Immobilization of proteins to biacore sensor chips using Staphylococcus aureus sortase A

The immobilization of proteins to surfaces is an active area of research due to strong interest in protein-based sensors. Here, we describe a novel method for immobilizing ligand proteins onto Biacore sensor chips using the transpeptidase activity of Staphylococcus aureus sortase A (SrtA). This method provides a robust and gentle approach for the site-directed, covalent coupling of proteins to biosensor chips. Notably, the high specificity of the sortase allows immobilization of proteins from less than pure protein samples allowing short cuts in protein purification protocols.     

A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli

Despite the fact that many genomes have been decoded, proteome chips comprising individually purified proteins have been reported only for budding yeast, mainly because of the complexity and difficulty of high-throughput protein purification. To facilitate proteomics studies in prokaryotes, we have developed a high-throughput protein purification protocol that allowed us to purify 4,256 proteins encoded by the Escherichia coli K12 strain within 10 h. The purified proteins were then spotted onto glass slides to create E. coli proteome chips. We used these chips to develop assays for identifying proteins involved in the recognition of potential base damage in DNA. By using a group of DNA probes, each containing a mismatched base pair or an abasic site, we found a small number of proteins that could recognize each type of probe with high affinity and specificity. We further evaluated two of these proteins, YbaZ and YbcN, by biochemical analyses. The assembly of libraries containing DNA probes with specific modifications and the availability of E. coli proteome chips have the potential to reveal important interactions between proteins and nucleic acids that are time-consuming and difficult to detect using other techniques.     

基因芯片技术和蛋白质组技术在神经科学中的应用及其研究进展

基因芯片技术和蛋白质组技术是最近发展起来的高通量技术,二者的出现使同时分析神经系统的大量基因的表达和基因产物蛋白质及其相互作用网络成为可能。它们在神经科学中的应用为了解脑功能提供了前所未有的机会。一个典型的基因芯片实验包括：芯片的准备或购买、靶DNA和探针的准备或标记、标记探针与靶DNA的杂交、芯片扫描和影象信息的数据分析。蛋白质组技术较为复杂,包括蛋白质分离、鉴定和信息分析三方面的内容。其中,分离技术多种多样。若分离技术以二维电泳为基础,则该实验通常由以下步骤组成：蛋白质样品的准备、电泳分离、染胶、分离蛋白点的切除、蛋白质的酶解(常用胰蛋白酶)、质谱分析(鉴定)和数据的信息处理。本文综述这两项技术的内容和实验步骤,然后着重叙述它们在神经科学中的应用,讨论其优缺点和面临的挑战,展望其发展前景。     

Protein micro- and macroarrays: digitizing the proteome

The early applications of microarrays and detection technologies have been centered on DNA-based applications. The application of array technologies to proteomics is now occurring at a rapid rate. Numerous researchers have begun to develop technologies for the creation of microarrays of protein-based screening tools. The stability of antibody molecules when bound to surfaces has made antibody arrays a starting point for proteomic microarray technology. To minimize disadvantages due to size and availability, some researchers have instead opted for antibody fragments, antibody mimics or phage display technology to create libraries for protein chips. Even further removed from antibodies are libraries of aptamers, which are single-stranded oligonucleotides that express high affinity for protein molecules. A variation on the theme of protein chips arrayed with antibody mimics or other protein capture ligand is that of affinity MS where the protein chips are directly placed in a mass spectrometer for detection. Other approaches include the creation of intact protein microarrays directly on glass slides or chips. Although many of the proteins may likely be denatured, successful screening has been demonstrated. The investigation of protein-protein interactions has formed the basis of a technique called yeast two-hybrid. In this method, yeast "bait" proteins can be probed with other yeast "prey" proteins fused to DNA binding domains. Although the current interpretation of protein arrays emphasizes microarray grids of proteins or ligands on glass slides or chips, 2-D gels are technically macroarrays of authentic proteins. In an innovative departure from the traditional concept of protein chips, some researchers are implementing microfluidic printing of arrayed chemistries on individual protein spots blotted onto membranes. Other researchers are using in-jet printing technology to create protein microarrays on chips. The rapid growth of proteomics and the active climate for new technology is driving a new generation of companies and academic efforts that are developing novel protein microarray techniques for the future.     

