Dolores Cahill

Nanoparticles in physiological environments are known to selectively adsorb proteins and other biomolecules forming a tightly bound biomolecular 'corona' on their surface. Where the exchange times of the proteins are sufficiently long, it is believed that the protein corona constitutes the particle identity in biological milieu. Here we show that proteins in the corona retain their functional characteristics and can specifically bind to cognate proteins on arrays of thousands of immobilised human proteins. The biological identity of the nanomaterial is seen to be specific to the blood plasma concentration in which they are exposed. We show that the resulting in situ nanoparticle interactome is dependent on the protein concentration in plasma, with the emergence of a small number of dominant protein-protein interactions. These interactions are those driven by proteins that are adsorbed onto the particle surface and whose binding epitopes are subsequently expressed or presented suitably on the particle surface. We suggest that, since specific tailored protein arrays for target systems and organs can be designed, their use may be an important element in an overall study of the biomolecular corona.

We report a rapid, highly sensitive microarray method for quantitative aflatoxin B1 (AFB1) detection in cereal samples. Following optimisation using an indirect competitive immunoassay, optimised amounts of AFB1-bovine serum albumin (AFB1-BSA)-conjugate were contact-printed onto 16 isolated sub-arrays on multi-pad nitrocellulose coated slides subsequently used in competitive binding assays.The toxin microarray working range for AFB1 was established in the range of 15pgg-1 to 3.04ngg-1, with a detection limit of 1pgg-1. To determine assay sensitivity in contaminated food models, wheat flour and barley grains samples were spiked with AFB1 standard dilutions. Following extraction, the working ranges of 0.11-4.15 and 0.18-4.31ngg-1 were determined, with detection limits of 30 and 90pgg-1, respectively. The sensitivity of the developed assay is below the European commission limit set for AFB1 detection and the assay procedure was completed in 3h time. Good recoveries (98%±11%) obtained demonstrate the suitability of the proposed method for rapid and sensitive quantification of AFB1 in contaminated cereal samples.

Metastasis is the most lethal step of cancer progression in patients with invasive melanoma. In most human cancers, including melanoma, tumor dissemination through the lymphatic vasculature provides a major route for tumor metastasis. Unfortunately, molecular mechanisms that facilitate interactions between melanoma cells and lymphatic vessels are unknown. Here, we developed an unbiased approach based on molecular mimicry to identify specific receptors that mediate lymphatic endothelial-melanoma cell interactions and metastasis. By screening combinatorial peptide libraries directly on afferent lymphatic vessels resected from melanoma patients during sentinel lymphatic mapping and lymph node biopsies, we identified a significant cohort of melanoma and lymphatic surface binding peptide sequences. The screening approach was designed so that lymphatic endothelium binding peptides mimic cell surface proteins on tumor cells. Therefore, relevant metastasis and lymphatic markers were biochemically identified, and a comprehensive molecular profile of the lymphatic endothelium during melanoma metastasis was generated. Our results identified expression of the phosphatase 2 regulatory subunit A, α-isoform (PPP2R1A) on the cell surfaces of both melanoma cells and lymphatic endothelial cells. Validation experiments showed that PPP2R1A is expressed on the cell surfaces of both melanoma and lymphatic endothelial cells in vitro as well as independent melanoma patient samples. More importantly, PPP2R1A-PPP2R1A homodimers occur at the cellular level to mediate cell-cell interactions at the lymphatic-tumor interface. Our results revealed that PPP2R1A is a new biomarker for melanoma metastasis and show, for the first time to our knowledge, an active interaction between the lymphatic vasculature and melanoma cells during tumor progression.

Autoantibodies represent an attractive biomarker for diagnostic assays principally due to the stability of immunoglobulin in patient serum facilitating measurement with conventional assays. Immune responses to tumorigenesis may facilitate detection of ovarian cancer in the early stages of the disease with identification of a panel of tumour specific autoantibodies. Despite the reporting of many tumour associated autoantibodies using arrays of tumour antigens, this has not led to the advance in diagnostic capability as rapidly as was initially expected. Here we examine the potential diagnostic utility of candidate autoantibody biomarkers identified via screening of serum samples on a high content human protein array from a unique cohort of early stage and late stage ovarian cancer patients. We analyse the performance of autoantibodies to the tumour suppressor protein p53 and the novel autoantigens alpha adducin and endosulfine alpha identified in our array screen. Each antigen has different performance characteristics using conventional ELISA format and Western blot immunoassay. The high attrition rate of promising autoantigens identified by array screening can in part be explained by the presentation of the epitope of the antigen in the subsequent method of validation and this study provides directions on maximising the potential of candidate biomarkers. This article is part of a Special Issue entitled: Translational Proteomics.

One of the deadly hallmarks of cancer is its ability to prosper within the constraints of the host immune system. Recent advances in immunoproteomics and high-throughput technologies have lead to profiling of the antibody repertoire in cancer patients. This in turn has lead to the identification of tumour associated antigens/autoantibodies. Autoantibodies are extremely attractive and promising biomarker entities, however there has been relatively little discussion on how to interpret the humoral immune response. It may be that autoantibody profiles hold the key to ultimately uncovering neoplastic associated pathways and through the process of immunosculpting the tumour may have yielded an immune response in the early stages of malignant tumour development. The aim of this review is to discuss the utility of the autoantibody response that is elicited as a result of malignancy and discuss the advantages and limitations of autoantibody profiling. This article is part of a Special Issue entitled: Translational Proteomics.

