substrate specificity for N-glycan hydrolysis from multiply occupied and natively folded proteins. Our results show that PNGase Rc is functional even under challenging, HDX quenching conditions (pH 2.5, 0 °C) and in the presence of 0.4 M tris(2-carboxyethyl)phosphine, 4 M urea, and 1 M guanidinium chloride. Most importantly, we successfully applied the PNGase Rc in an HDX-MS workflow to determine the epitope of a nanobody targeting the extracellular domain of human signal-regulating protein alpha (SIRPα).

HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1.

Annexin-A1 (ANXA1) belongs to a class of highly homologous Ca2+-dependent phospholipid-binding proteins. Its structure consists of a core region composed of four homologous repeats arranged in a compact, hydrolysis-resistant structure and an N-terminal region with a Ca2+-dependent conformation. ANXA1 is involved in several processes, including cell proliferation, apoptosis, metastasis, and the inflammatory response. Therefore, the development of antibodies blocking selected regions on ANXA1 holds great potential for the development of novel therapeutics treating inflammatory and cancer diseases. Here, we report the interaction site between an ANXA1-specific antibody known to inhibit T cell activation without adverse cytotoxic effects and ANXA1 using amide hydrogen-deuterium exchange mass spectrometry (HDX-MS). For the epitope determination, we applied two bottom-up HDX-MS approaches with pepsin digestion in solution and immobilized on beads. Both strategies revealed the interaction region within domain III of ANXA1 in Ca2+-bound conformation. The antibody-binding region correlates with the hydrophobic binding pocket of the N-terminal domain formed in the absence of calcium. This study demonstrates that even cryptic and flexible binding regions can be studied by HDX-MS, allowing a fast and efficient determination of the binding sites of antibodies which will help to define a mode of action profile for their use in therapy.

Toxicity of ionizing radiation (IR) in a human induced pluripotent stem cell (hiPSC)-derived 3D early neurodevelopmental model.

Prenatal brain development is a complex and sensitive process, highly susceptible to environmental influences such as pollutants, stress, malnutrition, drugs, tobacco exposure, or ionizing radiation (IR). Disturbances in development may cause life-long disabilities and diseases, such as ADHD, childhood cancers, cognitive problems, depression, anxiety and more severe developmental disabilities. Due to increasing medical imaging, radiation therapy, natural terrestrial radiation, radioactive pollution and long-distance flights, humans are increasingly exposed to IR. However, data on impact of IR on very early human brain development are scarce, particularly in the very first weeks of gestation. Here we investigated the effects of low-dose X-ray IR (1 Gy) in a 3D early brain developmental model derived from human pluripotent stem cells. In this model very early neural stem cells, neuroectodermal progenitor cells (NEP), were exposed to low-dose IR and direct as well as delayed effects were investigated. Expression of 20 different marker genes crucial for normal neural development was determined 48 h and 9 days post IR (pIR). All but one of the analyzed marker genes were reduced 48 h after IR, and all but seven genes normalized their expression by day 9 pIR. Among the seven markers were genes involved in neurodevelopmental and growth abnormalities. Moreover, we could show that stemness of the NEP was reduced after IR. We were thus able to identify a significant impact of radiation in cells surviving low-dose IR, suggesting that low-dose IR could have a negative impact on the early developing human brain, with potential later detrimental effects.

Highly Sensitive Matrix-Independent Quantification of Major Food Allergens Peanut and Soy by Competitive Real-Time PCR Targeting Mitochondrial DNA.

The development of two competitive real-time PCR assays for the quantitative detection of trace amounts of two major food allergens, peanut and soybean, is reported. In order to achieve very low detection levels for both allergens, we established PCR primers and probes targeting mitochondrial DNA sequences. We were able to demonstrate that this approach led to an increase in detection sensitivity in the range of at least 1 order of magnitude compared with published assays targeting nuclear DNA. Furthermore, we generated corresponding competitor molecules, which were used as internal standards to compete with matrix effects that are evident during DNA extraction and PCR amplification in heterogeneous analytical matrixes like food. According to the recently described competitive quantitative PCR method published by Holzhauser et al. (2014), we performed threshold calibration against milk powder spiked with 10 ppm peanut and soy. Matrix-independent quantitative determination of peanut and soy could be demonstrated for three different calibrated food matrix standards in a range between 1 and 100 ppm. The data presented indicate that both assay concepts are powerful analytical tools for the quantitative detection of trace amounts of peanut and soy in commercial food products.