Revisited BIA-MS combination: entire "on-a-chip" processing leading to the proteins identification at low femtomole to sub-femtomole levels

We present the results of a study in which biomolecular interaction analysis (BIA, Biacoretrade mark 2000) was combined with mass spectrometry (MS) using entire "on-a-chip" procedure. Most BIA-MS studies included an elution step of the analyte prior MS analysis. Here, we report a low-cost approach combining Biacore analysis with homemade chips and MS in situ identification onto the chips without elution step. First experiments have been made with rat serum albumin to determine the sensitivity and validation of the concept has been obtained with an antibody/antigen couple. Our "on-a-chip" procedure allowed complete analysis by MS/MS(2) of the biochip leading to protein identifications at low femtomole to sub-femtomole levels. Using this technique, identification of protein complexes were routinely obtained giving the opportunity to the "on-a-chip" processing to complete the BIA-MS approach in the discovery and analysis of protein complexes.     

Multiplexed protein profiling on antibody-based microarrays by rolling circle amplification

Multiplexed immunoassays on antibody-based protein microarrays are an attractive solution for analyzing biological responses in normal and diseased states. Recently, the feasibility and utility of these assays has been established as concerns about specificity and sensitivity are being overcome by careful quality control and amplification technologies such as rolling circle amplification (RCA). RCA-amplified protein chips can now profile up to 150 proteins in various substrates including serum, plasma, and supernatants with high sensitivity, broad dynamic range and good reproducibility. Diagnostic utility of RCA-amplified protein chips has been shown for multiplexed allergen testing. When allied with multivariate statistical analysis, RCA protein chips have the potential to identify multiplexed biomarker classifiers for disease diagnosis and drug response.     

Protocols of protein interactomics: Molecular fishing on optical chips and magnetic nanoparticles

In the living systems proteins function through interactions, which produce stable and dynamic protein complexes. Therefore necessity of profound studies of protein functions stipulates expansion of protein-protein interaction research. In the present review we describe experimental methods and protocols of protein interactomics, based on technology of molecular fishing on optical chips and paramagnetic nanoparticles. Both approaches are comparatively evaluated in the present review.     

Profiling of cytokine expression by biotin-labeled-based protein arrays

Global analysis of protein expression holds great promise in basic research and patient care. Previously we demonstrated that multiple cytokines could be detected simultaneously using an enzyme-linked immunosorbent assay protein array system with high sensitivity and specificity. In this paper, we described a biotin-labeled-based protein array system to detect multiple cytokines simultaneously from biological samples. In this new approach, proteins from a variety of biological sources are labeled with biotin. The biotin-labeled proteins are then incubated with antibody chips. Targeted proteins are captured by the array antibodies spotted on the antibody chips. The presence of targeted proteins is detected using Cy3- or Cy5-conjugated streptavidin and signals are imaged by laser scanner. The system also can be easily adapted to a two-color binding assay, allowing measurement of the levels of proteins in a test sample with respect to a reference sample at the same chip. To demonstrate its potential applications, we applied this technology to profile human cytokines, chemokines, growth factors, angiogenic factors and proteases in estrogen receptor (ER)+ and ER- cells. These results suggest that biotin-labeled-based antibody chip technology can provide a practical and powerful means of profiling hundreds or thousands of proteins for research and clinical purposes.     

Generation of bioreagents for protein chips

Protein microarrays have the potential to dramatically increase the throughput of proteomic analysis. Protein expression profiling chips with distinct spots of immobilized protein capture agents will allow the simultaneous measurement of hundreds to thousands of proteins from one sample. In contrast to DNA chips, for which the capture probes are easily designed and synthesized, the development of content for protein biochips is a long and laborious process. Careful consideration must be given to the specificities desired, the format of the assay, and the requirements of the capture agents, as well as to process optimization to minimize development time and cost. Monoclonal antibodies have been the prime choice as protein capture agents for the majority of protein chips developed to date. New technologies for the production of protein capture agents are more amenable to automation than traditional monoclonal antibody production and therefore carry the promise for industrialization.     

