Protein microarray on-demand: a novel protein microarray system

We describe a novel, simple and low-cost protein microarray strategy wherein the microarrays are generated by printing expression ready plasmid DNAs onto slides that can be converted into protein arrays on-demand. The printed expression plasmids serve dual purposes as they not only direct the synthesis of the protein of interest; they also serve to capture the newly synthesized proteins through a high affinity DNA-protein interaction. To accomplish this we have exploited the high-affinity binding (approximately 3-7 x 10 (-13) M) of E. coli Tus protein to Ter, a 20 bp DNA sequence involved in the regulation of E. coli DNA replication. In our system, each protein of interest is synthesized as a Tus fusion protein and each expression construct directing the protein synthesis contains embedded Ter DNA sequence. The embedded Ter sequence functions as a capture reagent for the newly synthesized Tus fusion protein. This "all DNA" microarray can be converted to a protein microarray on-demand without need for any additional capture reagent.     

RCA combined nanoparticle-based optical detection technique for protein microarray: a novel approach

Developing a readily available biosensor with excellent performances is the main focus of many research groups. Recently, major breakthroughs in miniaturization of molecular analysis have produced DNA and protein microarrays. The aim of our group is to develop a sensitive technique for analyzing signals on protein microarray by applying the surface plasmon resonance (SPR) method. This new detection technique for specific molecular binding utilizes rolling circles amplification (RCA) post-signal processing method [Nat. Genet. 19 (1998) 225-232] and optical visualization by nanogold particle-labeled molecules on a micro-structured chip surface. By covalent bonding of the RCA primer to the detection antibody guarantees that the linkage between the analyte and the amplified RCA product is maintained during the assay. Experimental results show that RCA has significantly enhanced sensitivity compared to conventional methods. This combination of an easily detectable signal with chip technology should have the potential to become a successful commercial application.     

A sol-gel-integrated protein array system for affinity analysis of aptamer-target protein interaction

A sol-gel microarray system was developed for a protein interaction assay with high activity. Comparing to 2-dimensional microarray surfaces, sol-gel can offer a more dynamic and broad range for proteins. In the present study, this sol-gel-integrated protein array was used in binding affinity analysis for aptamers. Six RNA aptamers and their target protein, yeast TBP (TATA-binding protein), were used to evaluate this method. A TBP-containing sol-gel mixture was spotted using a dispensing workstation under high-humidity conditions and each Cy-3-labeled aptamer was incubated. The dissociation constants (K(d)) were calculated by plotting the fluorescent intensity of the bound aptamers as a function of the TBP concentrations. The K(d) value of the control aptamer was found to be 8?nM, which agrees well with the values obtained using the conventional method, electric mobility shift assay. The sol-gel-based binding affinity measurements fit well with conventional binding affinity measurements, suggesting their possible use as an alternative to the conventional method. In addition, aptamer affinity measurements by the sol-gel-integrated protein chip make it possible to develop a simple high-throughput affinity method for screening high-affinity aptamers.     

A novel strategy for the targeted analysis of protein and peptide metabolites

In many biological applications such as epitope discovery or drug metabolism studies, the detection of naturally processed exogenous proteins (e.g. vaccines or peptide therapeutics) and their metabolites is frequently complicated by the presence of a complex endogenous mixture of closely related or even identical compounds. We describe a method that incorporates stable isotope labelling of the protein of interest, allowing the selective screening of the intact molecule and all metabolites using a modified precursor ion scan. This method involves monitoring the low-molecular-weight fragment ions produced during MS/MS that distinguish isotopically labelled peptides from related endogenous compounds. All isotopically labelled peptides can be selected using this method. The technique makes no assumptions about the processed or post-translational state of the peptide, and hence can selectively screen out modified peptides that would otherwise be missed by single reaction monitoring approaches. This method does not replace single reaction monitoring or regular precursor scanning techniques; instead, it is a method that can be used when the assumptions required for the former two techniques cannot be predicted. The potential for this technique to be used in metabolism and pharmacokinetic experiments is discussed with specific examples looking at the metabolism of α-synuclein in serum and the brain.     