Molecules differentially expressed in blood vessels among organs or between damaged and normal tissues, are attractive therapy targets; however, their identification within the human vasculature is challenging. Here we screened a peptide library in cancer patients to uncover ligand-receptors common or specific to certain vascular beds. Surveying ~2.35 x 10(6) motifs recovered from biopsies yielded a nonrandom distribution, indicating that systemic tissue targeting is feasible. High-throughput analysis by similarity search, protein arrays, and affinity chromatography revealed four native ligand-receptors, three of which were previously unrecognized. Two are shared among multiple tissues (integrin α4/annexin A4 and cathepsin B/apolipoprotein E3) and the other two have a restricted and specific distribution in normal tissue (prohibitin/annexin A2 in white adipose tissue) or cancer (RAGE/leukocyte proteinase-3 in bone metastases). These findings provide vascular molecular markers for biotechnology and medical applications.

Secretagogin is a calcium-binding protein whose expression is characterised in neuroendocrine, pancreatic, and retinal cells. We have used an array-based proteomic approach with the prokaryotically expressed human protein array (hEx1) and the eukaryotically expressed human protein array (Protoarray) to identify novel calcium-regulated interaction networks of secretagogin. Screening of these arrays with fluorophore-labelled secretagogin in the presence of Ca(2+) ions led to the identification of 12 (hEx1) and 6 (Protoarray) putative targets. A number of targets were identified in both array screens. The putative targets from the hEx1 array were expressed, purified, and subjected to binding analysis using surface plasmon resonance. This identified binding affinities for nine novel secretagogin targets with equilibrium dissociation constants in the 100 pM to 10 nM range. Six of the novel target proteins have important roles in vesicle trafficking; SNAP-23, ARFGAP2, and DOC2alpha are involved in regulating fusion of vesicles to membranes, kinesin 5B and tubulin are essential for transport of vesicles in the cell, and rootletin builds up the rootlet, which is believed to function as scaffold for vesicles. Among the targets are two enzymes, DDAH-2 and ATP-synthase, and one oncoprotein, myeloid leukaemia factor 2. This screening method identifies a role for secretagogin in secretion and vesicle trafficking interacting with several proteins integral to these processes.

The calcium ion (Ca(2+)) is a ubiquitous second messenger that is crucial for the regulation of a wide variety of cellular processes. The diverse transient signals transduced by Ca(2+) are mediated by intracellular -Ca(2+)-binding proteins. Calcium ions shuttle into and out of the cytosol, transported across membranes by channels, exchangers, and pumps that regulate flux across the ER, mitochondrial and plasma membranes. Calcium regulates both rapid events, such as cytoskeleton remodelling or release of vesicle contents, and slower ones, such as transcriptional changes. Moreover, sustained cytosolic calcium elevations can lead to unwanted cellular activation or apoptosis. Calmodulin represents the most significant of the Ca(2+)-binding proteins and is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile novel protein-protein interactions that calmodulin participates in, we probed a high-content recombinant human protein array with fluorophore-labelled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human proteins expressed from a human brain cDNA library. We describe the identification of a high affinity interaction between calmodulin and the single-pass transmembrane proteins STIM1 and STIM2 that localise to the ER. Translocation of STIM1 and STIM2 from the endoplasmic reticulum to the plasma membrane is a key step in store operated calcium entry in the cell.

Profiling the autoantibody (AAb) repertoire in serum has been routinely used for many years for the diagnosis of autoimmune diseases, including rheumatoid arthritis, scleroderma, and lupus. In recent years, AAb profiling of cancers has become a prominent field in oncology research. Protein arrays enable high-throughput screening of clinical samples, characterising the serum profile using low volumes of samples. This chapter describes the use of a protein array comprising 37,200 redundant proteins (containing over 10,000 non-redundant human recombinant proteins) for identification of the proteins bound by the antibodies in human sera using a test set of serum samples. The proteins identified have the potential to be candidate biomarkers. These recombinant proteins are expressed, purified, and robotically spotted on microarrays or chips to facilitate the screening of additional serum samples with the aim of identifying a candidate biomarker or panel of potential biomarkers for applications in disease diagnosis, stage, progression, or response to therapy.

Antibody-based microarrays are a rapidly evolving affinity-proteomic methodology that recently has shown great promise in clinical applications. The resolution of these proteomic analyses is, however, directly related to the number of data-points, i.e. antibodies, included on the array. Currently, this is a key bottleneck because of limited availability of numerous highly characterized antibodies. Here, we present a conceptually new method, denoted global proteome survey, opening up the possibility to probe any proteome in a species-independent manner while still using a limited set of antibodies. We use context-independent-motif-specific antibodies directed against short amino acid motifs, where each motif is present in up to a few hundred different proteins. First, the digested proteome is exposed to these antibodies, whereby motif-containing peptides are enriched, which then are detected and identified by mass spectrometry. In this study, we profiled extracts from human colon tissue, yeast cells lysate, and mouse liver tissue to demonstrate proof-of-concept.