Proteomic analyzes of copper metabolism in an in vitro model of Wilson disease using surface enhanced laser desorption/ionization-time of flight-mass spectrometry

In Wilson disease, mutations in the ATP7B-gene lead to hepatic accumulation of copper that becomes toxic when the hepatic binding capacity is exceeded, leading to oxidative stress and acute liver failure. Several proteins are probably involved in dealing with the excess copper and oxidative stress. As a first step towards biomarker discovery and analyzes of copper metabolism in Wilson disease patients we characterized copper-induced changes in protein expression in cell lysates and culture media from an in vitro copper-overload model using surface enhanced laser desorption/ionization (SELDI) proteomics technology. HepG2 cells were cultured for 48 h with a physiological (0.5 microM) or a pathological (100 microM) copper concentration. Samples were applied to weak cation exchange (WCX) proteinchip arrays and chips were analyzed by time of flight (TOF)-mass spectrometry. Copper-coated IMAC chips were used to detect copper-binding proteins in cell lysate of copper depleted cells using buffers with increasing imidazole concentrations. Data from the 2 to 50 kDa range indicate that high extra-cellular copper substantially altered both intra-cellular protein expression as well as the composition of the secretome. In the lysate 15 proteins were found up-regulated, while 6 proteins were down-regulated. In culture media 21 proteins were increased while 4 proteins were decreased in abundance. Copper-coated protein chips revealed the presence of 18 high-affinity copper-binding proteins. Further identification is necessary to determine the exact cellular roles of the discovered proteins.     

Improving immunosensor performances using an acoustic mixer on droplet microarray

A major drawback of protein microarrays is the lack of control of ligand immobilization at the surface of the chip which limits their performances and thus their impacts in in vitro diagnosis. To improve antibody (Ab) grafting during the spotting process on commercialized gold SPRi chips, we propose to produce a chaotic flow in every spotted droplet, by using an acoustic field, in order to disrupt the steady state of the reaction of Ab grafting. Our results show that acoustic mixing during Ab binding at the biochips surface increases their biorecognition performances of a mean factor of 2.7 in comparison with Ab layer grafted in a passive mode. Moreover, it increases statistically the homogeneity of the response over all the surface of the chips.     

Biomedical applications of protein chips

The development of microchips involving proteins has accelerated within the past few years. Although DNA chip technologies formed the precedent, many different strategies and technologies have been used because proteins are inherently a more complex type of molecule. This review covers the various biomedical applications of protein chips in diagnostics, drug screening and testing, disease monitoring, drug discovery (proteomics), and medical research. The proteomics and drug discovery section is further subdivided to cover drug discovery tools (on-chip separations, expression profiling, and antibody arrays), molecular interactions and signaling pathways, the identification of protein function, and the identification of novel therapeutic compounds. Although largely focused on protein chips, this review includes chips involving cells and tissues as a logical extension of the type of data that can be generated from these microchips.     

Protein chips: from concept to practice

A series of exciting reports over the past two years has established the usefulness of protein chips and made important advances in preparing protein arrays. However, several technical challenges must still be addressed to make these tools available to the wider community of researchers. Here, we discusses these challenges and survey recent opportunities for creating quantitative assays, preparing and immobilizing large numbers of proteins, using detection methods to analyze the results of chip-based experiments, and using informatics tools to interpret these results.     

水稻蛋白质组学研究进展

蛋白质组是一个基因组所表达蛋白质的总称,研究内容包括蛋白质基本氨基酸序列的鉴定到相关性状、修饰、功能及不同类型蛋白分子相互作用等等。本文简要介绍了蛋白质组学的产生背景,以及主要研究技术包括双向电泳(2D-PAGE)质谱、蛋白质芯片、酵母双杂交,并重点介绍了近期水稻蛋白质组学应用研究进展。     

Real time analysis of intact organelles using surface plasmon resonance

Membrane proteins remain refractory to standard protein chip analysis. They are typically expressed at low densities in distinct subcellular compartments, their biological activity can depend on assembly into macromolecular complexes in a specific lipid environment. We report here a real-time, label-free method to analyze membrane proteins inserted in isolated native synaptic vesicles. Using surface plasmon resonance-based biomolecular interaction analysis (Biacore), organelle capture from minute quantities of 10,000 g brain supernatant (1-10 microg) was monitored. Immunological and morphological characterization indicated that pure intact synaptic vesicles were immobilized on sensor chips. Vesicle chips were stable for days, allowing repetitive use with multiple analytes. This method provides an efficient way in which to characterize organelle membrane components in their native context. Organelle chips allow a broad range of measurements, including interactions of exogenous ligands with the organelle surface (kinetics, Kd), and protein profiling.     