Fine-tuned PEGylation of chitosan to maintain optimal siRNA-nanoplex bioactivity.

Polyethylene glycol (PEG) is a widely used modification for drug delivery systems. It reduces undesired interaction with biological components, aggregation of complexes and serves as a hydrophilic linker of ligands for targeted drug delivery. However, PEGylation can also lead to undesired changes in physicochemical characteristics of chitosan/siRNA nanoplexes and hamper gene silencing. To address this conflicting issue, PEG-chitosan copolymers were synthesized with stepwise increasing degrees of PEG substitution (1.5% to 8.0%). Subsequently formed PEG-chitosan/siRNA nanoplexes were characterized physicochemically and biologically. The results showed that small ratios of chitosan PEGylation did not affect nanoplex stability and density. However, higher PEGylation ratios reduced nanoplex size and charge, as well as cell uptake and final siRNA knockdown efficiency. Therefore, we recommend fine-tuning of PEGylation ratios to generate PEG-chitosan/siRNA delivery systems with maximum bioactivity. The degree of PEGylation for chitosan/siRNA nanoplexes should be kept low in order to maintain optimal nanoplex efficiency.

Plasma phosphatidylcholine alterations in cystic fibrosis patients: impaired metabolism and correlation with lung function and inflammation.

BACKGROUND: Liver impairment, ranging from steatosis to cirrhosis, is frequent in cystic fibrosis (CF) patients and is becoming increasingly significant due to their improved life expectancy. One aspect of hepatic alterations is caused by increased fecal loss of the essential nutrient choline, following enterohepatic bile phosphatidylcholine (PC) cycle impairment. Hepatic PC synthesis, both de novo and via phosphatidylethanolamine-N-methyl-transferase (PEMT), is essential for very low-density lipoprotein (VLDL) secretion. VLDL-PC in particular contributes to the organism's supply with polyunsaturated fatty acids (LC-PUFA), namely arachidonic (C20:4) and docosahexaenoic acid (C22:6). Consequently, choline deprivation and altered hepatic PC metabolism may affect plasma PC homeostasis and extrahepatic organ function. OBJECTIVES: To investigate relationships between altered plasma choline and PC homeostasis and markers of lung function and inflammation in CF. To assess alterations in hepatic choline and PC metabolism of CF patients. DESIGN: Quantification of plasma/serum choline and PC species in adult CF patients compared to controls. Correlation of PC with forced expiratory vital capacity (FEV1) and interleukin 6 (IL-6) concentrations. Analysis of choline and PC metabolism in CF compared to controls, using deuterated choline ([D9-methyl]-choline) labeling in vivo. RESULTS: Mean choline and PC concentrations in CF patients were lower than in controls. Choline and PC concentrations as well as fractions of C22:6-PC and C20:4-PC correlated directly with FEV1, but inversely with IL-6. Plasma concentrations of deuterated PC were decreased for both pathways, whereas only in PC synthesized via PEMT precursor enrichment was decreased. CONCLUSION: In CF patients, hepatic and plasma homeostasis of choline and PC correlate with lung function and inflammation. Impaired hepatic PC metabolism, exemplarily shown in three CF patients, provides an explanation for such correlations. Larger studies are required to understand the link between hepatic PC metabolism and overall clinical performance of CF patients, and the perspective of choline substitution of these patients.

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.

Hyaluronic acid/chitosan multilayer coatings on neuronal implants for localized delivery of siRNA nanoplexes.