Secretagogin is a hexa EF-hand Ca(2+)-binding protein expressed in neuroendocrine, pancreatic endocrine and retinal cells. The protein has been noted for its expression in specific neuronal subtypes in the support of hierarchical organizing principles in the mammalian brain. Secretagogin has previously been found to interact with SNAP25 involved in Ca(2+)-induced exocytosis. Here, the cellular interaction network of secretagogin has been expanded with nine proteins: SNAP-23, DOC2alpha, ARFGAP2, rootletin, KIF5B, β-tubulin, DDAH-2, ATP-synthase and myeloid leukemia factor 2, based on screening of a high content protein array and validation and quantification of binding with surface plasmon resonance and GST pulldown assays. All targets have association rate constants in the range 10(4)-10(6) M(-1) s(-1), dissociation rate constants in the range 10(-3)-10(-5) s(-1) and equilibrium dissociation constants in the 100 pM to 10 nM range. The novel target SNAP23 is an essential component of the high affinity receptor for the general membrane fusion machinery and an important regulator of transport vesicle docking and fusion. Complementary roles in vesicle trafficking are known for ARFGAP2 and DOC2alpha in regulating fusion of vesicles to membranes, kinesin 5B and tubulin for transport of vesicles in the cell, while rootletin builds up the rootlet believed to function as a scaffold for vesicles. The identification of a discrete network of interacting proteins that mediate secretion and vesicle trafficking suggests a regulatory role for secretagogin in these processes.

Off-target hits of drugs can lead to serious adverse effects or, conversely, to unforeseen alternative medical utility. Selectivity profiling against large panels of potential targets is essential for the drug discovery process to minimize attrition and maximize therapeutic utility. Lately, it has become apparent that drug promiscuity (polypharmacology) goes well beyond target families; therefore, lowering the profiling costs and expanding the coverage of targets is an industry-wide challenge to improve predictions. Here, we review current and promising drug profiling alternatives and commercial solutions in these exciting emerging fields.

Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labeled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human neural proteins from a human brain cDNA library. We designed a screen to find high affinity (K(D) < or = 1 microm) binding partners of calmodulin and identified 76 human proteins from all intracellular compartments of which 72 are novel. We measured the binding kinetics of 74 targets with calmodulin using a high throughput surface plasmon resonance assay. Most of the novel calmodulin-target complexes identified have low dissociation rates (k(off) < or = 10(-3) s(-1)) and high affinity (K(D) </= 1 mum), consistent with the design of the screen. Many of the identified proteins are known to assemble in neural tissue, forming assemblies such as the spectrin scaffold and the postsynaptic density. We developed a microarray of the identified target proteins with which we can characterize the biochemistry of calmodulin for all targets in parallel. Four novel targets were verified in neural cells by co-immunoprecipitation, and four were selected for exploration of the calmodulin-binding regions. Using synthetic peptides and isothermal titration calorimetry, calmodulin binding motifs were identified in the potassium voltage-gated channel Kv6.1 (residues 474-493), calmodulin kinase-like vesicle-associated protein (residues 302-316), EF-hand domain family member A2 (residues 202-216), and phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (residues 400-415).

Current clinical, laboratory or radiological parameters cannot accurately diagnose or predict disease outcomes in a range of autoimmune disorders. Biomarkers which can diagnose at an earlier time point, predict outcome or help guide therapeutic strategies in autoimmune diseases could improve clinical management of this broad group of debilitating disorders. Additionally, there is a growing need for a deeper understanding of multi-factorial autoimmune disorders. Proteomic platforms offering a multiplex approach are more likely to reflect the complexity of autoimmune disease processes. Findings from proteomic based studies of three distinct autoimmune diseases are presented and strategies compared. It is the authors' view that such approaches are likely to be fruitful in the movement of autoimmune disease treatment away from reactive decisions and towards a preventative stand point.

Translocation of STIM1 and STIM2 from the endoplasmic reticulum to the plasma membrane is a key step in store-operated calcium entry in the cell. We show by isothermal titration calorimetry that calmodulin binds in a calcium-dependent manner to the polybasic C-termini of STIM1 and STIM2, a region critical for their translocation to the plasma membrane ( K D < or = 1 microM in calcium). HSQC NMR spectroscopy shows this interaction is in the fast exchange regime. By binding STIM1 and STIM2, calmodulin may regulate store refilling, thereby ensuring the maintenance of its own action in intracellular signaling.

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination. NOTE: In the version of the article originally published, Manfred Koegl’s name was misspelled. Additionally, Zoltan Konthur's affiliation was listed incorrectly; it should be Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany. These errors have been corrected in the HTML and PDF versions of the article.

High density DNA microarray technology has played a key role in the analysis of whole genomes and their gene expression patterns. The ability to study many thousands of individual genes using oligonucleotide or cDNA arrays is now very widespread, with its uses ranging from the profiling of gene expression patterns in whole organisms or tissues to the comparison of healthy and pathological samples. However, despite the success of DNA microarrays, it is obvious that the biological function is executed by biomolecules such as proteins. Protein biochips are therefore emerging to follow DNA microarrays as a possible screening tool. We will present different types of biochips including protein and antibody arrays, as well as carbohydrate, peptide and living cell arrays. Recent progress and current bottlenecks in high-throughput generation of chip content, surface chemistry, molecule attachment, detection methods, and applications in the proteomics field and in drug discovery will be discussed.