Zeptosens' protein microarrays: a novel high performance microarray platform for low abundance protein analysis

Protein microarrays are considered an enabling technology, which will significantly expand the scope of current protein expression and protein interaction analysis. Current technologies, such as two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry, allowing the identification of biologically relevant proteins, have a high resolving power, but also considerable limitations. As was demonstrated by Gygi et al. (Proc. Nat. Acad. Sci. USA 2000,97, 9390-9395), most spots in 2-DE, observed from whole cell extracts, are from high abundance proteins, whereas low abundance proteins, such as signaling molecules or kinases, are only poorly represented. Protein microarrays are expected to significantly expedite the discovery of new markers and targets of pharmaceutical interest, and to have the potential for high-throughput applications. Key factors to reach this goal are: high read-out sensitivity for quantification also of low abundance proteins, functional analysis of proteins, short assay analysis times, ease of handling and the ability to integrate a variety of different targets and new assays. Zeptosens has developed a revolutionary new bioanalytical system based on the proprietary planar waveguide technology which allows us to perform multiplexed, quantitative biomolecular interaction analysis with highest sensitivity in a microarray format upon utilizing the specific advantages of the evanescent field fluorescence detection. The analytical system, comprising an ultrasensitive fluorescence reader and microarray chips with integrated microfluidics, enables the user to generate a multitude of high fidelity data in applications such as protein expression profiling or investigating protein-protein interactions. In this paper, the important factors for developing high performance protein microarray systems, especially for targeting low abundant messengers of relevant biological information, will be discussed and the performance of the system will be demonstrated in experimental examples.     

A novel system for large-scale gene expression analysis: bacterial colonies array

In the present work, we report the use of bacterial colonies to optimize macroarray technique. The devised system is significantly cheaper than other methods available to detect large-scale differential gene expression. Recombinant Escherichia coli clones containing plasmid-encoded copies of 4,608 individual expressed sequence tag (ESTs) were robotically spotted onto nylon membranes that were incubated for 6 and 12 h to allow the bacteria to grow and, consequently, amplify the cloned ESTs. The membranes were then hybridized with a beta-lactamase gene specific probe from the recombinant plasmid and, subsequently, phosphorimaged to quantify the microbial cells. Variance analysis demonstrated that the spot hybridization signal intensity was similar for 3,954 ESTs (85.8%) after 6 h of bacterial growth. Membranes spotted with bacteria colonies grown for 12 h had 4,017 ESTs (87.2%) with comparable signal intensity but the signal to noise ratio was fivefold higher. Taken together, the results of this study indicate that it is possible to investigate large-scale gene expression using macroarrays based on bacterial colonies grown for 6 h onto membranes.     

A quantitative peptide array for evaluation of protein kinase activity

Peptide array, which is known as an emerging technology, has been developed for identification of protein kinase activity. For this purpose, the ability of quantitative analysis is very important because the absolute change in protein kinase activity is critical for the determination of cellular function. Here we report an original type of peptide array for quantitative evaluation of protein kinase activity by fluorescence imaging. We used the peptide array for the quantitative evaluation of the nonreceptor tyrosine kinase c-Src activity as a model for detecting protein kinase activities. By using positive and negative control peptides, we obtained the actual ratio of tyrosine phosphorylation of substrate peptide not only by purified c-Src but also by c-Src in cell lysate. In addition, the experimental approach provided simple immobilization of peptide. Our sensitive, specific, and high-throughput peptide array can be used for quantitative evaluation of kinase activity and potentially can be applied to drug discovery and screening.     

Fluorescent "Turn-on" system utilizing a quencher-conjugated peptide for specific protein labeling of living cells

A specific protein fluorescent labeling method has been used as a tool for bio-imaging in living cells. We developed a novel system of switching “fluorescent turn on” by the recognition of a fluorescent probe to a hexahistidine-tagged (His-tag) protein. The tetramethyl rhodamine bearing three nitrilotriacetic acids, which was used as a fluorescent probe to target a His-tagged protein, formed a reversible complex with the quencher, (Dabcyl)-conjugated oligohistidines, in the homogeneous solution, causing fluorescence of the fluorophore to be quenched. The complex when applied to living cells (COS-7) expressing His-tagged proteins on the cell surface caused the quencher-conjugated oligohistidines to be dissociated from the complex by specific binding of the fluorescent probe to the tagged protein, resulting in the fluorescent emission. The complex that did not participate in the binding event remained in the quenched state to maintain a low level of background fluorescence.     