Binding, stabilizing and promoting cellular uptake of siRNA are all critical efforts in creating matrices for the localized delivery of siRNA molecules to target cells. In this study, we describe the generation of chitosan imidazole/siRNA nanoplexes (NPs) embedded in nano scope polyelectrolyte multilayers (PEMs) composed of hyaluronic acid and chitosan for sustained and localized drug delivery. Regular PEM build-up, successful integration of NPs and controlled release under physiological conditions were shown. Biological efficacy was evaluated in neuronal cell culture concerning cell adhesion, viability, NPs uptake and gene silencing. The additionally shown biological functionalization of neuronal implants possesses potential for future applications in the field of regenerative medicine and treatment of spinal cord injuries.

G protein-coupled receptor quantification using peptide group-specific enrichment combined with internal peptide standard reporter calibration.

The G protein-coupled receptor (GPCR) super-family comprises the largest and most diverse group of membrane receptors in eukaryotes. GPCRs are involved in a plethora of physiological functions in all kinds of tissues. Detailed knowledge about GPCR presence and expression levels in tissues can be very helpful for drug development as the majority of drugs are designed to modulate membrane receptors. Furthermore, it is known that many adverse drug effects result from GPCR interactions. However, very few satisfactory methods are currently available for the detection and quantification of GPCRs. The detection is complicated by their three-dimensional structure, their hydrophobic properties, and their localization in the plasma membrane with 7-trans-membrane domains and small cytosolic and extracellular domains. Due to these properties it is very difficult to generate specific antibodies directed against GPCRs for sandwich immunoassays and Western blot. We therefore designed an immunoaffinity- and mass spectrometry-based approach to analyze GPCR-specific signature peptides in tryptic digests in rat tissue lysates. The expression levels of four different GPCRs were determined using chemically labeled synthetic standard peptides. Here, we demonstrate for the first time, that peptide immunoaffinity MS-based methods can render a reliable and quantitative analysis of multi-membrane spanning receptor molecules.

Impact of polyelectrolytes and their corresponding multilayers to human primary endothelial cells.

The layer-by-layer technique, which allows simple preparation of polyelectrolyte multilayers, came into the focus of research for development of functionalized medical devices. Numerous literature exist that concentrate on the film build-up and the behaviour of cells on polyelectrolyte multilayers. However, in case of very soft polyelectrolyte multilayers, studies of the cell behaviour on these films are sometimes misleading with regard to clinical applications because cells do not die due to cytotoxicity but due to apoptosis by missing cell adhesion. It turns out that the adhesion in vitro, and thus, the viability of cells on polyelectrolyte multilayers is mostly influenced by their mechanical properties. In order to decide, which polyelectrolyte multilayers are suitable for implants, we take this problem into account by putting the substrates with soft films on top of pre-cultured human primary endothelial cells ('reverse assay'). Hence, the present work aims giving a more complete and reliable study of typical polyelectrolyte multilayers with regard to clinical applications. In particular, coatings consisting of hyaluronic acid and chitosan as natural polymers and sulfonated polystyrene and polyallylamine hydrochlorite as synthetic polymers were studied. The adsorption of polyelectrolytes was characterized by physico-chemical methods which show regular buildup. Biological examination of the native or modified polyelectrolyte multilayers was based on their effect to cell adhesion and morphology of endothelial cells by viability assays, immunostaining and scanning electron microscopy. Using the standard method, which is typically applied in literature--seeding cells on top of films--shows that the best adhesion and thus, viability can be achieved using sulfonated polystyrene/polyallylamine hydrochlorite. However, putting the films on top of endothelial cells reveals that hyaluronic acid/chitosan may also be suitable for clinical applications: This result is especially remarkable, since hyaluronic acid and chitosan mediate per se no cytotoxic effects, whereas the individual polyelectrolytes, sulfonated polystyrene and polyallylamine hydrochlorite, and their complexes show slight cytotoxicity.

Five years later: the current status of the use of proteomics and transcriptomics in EMF research.