Dilated cardiomyopathy (DCM) is a myocardial disease characterized by progressive depression of myocardial contractile function and ventricular dilatation. Thirty percent of DCM patients belong to the inherited genetic form; the rest may be idiopathic, viral, autoimmune, or immune-mediated associated with a viral infection. Disturbances in humoral and cellular immunity have been described in cases of myocarditis and DCM. A number of autoantibodies against cardiac cell proteins have been identified in DCM. In this study, we have profiled the autoantibody repertoire of plasma from DCM patients against a human protein array consisting of 37,200 redundant, recombinant human proteins and performed qualitative and quantitative validation of these putative autoantigens on protein microarrays to identify novel putative DCM specific autoantigens. In addition to analyzing the whole IgG autoantibody repertoire, we have also analyzed the IgG3 antibody repertoire in the plasma samples to study the characteristics of IgG3 subclass antibodies. By combining screening of a protein expression library with protein microarray technology, we have detected 26 proteins identified by the IgG antibody repertoire and 6 proteins bound by the IgG3 subclass. Several of these autoantibodies found in plasma of DCM patients, such as the autoantibody against the Kv channel-interacting protein, are associated with heart failure.

During the course of raising monoclonal antibodies, reagents are often produced that are not directed against the immunising antigen. These may pass unnoticed unless a screening step based on immunostaining of human tissue is included. Many of these reagents are auto-antibodies, often directed against intracellular targets (e.g. nuclear components), and the process of hybridoma production serves to "rescue" these self-reactive B cell clones. Such unexpected antibodies of this sort may prove of interest if their distribution is analysed on normal tissues and within the spectrum of leukaemia/lymphoma samples showing specific patterns of staining. However, before they can be used diagnostically or therapeutically, their target molecule needs to be identified, and this can be technically demanding. We describe a novel approach that can be used to define the targets of new monoclonal antibodies to intracellular molecules. The technique involves screening against protein arrays comprising thousands of recomb

Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid synthase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, histones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-activated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance.

Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases.

Protein array technology is becoming an increasingly important tool in the drive toward proteome-scale analysis of protein activity and interactions. Presently, this technology compliments the more traditional methods for proteomic analysis, including two-dimensional gel electrophoresis/chromatography and mass spectrometry. While the task of producing a “whole-proteome” chip, containing active proteins, is a daunting one, current protein and antibody arrays represent the first steps toward that goal. In this review, we discuss current approaches for the generation of protein arrays, and their applications, including their use in the study of protein–protein, protein–nucleic acid, enzyme–substrate, and so on, interactions. Potential applications of protein arrays in interaction screening, such as compound–protein interactions are also discussed.

Protein array technology is becoming an increasingly important tool in the drive toward proteome-scale analysis of protein activity and interactions. Presently, this technology compliments the more traditional methods for proteomic analysis, including two-dimensional gel electrophoresis/chromatography and mass spectrometry. While the task of producing a “whole-proteome” chip, containing active proteins, is a daunting one, current protein and antibody arrays represent the first steps toward that goal. In this review, we discuss current approaches for the generation of protein arrays, and their applications, including their use in the study of protein–protein, protein–nucleic acid, enzyme–substrate, and so on, interactions. Potential applications of protein arrays in interaction screening, such as compound–protein interactions are also discussed.

The human genome has been sequenced and the challenges of understanding the function of the newly discovered genes have been addressed. High-throughput technologies such as DNA microarrays have been developed for the profiling of gene expression patterns in whole organisms or tissues. Protein arrays are emerging to follow DNA chips as possible screening tools. Here, we review the generation and application of microarray technology to obtain more information on the regulation of proteins, their biochemical functions and their potential interaction partners. Already, a large variety of assays based on antibody-antigen interactions exists. In addition, the medical relevance of protein arrays will be discussed.

Neisseria meningitidis is the most common cause of meningitis and causes epidemic outbreaks. One trait of N. meningitidis, which is associated with most of the currently recognized virulence determinants, is the presence of phase-variable genes that are suspected to enhance its ability to cause an invasive disease. To detect the immune responses to phase-variable expressed proteins, we applied protein microarray technology for the screening of meningitis patient sera. We amplified all 102 known phase-variable genes from N. meningitidis serogroup B strain MC58 by polymerase chain reaction and subcloned them for expression in Escherichia coli. With this approach, we were able to express and purify 67 recombinant proteins representing 66% of the annotated genes. These were spotted robotically onto coated glass slides to generate protein microarrays, which were screened using 20 sera of patients suffering from meningitis, as well as healthy controls. From these screening experiments, 47 proteins emerged as immunogenic, exhibiting a variable degree of seroreactivity with some of the patient sera. Nine proteins elicited an immune response in more than three patients, with one of them, the phase-variable opacity protein OpaV (NMB0442), showing responses in 11 patient sera. This is the first time that protein microarray technology has been applied for the investigation of genetic phase variation in pathogens. The identification of disease-specific proteins is a significant target in biomedical research, as such proteins may have medical, diagnostic, and commercial potential as disease markers.

The mouse is the premier genetic model organism for the study of disease and development. We describe the establishment of a mouse T helper cell type 1 (T(H)1) protein expression library that provides direct access to thousands of recombinant mouse proteins, in particular those associated with immune responses. The advantage of a system based on the combination of large cDNA expression libraries with microarray technology is the direct connection of the DNA sequence information from a particular clone to its recombinant, expressed protein. We have generated a mouse T(H)1 expression cDNA library and used protein arrays of this library to characterize the specificity and cross-reactivity of antibodies. Additionally, we have profiled the autoantibody repertoire in serum of a mouse model for systemic lupus erythematosus on these protein arrays and validated the putative autoantigens on highly sensitive protein microarrays.

Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction mod- ules in eucaryotes, including plants. These protein phos- phorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identify- ing potential substrates for Arabidopsis thaliana mitogen- activated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of pro- tein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were ob- tained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid syn- thase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological rele- vance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, his- tones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-ac- tivated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance. Molecular & Cellular Proteomics 4:1558-1568, 2005.

Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown, as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task to identify potential substrates for Arabidopsis thaliana mitogen-activated protein kinases 3 (MPK3) and 6 (MPK6), which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomics method allowing high-throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-Aminocyclopropane-1-carboxylic acid synthase-6 (ACS-6) was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore, we revealed transcription factors, transcription regulators, splicing factors, receptors, histones and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach allows to shortlist candidate substrates of MAP kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance

A critical role for the conserved alpha-integrin cytoplasmic motif, KVGFFKR, is recognized in the regulation of activation of the platelet integrin alpha(IIb)beta(3). To understand the molecular mechanisms of this regulation, we sought to determine the nature of the protein interactions with this cytoplasmic motif. We used a tagged synthetic peptide, biotin-KVGFFKR, to probe a high density protein expression array (37,200 recombinant human proteins) for high affinity interactions. A number of potential integrin-binding proteins were identified. One such protein, a chloride channel regulatory protein, ICln, was characterized further because its affinity for the integrin peptide was highest as was its expression in platelets. We verified the presence of ICln in human platelets by PCR, Western blots, immunohistochemistry, and its co-association with alpha(IIb)beta(3) by surface plasmon resonance. The affinity of this interaction was 82.2 +/- 24.4 nm in a cell free assay. ICln co-immunoprecipitates with alpha(IIb)beta(3) in platelet lysates demonstrating that this interaction is physiologically relevant. Furthermore, immobilized KVGFFKR peptides, but not control KAAAAAR peptides, specifically extract ICln from platelet lysates. Acyclovir (100 microm to 5 mm), a pharmacological inhibitor of the ICln chloride channel, specifically inhibits integrin activation (PAC-1 expression) and platelet aggregation without affecting CD62 P expression confirming a specific role for ICln in integrin activation. In parallel, a cell-permeable peptide corresponding to the potential integrin-recognition domain on ICln (AKFEEE, 10-100 microm) also inhibits platelet function. Thus, we have identified, verified, and characterized a novel functional interaction between the platelet integrin and ICln, in the platelet membrane.

Automation is the key approach for genomewide and proteomewide screening of function and interaction. Especially for proteomics, antibody microarrays are a useful tool for massive parallel profiling of complex samples. To meet the requirements of antibody microarrays and to obtain a great variety of antibodies, new technologies such as phage display have partly replaced the classical hybridoma method. While the selection process for phage-displayed antibody fragments itself has been automated, the bottleneck was shifted further downstream to the identification of monoclonal binders obtained from the selections. Here, we present a new approach to reduce time, material, and waste to extend automation beyond the selection process by application of conventional microarray machinery. We were able to express recombinant antibody fragments in a single inoculation and expression step and subjected them without purification directly to an automated high-throughput screening procedure based on the multiple spotting technique (MIST). While obtaining comparable sensitivities to enzyme-linked immunosorbent assays, we minimized manual interaction steps and streamlined the technique to be accessible within the automated selection procedure.

The expression and characterization of large protein libraries requires high-throughput tools for rapid and cost-effective expression and screening. A promising tool to meet these requirements is miniaturized high-density plates in chip format, consisting of an array of wells with submicroliter volumes. Here, we show the combination of nanowell chip technology and cell-free transcription and translation of proteins. Using piezoelectric dispensers, we transferred proteins into nanowells down to volumes of 100 nL and successfully detected fluorescence using confocal laser scanning. Moreover, we showed cell-free expression of proteins on a nanoliter scale using commercially available coupled transcription and translation systems. To reduce costs, we demonstrated the feasibility of diluting the coupled in vitro transcription and translation mix prior to expression. Additionally, we present an enzymatic inhibition assay in nanowells to anticipate further applications, such as the high-throughput screening of drug candidates or the identification of novel enzymes for biotechnology.

Proteins secreted by activated platelets can adhere to the vessel wall and promote the development of atherosclerosis and thrombosis. Despite this biologic significance, however, the complement of proteins comprising the platelet releasate is largely unknown. Using a proteomics approach, we have identified more than 300 proteins released by human platelets following thrombin activation. Many of the proteins identified were not previously attributed to platelets, including secretogranin III, a potential monocyte chemoattractant precursor; cyclophilin A, a vascular smooth muscle cell growth factor; calumenin, an inhibitor of the vitamin K epoxide reductase-warfarin interaction, as well as proteins of unknown function that map to expressed sequence tags. Secretogranin III, cyclophilin A, and calumenin were confirmed to localize in platelets and to be released upon activation. Furthermore, while absent in normal vasculature, they were identified in human atherosclerotic lesions. Therefore, these and other proteins released from platelets may contribute to atherosclerosis and to the thrombosis that complicates the disease. Moreover, as soluble extracellular proteins, they may prove suitable as novel therapeutic targets.

