Proteomics strategies for protein identification

The information from genome sequencing provides new approaches for systems-wide understanding of protein networks and cellular function. DNA microarray technologies have advanced to the point where nearly complete monitoring of gene expression is feasible in several organisms. An equally important goal is to comprehensive survey cellular proteomes and profile protein changes under different cellular states. This presents a complex analytical problem, due to the chemical variability between proteins and peptides. Here, we discuss strategies to improve accuracy and sensitivity of peptide identification, distinguish represented protein isoforms, and quantify relative changes in protein abundance.     

Ampicillin/penicillin-binding protein interactions as a model drug-target system to optimize affinity pull-down and mass spectrometric strategies for target and pathway identification

The identification and validation of the targets of active compounds identified in cell-based assays is an important step in preclinical drug development. New analytical approaches that combine drug affinity pull-down assays with mass spectrometry (MS) could lead to the identification of new targets and druggable pathways. In this work, we investigate a drug-target system consisting of ampicillin- and penicillin-binding proteins (PBPs) to evaluate and compare different amino-reactive resins for the immobilization of the affinity compound and mass spectrometric methods to identify proteins from drug affinity pull-down assays. First, ampicillin was immobilized onto various amino-reactive resins, which were compared in the ampicillin-PBP model with respect to their nonspecific binding of proteins from an Escherichia coli membrane extract. Dynal M-270 magnetic beads were chosen to further study the system as a model for capturing and identifying the targets of ampicillin, PBPs that were specifically and covalently bound to the immobilized ampicillin. The PBPs were identified, after in situ digestion of proteins bound to ampicillin directly on the beads, by using either one-dimensional (1-D) or two-dimensional (2-D) liquid chromatography (LC) separation techniques followed by tandem mass spectrometry (MS/MS) analysis. Alternatively, an elution with N-lauroylsarcosine (sarcosyl) from the ampicillin beads followed by in situ digestion and 2-D LC-MS/MS analysis identified proteins potentially interacting noncovalently with the PBPs or the ampicillin. The in situ approach required only little time, resources, and sample for the analysis. The combination of drug affinity pull-down assays with in situ digestion and 2-D LC-MS/MS analysis is a useful tool in obtaining complex information about a primary drug target as well as its protein interactors.     

Computer-aided model-building strategies for protein design

Model-building strategies for protein modification and design are developed. These strategies emphasize simple geometric aspects of protein structure, use local coordinate systems defined at particular residues, and systematically consider a large number of alternative sequences and conformations. We have written a computer program, PROTEUS, to implement these search methods. PROTEUS has been used to find positions where disulfide bonds could be added to the N-terminal domain of the lambda repressor and to predict how a loop on the surface of repressor could be shortened.     

Gradient design to optimize rate zonal separations

An approach to the design of gradients which maximize resolution is developed by analyzing the sedimentation of particles in linear sucrose gradients. Our analysis establishes the fundamental principles of rate separations. These principles can assist in the successful design of preparative centrifugation procedures. Rate separations are always optimal in homogeneous media or very shallow gradients of low density. In homogeneous media, resolution of particles which differ only in sedimentation coefficients is determined by the ratio of their sedimentation coefficients. Particles whose sedimentation properties oppose each other can, under certain conditions, not separate or barely separate unless conditions are carefully selected. Particle populations which differ more in density than in sedimentation coefficients clearly separate better by rate than by isopycnic banding. Rate separations in gradients are considerably improved in a type of gradient where the viscosity decreased as the density increased.     

Type II beta regulatory subunit of cAMP-dependent protein kinase: purification strategies to optimize crystallization

To elucidate the structural basis for important differences between types I and II regulatory subunit isoforms (RI and RII) of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase, the full-length RII beta isoform and five RII beta deletion mutants were constructed, expressed, purified, and screened for crystallization. Only one of these six proteins yielded diffraction quality crystals. Crystals were grown of the RII beta deletion mutant (delta 1-111) monomer potentially in complex with two cAMP molecules. X-ray diffraction quality data were obtained only after significant modification to existing purification procedures. Modifications required a Sepharose, not agarose, support for cAMP affinity chromatography followed by rapid, quantitative removal of free cAMP by size-exclusion chromatography under reducing conditions. Data to 2.4 A resolution were collected at 29 degrees C using synchrotron radiation on a single crystal measuring 0.2 x 0.3 x 1.2 mm(3). Data were 99% complete. The hexagonal crystal belonged to space group P6((1)) or P6((5)) with unit cell dimensions a = b = 161.62 A and c = 39.66 A.     

Evaluation of alternative strategies to optimize ketorolac transdermal delivery

In the present study, 2 alternative strategies to optimize ketorolac transdermal delivery, namely, prodrugs (polyoxyethylene glycol ester derivatives, I–IV) and nanostructured lipid carriers (NLC) were investigated. The synthesized prodrugs were chemically stable and easily degraded to the parent drug in human plasma. Ketorolac-loaded NLC with high drug content could be successfully prepared. The obtained products formulated into gels showed a different trend of drug permeation through human stratum corneum and epidermis. Particularly, skin permeation of ester prodrugs was significantly enhanced, apart from ester IV, compared with ketorolac, while the results of drug release from NLC outlined that these carriers were ineffective in increasing ketorolac percutaneous absorption owing to a higher degree of mutual interaction between the drug and carrier lipid matrix. Polyoxyethylene glycol esterification confirmed to be a suitable approach to enhance ketorolac transdermal delivery, while NLC seemed more appropriate for sustained release owing to the possible formation of a drug reservoir into the skin. Published: August 4, 2006     

Improved Pruning algorithms and Divide-and-Conquer strategies for Dead-End Elimination, with application to protein design