The World Health Organization's and Radiation and Nuclear Safety Authority's "Workshop on Application of Proteomics and Transcriptomics in Electromagnetic Fields Research" was held in Helsinki in the October/November 2005. As a consequence of this meeting, Proteomics journal published in 2006 a special issue "Application of Proteomics and Transcriptomics in EMF Research" (Vol. 6 No. 17; Guest Editor: D. Leszczynski). This Proteomics issue presented the status of research, of the effects of electromagnetic fields (EMF) using proteomics and transcriptomics methods, present in 2005. The current overview/opinion article presents the status of research in this area by reviewing all studies that were published by the end of 2010. The review work was a part of the European Cooperation in the Field of Scientific and Technical Research (COST) Action BM0704 that created a structure in which researchers in the field of EMF and health shared knowledge and information. The review was prepared by the members of the COST Action BM0704 task group on the high-throughput screening techniques and electromagnetic fields (TG-HTST-EMF).

Combining ultracentrifugation and peptide termini group-specific immunoprecipitation for multiplex plasma protein analysis.

Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques.

Increased palmitoyl-myristoyl-phosphatidylcholine in neonatal rat surfactant is lung specific and correlates with oral myristic acid supply.

Surfactant predominantly comprises phosphatidylcholine (PC) species, together with phosphatidylglycerols, phosphatidylinositols, neutral lipids, and surfactant proteins-A to -D. Together, dipalmitoyl-PC (PC16:0/16:0), palmitoyl-myristoyl-PC (PC16:0/14:0), and palmitoyl-palmitoleoyl-PC (PC16:0/16:1) make up 75-80% of mammalian surfactant PC, the proportions of which vary during development and in chronic lung diseases. PC16:0/14:0, which exerts specific effects on macrophage differentiation in vitro, increases in surfactant during alveolarization (at the expense of PC16:0/16:0), a prenatal event in humans but postnatal in rats. The mechanisms responsible and the significance of this reversible increase are, however, not understood. We hypothesized that, in rats, myristic acid (C14:0) enriched milk is key to lung-specific PC16:0/14:0 increases in surfactant. We found that surfactant PC16:0/14:0 in suckling rats correlates with C14:0 concentration in plasma chylomicrons and lung tissue triglycerides, and that PC16:0/14:0 fractions reflect exogenous C14:0 supply. Significantly, C14:0 was increased neither in plasma PC, nor in liver triglycerides, free fatty acids, or PC. Lauric acid was also abundant in triglycerides, but was not incorporated into surfactant PC. Comparing a C14:0-rich milk diet with a C14:0-poor carbohydrate diet revealed increased C14:0 and decreased C16:0 in plasma and lung triglycerides, respectively. PC16:0/14:0 enrichment at the expense of PC16:0/16:0 did not impair surfactant surface tension function. However, the PC profile of the alveolar macrophages from the milk-fed animals changed from PC16:0/16:0 rich to PC16:0/14:0 rich. This was accompanied by reduced reactive oxygen species production. We propose that nutritional supply with C14:0 and its lung-specific enrichment may contribute to decreased reactive oxygen species production during alveolarization.

Targeting peptide termini, a novel immunoaffinity approach to reduce complexity in mass spectrometric protein identification.

Mass spectrometry and peptide-centric approaches are powerful techniques for the identification of differentially expressed proteins. Despite enormous improvements in MS technologies, sample preparation and efficient fractionation of target analytes are still major bottlenecks in MS-based protein analysis. The complexity of tryptically digested whole proteomes needs to be considerably reduced before low abundance proteins can be effectively analyzed using MS/MS. Sample preparation strategies that use peptide-specific antibodies are able to reduce the complexity of tryptic digests and lead to a substantial increase in throughput and sensitivity; however, the number of peptide-specific capture reagents is low, and consequently immunoaffinity-based approaches are only capable of detecting small sets of protein-derived peptides. In this proof-of-principle study, special anti-peptide antibodies were used to enrich peptides from a complex mixture. These antibodies recognize short amino acid sequences that are found directly at the termini of the peptides. The recognized epitopes consist of three or four amino acids only and include the terminally charged group of the peptide. Because of its limited length, antibodies recognizing the epitope will enrich not only one peptide but a whole class of peptides that share this terminal epitope. In this study, ß-catenin-derived peptides were used to demonstrate that it is possible (i) to effectively generate antibodies that recognize short C-terminal peptide epitopes and (ii) to enrich and identify peptide classes from a complex mixture using these antibodies in an immunoaffinity MS approach. The expected ß-catenin peptides and a set of 38 epitope-containing peptides were identified from trypsin-digested cell lysates. This might be a first step in the development of proteomics applications that are based on the use of peptide class-specific antibodies.