There is burgeoning interest in protein microarrays, but a source of thousands of nonredundant, purified proteins was not previously available. Here we show a glass chip containing 2413 nonredundant purified human fusion proteins on a polymer surface, where densities up to 1600 proteins/cm(2) on a microscope slide can be realized. In addition, the polymer coating of the glass slide enables screening of protein interactions under nondenaturing conditions. Such screenings require only 200-microl sample volumes, illustrating their potential for high-throughput applications. Here we demonstrate two applications: the characterization of antibody binding, specificity, and cross-reactivity; and profiling the antibody repertoire in body fluids, such as serum from patients with autoimmune diseases. For the first application, we have incubated these protein chips with anti-RGSHis(6), anti-GAPDH, and anti-HSP90beta antibodies. In an initial proof of principle study for the second application, we have screened serum from alopecia and arthritis patients. With analysis of large sample numbers, identification of disease-associated proteins to generate novel diagnostic markers may be possible.

The enzyme-linked immunosorbent assay (ELISA) is typically applied in the format of microtiter plates. To increase throughput and reduce consumption of precious samples, efforts have been made to transfer ELISA to the microchip format using conventional microarrays, microfluidic systems, and chips bearing microwells. However, all three formats lack the possibility to screen several analytes on several immobilized binders at a time or require complicated liquid handling, surface modifications, and additional equipment. Here, we describe an immunoassay performed on a standard microscope slide without the requirement for wells or tubes to separate the samples using standard surfaces and machinery already available for microarray technology. The new multiple spotting technique (MIST) comprises immobilization of a binder onto a surface and subsequent spotting of the second compound on the same spot, on top of the immobilized binder. We show that the analytes bind their ligands immediately within the confined space of separate droplets on the chip surface, thereby eliminating the need for extra incubation time. We illustrate the feasibility of the new technique by spotting dilution rows of proteins or monoclonal and polyclonal antibodies on top of their immobilized binders. Moreover, we demonstrate specificity by applying a mixture of antibodies in a multiplex format and demonstrate that the technique is compatible with conventional microarray protocols, such as total incubation. Finally, we indicate that the technique is capable of quantifying as little as 400 zmol (240,000 molecules) of analyte.

The performance of protein and antibody microarrays is dependent on various factors, one of which is the use of an appropriate microarray surface for the immobilisation of either protein or antibody samples. We have investigated the properties of seven new surfaces in the context of both protein and antibody microarray technology. We have demonstrated the functionality of all new slide coatings and investigated the mean signal to spotted concentration ratio, determined detection limits and calculated coefficients of variation. Moreover, new concepts for slide coatings such as dendrimer and poly(ethylene glycol)-epoxy slides were evaluated and improved qualities of novel slide surfaces were observed. Optimal slide coatings for antibody and protein chips were proposed and the requirements for both technologies were discussed.

Arraying technologies have shown the way to smaller sample volumes, more efficient analyses and higher throughput. Proteomics is a field, which has grown in significance in the last five years. This review outlines recent developments in protein arrays and their applications in proteomics, and discusses the requirements, current limitations and the potential and future perspectives of the technology.

Starting with the discovery of penicillin, other antibiotics, and insulin, the quest for understanding and use of biological systems, i. e. , microorganisms and ani mal tissue, for the production of value products has lead to a dramatic increase in microbiological and bioengineering research in the last decades. Chemical and pharmaceutical companies quickly realized the huge commercial potential of these bioproducts and have spent millions of US dollars on R &D as well as on a build up of production facilities. Although there was limited knowledge about the cell's molecular mechanisms, which are the basis for the formation of the desired products, products from fermentation and extraction of biological matrices were a success right from the start. R&D projects within industry and academia on the continuous improvement of production processes, especially microbial productivity and down stream processing, allowed a fast return of investment and secured competitiveness in the market. Whereas the focus of such research projects was mainly on the discovery of strains with higher pro ductivity for the product of interest, e. g. , antibiotics, a lot of expertise and knowledge was generated allowing the use of biotechnological products and processes outside the pharmaceutical arena. The tremendous increase in knowl edge and the technological developments in microbial genetics where driven by these research projects and, accompanied with the advancements in nucleotide chemistry leading to a much better understanding of intracellular processes, served as a basis for modern molecular biology and recombinant biotech nology.

With the advent of protein and antibody microarray technology several different coatings and protocols have been published, which may be broadly divided into two types: gel-coated surfaces and plain non-gel-coated glass or plastic surfaces, some with chemical groups attached. We have screened 11 different array surfaces of both types and compared them with respect to their detection limit, inter- and intrachip variation, and storage characteristics. Five different antibodies were immobilized onto each type of microarray support, with total protein concentrations ranging from 40 fmol to 25 amol per spot. From these results, it was seen that some antibodies were more suited for use on antibody arrays. All measurements were performed in quadruplicate, and the results revealed high signal uniformity and reproducibility of most plain glass and plastic slides. Lower detection limits were obtained with polyacrylamide-coated slides, making them more suitable for the detection of very low concentrations of antigen. All microarray coatings could be stored for a period of 8 weeks; however, improved results were seen after 2 weeks of storage. In conclusion, the results indicate the need to test each antibody to be used on an antibody array and to select the microarray coating based on experimental requirements.

With the genome sequences of several organisms now in public databases, the scientific community has realized that it is time to prepare for the next step: the understanding of biological systems or systems biology. Whereas genes contain the information for life, the encoded proteins and RNAs fulfill nearly all the functions, from replication to regulation. At present, there is a perceived demand for high-throughput and parallel analytical devices as research tools in systems biology, and, in addition, for new concepts to extract knowledge and value from these data. Protein biochips will play a decisive role in meeting this need in the future.