A novel real-time driving fatigue detection system based on wireless dry EEG

Development of techniques for detection of mental fatigue has varied applications in areas where sustaining attention is of critical importance like security and transportation. The objective of this study is to develop a novel real-time driving fatigue detection methodology based on dry Electroencephalographic (EEG) signals. The study has employed two methods in the online detection of mental fatigue: power spectrum density (PSD) and sample entropy (SE). The wavelet packets transform (WPT) method was utilized to obtain the \(\theta \) (4–7 Hz), \(\alpha \) (8–12 Hz) and \(\beta \) (13–30 Hz) bands frequency components for calculating corresponding PSD of the selected channels. In order to improve the fatigue detection performance, the system was individually calibrated for each subject in terms of fatigue-sensitive channels selection. Two fatigue-related indexes: (\(\theta +\alpha \))/\(\beta \) and \(\theta \)/\(\beta \) were computed and then fused into an integrated metric to predict the degree of driving fatigue. In the case of SE extraction, the mean of SE averaged across two EEG channels (‘O1h’ and ‘O2h’) was used for fatigue detection. Ten healthy subjects participated in our study and each of them performed two sessions of simulated driving. In each session, subjects were required to drive simulated car for 90 min without any break. The results demonstrate that our proposed methods are effective for fatigue detection. The prediction of fatigue is consistent with the observation of reaction time that was recorded during simulated driving, which is considered as an objective behavioral measure.     

Discovery of cellular substrates for protein kinase A using a peptide array screening protocol

Post-translational modification of proteins is a universal form of cellular regulation. Phosphorylation on serine, threonine, tyrosine or histidine residues by protein kinases is the most widespread and versatile form of covalent modification. Resultant changes in activity, localization or stability of phosphoproteins drives cellular events. MS and bioinformatic analyses estimate that ~30% of intracellular proteins are phosphorylated at any given time. Multiple approaches have been developed to systematically define targets of protein kinases; however, it is likely that we have yet to catalogue the full complement of the phosphoproteome. The amino acids that surround a phosphoacceptor site are substrate determinants for protein kinases. For example, basophilic enzymes such as PKA (protein kinase A), protein kinase C and calmodulin-dependent kinases recognize basic side chains preceding the target serine or threonine residues. In the present paper we describe a strategy using peptide arrays and motif-specific antibodies to identify and characterize previously unrecognized substrate sequences for protein kinase A. We found that the protein kinases PKD (protein kinase D) and MARK3 [MAP (microtubule-associated protein)-regulating kinase 3] can both be phosphorylated by PKA. Furthermore, we show that the adapter protein RIL [a product of PDLIM4 (PDZ and LIM domain protein 4)] is a PKA substrate that is phosphorylated on Ser(119) inside cells and that this mode of regulation may control its ability to affect cell growth.     

Sensitive single-molecule protein quantification and protein complex detection in a microarray format

Single-molecule protein analysis provides sensitive protein quantitation with a digital read-out and is promising for studying biological systems and detecting biomarkers clinically. However, current single-molecule platforms rely on the quantification of one protein at a time. Conventional antibody microarrays are scalable to detect many proteins simultaneously, but they rely on less sensitive and less quantitative quantification by the ensemble averaging of fluorescent molecules. Here, we demonstrate a single-molecule protein assay in a microarray format enabled by an ultra-low background surface and single-molecule imaging. The digital read-out provides a highly sensitive, low femtomolar limit of detection and four orders of magnitude of dynamic range through the use of hybrid digital-analog quantification. From crude cell lysate, we measured levels of p53 and MDM2 in parallel, proving the concept of a digital antibody microarray for use in proteomic profiling. We also applied the single-molecule microarray to detect the p53-MDM2 protein complex in cell lysate. Our study is promising for development and application of single-molecule protein methods because it represents a technological bridge between single-plex and highly multiplex studies.     