Amplification of fluorescence with packed beads to enhance the sensitivity of miniaturized detection in microfluidic chip

This paper reports the pre-concentration of C-reactive protein (CRP) antigen with packed beads in a microfluidic chamber to enhance the sensitivity of the miniaturized fluorescence detection system for portable point-of-care testing devices. Although integrated optical systems in microfluidic chips have been demonstrated by many groups to replace bulky optical systems, the problem of low sensitivity is a hurdle for on-site clinical applications. Hence we integrated the pre-concentration module with miniaturized detection in microfluidic chips (MDMC) to improve analytical sensitivity. Cheap silicon-based photodiodes with optical filter were packaged in PDMS microfluidic chips and beads were packed by a frit structure for pre-concentration. The beads were coated with CRP antibodies to capture antigens and the concentrated antigens were eluted by an acid buffer. The pre-concentration amplified the fluorescence intensity by about 20-fold and the fluorescence signal was linearly proportional to the concentration of antigens. Then the CRP antigen was analyzed by competitive immunoassay with an MDMC. The experimental result demonstrated that the analytical sensitivity was enhanced up to 1.4 nM owing to the higher signal-to-noise ratio. The amplification of fluorescence by pre-concentration of bead-based immunoassay is expected to be one of the methods for portable fluorescence detection system.     

Effects of chipping, grinding, and heat on survival of emerald ash borer, Agrilus planipennis (Coleoptera: Buprestidae), in chips

The emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), a phloem-feeding insect from Asia, was identified in 2002 as the cause of widespread ash (Fraxinus sp.) mortality in southeastern Michigan and Essex County, Ontario. Most larvae overwinter as nonfeeding prepupae in the outer sapwood or thick bark of large trees. In a series of studies, we evaluated effects of grinding, chipping, and heat treatment on survival of A. planipennis prepupae in ash material. Heavily infested ash bolts containing roughly 8,700 prepupae were processed by a horizontal grinder with either a 2.5- or 10-cm screen. There was no evidence of A. planipennis survival in chips processed with the 2.5-cm screen, but eight viable prepupae were recovered from chips processed with the 10-cm screen. We chiseled additional sentinel chips with prepupae from ash logs and buried 45 in each chip pile. In total, six prepupae in sentinel chips survived the winter, but we found no sign of adult A. planipennis emergence from the processed chips. Subsequently, we assessed prepupal survival in chips processed by a chipper or a horizontal grinder fit with 5-, 10-, or 12.7-cm screens. An estimated 1,565 A. planipennis prepupae were processed by each treatment. Chips from the chipper were shorter than chips from the grinder regardless of the screen size used. No live prepupae were found in chips produced by the chipper, but 21 viable prepupae were found in chips from the grinder. Infested wood and bark chips chiseled from logs were held in ovens at 25, 40, or 60 degrees C for 8, 24, or 48 h. Prepupal survival was consistently higher in wood chips than bark chips at 40 degrees C, whereas no prepupae survived exposure to 60 degrees C for eight or more hours. In a second study, prepupae in wood chips were exposed to 40, 45, 50, 55, or 60 degrees C for 20 or 120 min. Some prepupae survived 20 min of exposure to all temperatures. No prepupae survived exposure to 60 degrees C for 120 min, but 17% survived exposure to 55 degrees C for 120 min, suggesting that some fraction of the population may survive internationally recognized phytosanitary standards (ISPM-15) for treatment of wood packing material.     

Serological protein profiling of neuroblastoma by ProteinChip SELDI-TOF technology

Serological proteins of neuroblastoma were profiled and analyzed by ProteinChip-SELDI-TOF MS technology with five types of protein chips. By comparing with normal control, a number of protein or polypeptide signals were found significantly and consistently different in their intensities (expression levels) in tumor sera. Interestingly, nine polypeptide peaks in these proteomic features can be simultaneously detected with consistent variations by more than one type of protein chips. None of the expression differences of these nine polypeptides was found in similar comparisons between healthy controls and hepatomas. Preliminary protein identification showed hints for that some of these proteomic alterations may be closely related to the tumorigenesis of neuroblastoma. These results demonstrated the potential of serological biomarker identification for neuroblastoma by ProteinChip-SELDI technology.