µFBI: a microfluidic bead-based immunoassay for multiplexed detection of proteins from a µL sample volume.

BACKGROUND: Over the last ten years, miniaturized multiplexed immunoassays have become robust, reliable research tools that enable researchers to simultaneously determine a multitude of parameters. Among the numerous analytical protein arrays available, bead-based assay systems have evolved into a key technology that enables the quantitative protein profiling of biological samples whilst requiring only a minimal amount of sample material. METHODOLOGY/PRINCIPAL FINDINGS: A microfluidic bead-based immunoassay, µFBI, was developed to perform bead-based multiplexed sandwich immunoassays in a capillary. This setup allows the simultaneous detection of several parameters and only requires 200 ng of tissue lysate in a 1 µL assay volume. In addition, only 1 µL of detection antibodies and 1 µL of the reporter molecule Streptavidin-Phycoerythrin were required. The µFBI was used to compare the expression of seven receptor tyrosine kinases and their degree of tyrosine phosphorylation in breast cancer tissue and in normal tissue lysates. The total amount of HER-2, as well the degree of tyrosine phosphorylation was much higher in breast cancer tissue than in normal tissue. µFBI and a standard bead-based assay led to identical protein expression data. Moreover, it was possible to reduce the quantity of sample material required by a factor of 100 and the quantity of reagents by a factor of 30. CONCLUSIONS/SIGNIFICANCE: The µFBI, microfluidic bead-based immunoassay, allows the analysis of multiple parameters from a very small amount of sample material, such as tumor biopsies or tissue sections.

MicroPrep: chip-based dielectrophoretic purification of mitochondria.

We have developed a microfluidic system--microPrep--for subcellular fractionation of cell homogenates based on dielectrophoretic sorting. Separation of mitochondria isolated from a human lymphoblastoid cell line was monitored by fluorescence microscopy and further characterized by western blot analysis. Robust high throughput and continuous long-term operation for up to 60 h of the microPrep chip system with complex biological samples became feasible as a result of a comprehensive set of technical measures: (i) coating of the inner surfaces of the chip with BSA, (ii) application of mechanical actuators to induce periodic flow patterns, (iii) efficient cooling of the device to ensure integrity of organelle, (iv) a wide channel to provide for high fluidic throughput, and (v) integration of a serial arrangement of 10 dielectrophoretic deflector units to enable separation of samples with a high particle load without clogging. Hence, microPrep yields tens of micrograms of enriched and purified mitochondria within hours. Western blots of mitochondria fractions showed that contaminating endoplasmatic reticulum was reduced by a factor 6 when compared with samples prepared by state of the art centrifugation.

Optimal selection of epitopes for TXP-immunoaffinity mass spectrometry.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Proteome wide screening using peptide affinity capture.

MS-based strategies are key technologies for identifying proteins in proteomic research. Despite significant improvements in recent years efficient fractionation processes of target analytes remain major bottlenecks in MS-based protein analysis. Immunoaffinity-based sample fractionation strategies have shown their potential for the enrichment of analyte peptides of interest, but only small numbers of analytes can be quantified in one experiment. The lack of appropriate capture reagents limits the application of immunoaffinity-based approaches and only biased biomarker discovery approaches are possible. This perspective discusses the current status of immunoaffinity MS-based approaches and introduces a novel concept that uses group specific anti-peptide antibodies -- Triple X Proteomics Antibodies -- for the enrichment of signature peptides. Classes of peptides with identical termini can be fractionated based on TXP immunoaffinity enrichment steps and can subsequently be identified using established tandem MS procedures. Based on bioinformatic algorithms minimal sets of TXP epitopes can be specified, that cover a wide range of given proteome landscapes of one or even several different species. This opens the possibility to use a minimal number of TXP antibodies as a universal toolbox for general immunoaffinity-based approaches in proteome analysis.