Heterologous gene expression is often treated empirically and a number of host organisms are systematically tested. Early successes in the expression of recombinant proteins were achieved using the well-studied bacterium Escherichia coli (1). This prokaryotic expression system is simple to handle, costeffective, and produces large amounts of heterologous proteins (2). However, when expressing many different genes, especially eukaryotic genes, this often leads to the production of aggregated and denatured proteins, localized in inclusion bodies, and only a small fraction matures into the desired native form (3–5). Alternatively, eukaryotic expression systems have been developed to obtain more soluble protein, which in addition, may undergo some eukaryotic post-translational modifications. Yeast expression systems, including the methylotrophic yeast Pichia pastoris, have been used over the last few years as powerful expression systems for a number of heterologous genes (6–10). However, both eukaryotic and prokaryotic systems have their advantages and disadvantages. Therefore, choosing a suitable expression system for a particular protein is a compromise, depending primarily on the properties of the protein, the amounts required, and its intended purpose.

The molecular diversity of receptors in human blood vessels remains largely unexplored. We developed a selection method in which peptides that home to specific vascular beds are identified after administration of a peptide library. Here we report the first in vivo screening of a peptide library in a patient. We surveyed 47,160 motifs that localized to different organs. This large-scale screening indicates that the tissue distribution of circulating peptides is nonrandom. High-throughput analysis of the motifs revealed similarities to ligands for differentially expressed cell-surface proteins, and a candidate ligand-receptor pair was validated. These data represent a step toward the construction of a molecular map of human vasculature and may have broad implications for the development of targeted therapies.

We describe the technical feasibility and methodology to characterize a protein by a minimal set of structural information generated by matrix assisted laser desorption/ionization (MALDI)-mass spectrometry, termed a "minimal protein Identifier" (MPI). MPIs can be determined for proteins from two-dimensional gels and recombinant proteins and can be used to compare and identify proteins from these sources.

A major interest of Jörg is the establishment and sensible use of high-throughput analysis processes — with some emphasis on array-based assays — for the understanding and evaluation of the complex molecular interactions in living organisms.

We developed a high-throughput technique for the generation of cDNA libraries in the yeast Saccharomyces cerevisiae which enables the selection of cloned cDNA inserts containing open reading frames (ORFs). For direct screening of random-primed cDNA libraries, we have constructed a yeast shuttle/expression vector, the so-called ORF vector pYEXTSH3, which allows the enriched growth of protein expression clones. The selection system is based on the HIS3 marker gene fused to the C terminus of the cDNA insert. The cDNAs cloned in-frame result in histidine prototrophic yeast cells growing on minimal medium, whereas clones bearing the vector without insert or out-of-frame inserts should not grow on this medium. A randomly primed cDNA library from human fetal brain tissue was cloned in this novel vector, and using robot technology the selected clones were arrayed in microtiter plates and were analyzed by sequencing and for protein expression. In the constructed cDNA expression library, about 60% of clones bear an insert in the correct reading frame. In comparison to unselected libraries it was possible to increase the clones with inserts in the correct reading frame more than fourfold, from 14% to 60%. With the expression system described here, we could avoid time-consuming and costly techniques for identification of clones expressing protein by using antibody screening on high-density filters and subsequently rearraying the selected clones in a new "daughter" library. The advantage of this ORF vector is that, in a one-step screening procedure, it allows the generation of expression libraries enriched for clones with correct reading frames as sources of recombinant proteins.

The human genome is sequenced and the challenges of understanding the function of the newly discovered genes have been addressed. For this purpose, high-throughput technologies have been developed that allow the monitoring of gene activity at the transcriptional level by analysis of complex expression patterns of a specific tissue. Differential gene expression can be most efficiently monitored by oligonucleotide or cDNA hybridization on DNA arrays. Recently, protein arrays are emerging to follow DNA chips as a tool to profile protein products encoded by globally or differentially expressed cDNA clones. Array technology was enabled by the development of devices that could array biological samples at high density with high precision onto immobilizing surfaces, ranging from the classic microtiter plate to new chip-sized supports. In addition, the introduction of automated technology to the protein level involves the simultaneous expression of a large number of cDNA clones in an appropriate vector and expression system, allowing the specific detection and purification of all the recombinant proteins. With the ordered arrangement of recombinantly expressed proteins, a direct link to the corresponding DNA sequence information is possible and consequently, clone libraries become amenable to be integrated in a database including all steps from DNA sequencing to functional assays of the translated gene product. Here, we review the generation and application of microarray technology as a highly parallel approach to obtain more information on the regulation of proteins, their biochemical function and potential interaction partners. Already, a large variety of assays based on antibody-antigen interaction exists and in addition, the medical relevance of protein arrays will be discussed. Also, further applications such as protein-DNA, protein-RNA and protein-substrate interactions will be presented, since initial studies on immobilized proteins were reported. Proteomics is an emerging field to profile protein repertoires. Because there is no reliable correlation between gene activity monitored by genomic studies and cellular protein abundance, application of protein arrays will link both genomics and proteomics.