Real-time detection of DNA hybridization on microarray using a CCD-based imaging system equipped with a rotated microlens array disk

This work describes a novel charge-coupled device (CCD)-based imaging system (MB Biochip Reader?) for real-time detection of DNA hybridization to DNA microarrays. The MB Biochip Reader? consisted of a laser light source (532 nm), a microlens array for generation of a multi-beam laser, and a CCD for 2-D signal imaging. The MB Biochip Reader? with a rotated microlens array, allowed large-field imaging (6.2 mm × 7.6 mm with 6.45 μm resolution) with fast time-resolution at 0.2 s without speckle noise. Furthermore, real-time detection of DNA hybridization, which is sufficient to obtain accurate data from tens of thousands of array element per field, was successfully performed without the need for laser scanning. The performance of the MB Biochip Reader? for DNA microarray imaging was similar to the commercially available photomultiplier tube (PMT)-based microarray scanner, ScanArray Lite. The system potentially could be applied toward real-time analysis in many other fluorescent techniques in addition to real-time DNA microarray analysis.     

A novel expression system for recombinant marine mussel adhesive protein Mefp1 using a truncated OmpA signal peptide

To express an increased level of recombinant Mefp1 (marine mussel adhesive protein) in soluble form, we constructed expression vectors encoding truncated OmpA signal peptide-Mefp1 fusion proteins. OmpA signal peptide (OmpASP) is the 21 residue peptide fragment of the 23 residue OmpA signal sequence cleavable by signal peptidase I. We successfully produced increased levels of soluble recombinant Mefp1 (rMefp1) with various deletions of OmpASP, and found that the increased expression was caused by the increased pI of the N-terminus of the fusion proteins (> or = 10.55). All the OmpA signal peptide segments of 3-21 amino acids in length had the same pI value (10.55). Our results suggest that the pI value of the truncated OmpASP (OmpASP(tr)) play an important role in directional signaling for the fusion protein, but we found no evidence for the presence of a secretion enhancer in OmpASP. For practical applications, we increased the expression of soluble rMefp1 with OmpASP(tr) peptides as directional signals, and obtained rMefp1 with the native amino terminus (nN-rMefp1) using an OmpASP(tr)| Xa leader sequence that contains the recognition site for Xa protease.     

Antibody-based fluorescence detection of kinase activity on a peptide array

Peptide-based microarrays allow for high-throughput identification of protein kinase substrates. However, current methods of detecting kinase activity require the use of radioisotopes. We have developed a novel fluorescence-based approach for quantitative detection of peptide phosphorylation on chip using fluorescently-labeled anti-phosphoserine and anti-phosphotyrosine antibodies. This method is sensitive, specific and extremely fast, presenting obvious advantages and may find wider uses in high-throughput kinase screenings.     

A novel methodology for double protein A-gold immunolabeling utilizing the monovalent fragment of protein A.

A method for sequential protein A-gold immunolabeling is described whereby the binding of second gold probe to the first antibody-protein A-gold complex is reduced to acceptably minimal levels. Immunolabeling of thin sections of embedded pituitary tissue was used as a model system. After an initial immunolabeling for prolactin, sections were incubated in normal serum (rabbit) followed by a monovalent fragment of protein A. These latter two incubations reduced artifactual second gold probe label over prolactin-labeled secretory granules to minimal levels (much less than 1 particle per granule) when sections were subsequently immunolabeled with normal serum. The combination of normal serum and protein A fragment incubations saturates IgG and protein A binding sites on the first antibody-gold probe complex. The latter is thereafter unable to bind further IgG (and thus gold probe) because of the monovalent nature of the protein A fragment. It is suggested that this methodology may be extended to multiple immunolabeling procedures for electron microscopy. In addition, when used before single labeling this method may be an effective way to minimize nonspecific IgG binding in cases where the tissue or antibody under study may be a problem.     