Many new gene products are being discovered by large-scale genomics and proteomics strategies, the challenge is now to develop high throughput approaches to systematically analyse these proteins and to assign a biological function to them. Having access to these gene products as recombinantly expressed proteins, would allow them to be robotically arrayed to generate protein chips. Other applications include using these proteins for the generation of specific antibodies, which can also be arrayed to produce antibody chips. The availability of such protein and antibody arrays would facilitate the simultaneous analysis of thousands of interactions within a single experiment. This chapter will focus on current strategies used to generate protein and antibody arrays and their current applications in biological research, medicine and diagnostics. The shortcomings of these approaches, the developments required, as well as the potential applications of protein and antibody arrays will be discussed.

We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning.

High-density DNA and protein arrays, also known as microarrays or chips, are miniaturised devices, which comprise small flat surfaces, onto which are ordered arrangements of individual samples are positioned, enabling their parallel analysis, which can be used for biological experimentation. High density array technologies allow the use of smaller sample volumes, more efficient analyses and higher throughput. This review outlines recent developments in the generation of high density DNA and protein arrays and their potential applications in the areas such as diagnostics and proteomics.

The array format has revolutionised biomedical experimentation and diagnostics, enabling ordered high-throughput analysis. During the past decade, classic solid phase substrates, such as microtitre plates, membrane filters and microscopic slides, were turned into high-density, chip-like structures. The concept of the arrayed library was central to this development which now extends from DNA to protein. The new and versatile protein array technology allows high-throughput screening for gene expression and molecular interactions. As a major platform for functional genomics, it is already on its way into medical diagnostics.

High-density DNA and protein arrays are small flat surfaces that allow the simultaneous analysis of thousands of molecular parameters within a single experiment. DNA array technologies have resulted in smaller sample volumes, more efficient analyses and higher throughput. As proteins are more complex and more diverse compared with nucleic acids, development of similar platforms for proteomics has proved difficult. This review outlines current techniques used in the generation and applications of high-density protein arrays, with emphasis on recent developments and applications in proteomics.

The present invention relates to a method for identifying and/or characterizing a (poly)peptide comprising: (a) analyzing a peptide map of said (poly)peptide, comprising at least 1 peptide, and its peptide primary structure fingerprint by mass spectrometry; and (b) comparing data obtained in step (a) with a reference (poly)peptide database, said database comprising mass spectrometric data of peptide maps, comprising at least 1 peptide, and of its peptide primary structure fingerprint, of a (poly)peptide or of a variety of (poly)peptides.

For any attempt to understand the biology of an organism the incorporation of a cDNA-based approach is unavoidable, because it is a major approach to studying gene function. The complete sequence of the genome alone is not sufficient to understand any organism; its gene regulation, expression, splice variation, posttranslational modifications, and proteinprotein interactions all need to be addressed. Because the majority of vertebrate genes have probably been identified as ESTs44,52 the next stage of the Human Genome Project is attributing functional information to these sequences. In most cases hybridization-based approaches on arrayed pieces of DNA represent the most efficient way to study the expression level and splicing of a gene in a given tissue. Similar technology, now being applied at the protein level using protein expression libraries, high-density protein membranes, and antibody screening, should allow studies of protein localization and modifications.

We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6 sequence of expressed proteins (RGS·His antibody, Qiagen) detected 20% of the library as putative expression clones. Two example genes, GAPDH and HSP90α, were identified on high-density filters using DNA probes and antibodies against their proteins.

The GTP cyclohydrolase I (GTP-CH) gene of the cellular slime mould Dictyostelium discoideum has been cloned and sequenced. The 855 bp cDNA of this gene contains the open reading frame (ORF) encoding 232 amino acids with a predicted molecular mass of approx. 26 kDa. Southern blot analysis indicated the presence of a single gene for GTP-CH in Dictyostelium. PCR amplification of the ORF from chromosomal DNA and sequencing showed the existence of a 101 bp intron in the GTP-CH gene of Dictyostelium discoideum. The amino acid sequence has 47% and 49% positional identity to those of the human and yeast enzymes respectively. Most of the sequence variation between species is located in the N-terminal part of the protein. The overall identity with the E. coli protein is markedly lower. The enzyme was expressed in E. coli and purified as a 68 kDa fusion protein with the maltose-binding protein of E. coli. GTP-CH of Dictyostelium is heat-stable and showed maximal activity at 60 degrees C. The Km value for GTP is 50 microM.

To examine the cause of altered follicular fluid steroid levels and lower in vitro fertilization rate observed in infertile women with minor endometriosis, we have compared the production of estradiol (aromatase activity) and progesterone of freshly isolated granulosa cells (3h. incubation) from such women and a control group with tubal or unexplained infertility, having IVF during unstimulated or gonadotropin-stimulated cycles. As previously observed, mature oocytes from women with endometriosis had a reduced fertilization and cleavage rate in vitro in unstimulated cycles (19/37[51%] vs. 69/94[73%], p < 0.05) and stimulated cycles (20/37[57%] vs. 32/39[82%], p < 0.01). Median [95%CI] basal aromatase activity was lower in endometriosis compared with control in unstimulated cycles (2.84[2.03-3.49] pmol E2/10(3) cells/3h, n = 31 vs. 3.63[2.72-3.49], n = 55, p = 0.057) and stimulated cycles (0.31[0.16-0.50], n = 14 vs. 0.99[0.70-1.52], n = 20, p < 0.001). Progesterone production followed a similar pattern in...