Antibody array analysis with label-based detection and resolution of protein size

Elements from DNA microarray analysis, such as sample labeling and micro-spotting of capture reagents, have been successfully adapted to multiplex measurements of soluble cytokines. Application in cell biology is hampered by the lack of mono-specific antibodies and the fact that many proteins occur in complexes. Here, we incorporated a principle from Western blotting and resolved protein size as an additional parameter. Proteins from different cellular compartments were labeled and separated by size exclusion chromatography into 20 fractions. All were analyzed with replicate antibody arrays. The elution profiles of all antibody targets were compiled to color maps that resemble Western blots with bands of antibody reactivity across the size separation range (670-10 kDa). A new solid phase designed for processing in microwell plates was developed to handle the large number of samples. Antibodies were bound to protein G-coupled microspheres surface-labeled with 300 combinations of four fluorescent dyes. Fluorescence from particle color codes and the protein label were measured by high-speed flow cytometry. Cytoplasmic protein kinases were detected as bands near predictable elution points. For proteins with atypical elution characteristics or multiple contexts, two or more antibodies were used as internal references of specificity. Membrane proteins eluted near the void volume, and additional bands corresponding to intracellular forms were detected for several targets. Elution profiles of cyclin-dependent kinases (cdks), cyclins, and cyclin-dependent kinase inhibitors, were compatible with their occurrence in complexes that vary with the cell cycle phase and subcellular localization. A two-dimensional platform circumvents the need for mono-specific capture antibodies and extends the utility of antibody array analysis to studies of protein complexes.     

A novel peptide tag for detection and purification of recombinant expressed proteins

Peptide tags have proven useful for the detection and purification of recombinant proteins. However cross reactions of antibodies raised to the tag are frequently observed due to the presence of host proteins containing all or parts of the tag. In this report we have identified a unique viral peptide sequence, R-tag, that by blast searches is absent from the commonly expression hosts Arabidopsis thaliana, Escherichia coli, Pichia pastoris and mouse myeloma cell NSO. We have prepared monoclonal antibodies to this peptide and confirmed the absence of this peptide sequence from the above genomes by Western blotting. We have also modified protein expression vectors to incorporate this sequence as a fusion tag in expressed proteins and shown its use to successfully purify recombinant proteins by immunoaffinity procedures.     

A novel cell array technique for high-throughput,cell-based analysis

Summary Microarray technology has burgeoned over the past few years from nucleic acid-based arrays to tissue microarrays (TMAs). This study aimed to develop a technique to incorporate cell lines into an array and to demonstrate the usefulness of this technique by performing immunohistochemistry for β-catenin. Cell suspensions were prepared from 23 tumor cell lines. These were fixed in formalin, suspended in agar, and embedded in paraffin to produce a cell block. A “tissue microarrayer” was used to remove triplicate, 0.6 mm-cores from each cell block and to transfer these into a recipient paraffin block at precise coordinates. Immunohistochemistry was used to identify cell lines positive for β-catenin. Cultured cells were successfully incorporated into the microarray, with preservation of cell architecture and even distribution of cells within each core. A total of 18 of 69 cores (26%) were lost in processing. A total of 16 of 23 cell lines were identified as positive for membrane and cytoplasmic β-catenin, and 6 of 23 were negative. Only one cell line was unscorable because of complete core loss. We have developed a “cell microarray” technique for analyzing antigen expression by immunohistochemistry in multiple cell lines in a single expriment. This novel application of microarrays permits high-throughput, cost-efficient analysis, with the potential to rapidly identify markers with potential diagnostic and therapeutic implications in human disease.     

pFind: a novel database-searching software system for automated peptide and protein identification via tandem mass spectrometry

SUMMARY: Research in proteomics requires powerful database-searching software to automatically identify protein sequences in a complex protein mixture via tandem mass spectrometry. In this paper, we describe a novel database-searching software system called pFind (peptide/protein Finder), which employs an effective peptide-scoring algorithm that we reported earlier. The pFind server is implemented with the C++ STL, .Net and XML technologies. As a result, high speed and good usability of the software are achieved.