MOTIVATION: Structure-based protein redesign can help engineer proteins with desired novel function. Improving computational efficiency while still maintaining the accuracy of the design predictions has been a major goal for protein design algorithms. The combinatorial nature of protein design results both from allowing residue mutations and from the incorporation of protein side-chain flexibility. Under the assumption that a single conformation can model protein folding and binding, the goal of many algorithms is the identification of the Global Minimum Energy Conformation (GMEC). A dominant theorem for the identification of the GMEC is Dead-End Elimination (DEE). DEE-based algorithms have proven capable of eliminating the majority of candidate conformations, while guaranteeing that only rotamers not belonging to the GMEC are pruned. However, when the protein design process incorporates rotameric energy minimization, DEE is no longer provably-accurate. Hence, with energy minimization, the minimized-DEE (MinDEE) criterion must be used instead. RESULTS: In this paper, we present provably-accurate improvements to both the DEE and MinDEE criteria. We show that our novel enhancements result in a speedup of up to a factor of more than 1000 when applied in redesign for three different proteins: Gramicidin Synthetase A, plastocyanin, and protein G. AVAILABILITY: Contact authors for source code.     

Protein engineering to optimize recombinant protein purification

Genetic approaches have been used to facilitate purification of recombinant proteins, on both a large and a small scale. Based on developments in three different areas: (i) affinity chromatography; (ii) specific cleavage of fusion proteins and (iii) secretion of fusion proteins, a coupled expression/secretion system was designed. It was further improved by protein engineering. Using a synthetic DNA fragment, encoding two IgG-binding domains derived from staphylococcal protein A, gene products were secreted to the culture medium of Escherichia coli and purified with a one-step affinity procedure. The system has been used for large-scale production of biologically active human peptide hormones, to generate peptides for antibody production and to immobilize proteins on solid supports.     

Crystallization data mining in structural genomics: using positive and negative results to optimize protein crystallization screens

Recent efforts to collect and mine crystallization data from structural genomics (SG) consortia have led to the identification of minimal screens and novel screening strategies that can be used to streamline the crystallization process. Two groups, the Joint Center for Structural Genomics and the University of Toronto, carried out large-scale crystallization trials on different sets of bacterial targets (539, JCSG and 755, Toronto), using different sample processing and crystallization methods, and then analyzed their results to identify the smallest subset of conditions that would have crystallized the maximum number of protein targets. The JCSG Core Screen contains 67 conditions (from 480) while the Toronto Minimal Screen contains 6 (from 48). While the exact conditions included in the two screens do not overlap, the major precipitants of the conditions are similar and thus both screens can be used to determine if a protein has a natural propensity to crystallize. In addition, studies from other groups including the University of Queensland, the Mycobacterium tuberculosis SG group, the Southeast Collaboratory for SG, and the York Structural Biology Laboratory indicate that alternative crystallization strategies may be more successful at identifying initial crystallization conditions than typical sparse matrix screens. These minimal screens and alternative screening strategies are already being used to optimize the crystallization processes within large SG efforts. The differences between these results, however, demonstrate that additional studies which examine the influence of protein biophysical properties and sample preparation methods on crystal formation must also be carried out before more robust screens can be identified.     

Chemical proteomics and functional proteomics strategies for protein kinase inhibitor validation and protein kinase substrate identification: Applications to protein kinase CK2

Since protein kinases have been implicated in numerous human diseases, kinase inhibitors have emerged as promising therapeutic agents. Despite this promise, there has been a relative lag in the development of unbiased strategies to validate both inhibitor specificity and the ability to inhibit target activity within living cells. To overcome these limitations, our efforts have been focused on the development of systematic strategies that employ chemical and functional proteomics. We utilized these strategies to evaluate small molecule inhibitors of protein kinase CK2, a constitutively active kinase that has recently emerged as target for anti-cancer therapy in clinical trials. Our chemical proteomics strategies used ATP or CK2 inhibitors immobilized on sepharose beads together with mass spectrometry to capture and identify binding partners from cell extracts. These studies have verified that interactions between CK2 and its inhibitors occur in complex mixtures. However, in the case of CK2 inhibitors related to 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), our work has also revealed off-targets for the inhibitors. To complement these studies, we devised functional proteomics approaches to identify proteins that exhibit decreases in phosphorylation when cells are treated with CK2 inhibitors. To identify and validate those proteins that are direct substrates for CK2, we have also employed mutants of CK2 with decreased inhibitor sensitivity. Overall, our studies have yielded systematic platforms for studying CK2 inhibitors which we believe will foster efforts to define the biological functions of CK2 and to rigorously investigate its potential as a candidate for molecular-targeted therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Use of a sequential strategy of experimental design to optimize the inulinase production in a batch bioreactor

A potential application of inulinase in the food industry is the production of fructoligosaccharides (FOS) by the transfructosilation of sucrose. The FOSs present many interesting functional properties besides their ability to increase the shelf-life and flavor of many products. The use of an industrial medium represents a good alternative to producing inulinase at low cost, since the activity may improve, or at least remain the same, as that obtained using a synthetic medium. This work was an optimization study of the inulinase production by Kluyveromyces marxianus NRRL Y-7571 using industrial pre-treated culture medium in a bioreactor employing a sequential strategy of experimental design. Initially, a Plackett–Burman (Screening Design) design was used, where the studied variables were molasses, corn steep liquor, yeast extract concentration, and agitation and aeration rates. After the analysis of the effects, a central composite rotational design (CCRD) was carried out. The optimized condition for the inulinase production was: 250 g/l of molasses, 80 g/l of corn steep liquor, 6 g/l of yeast extract, 300 rpm of agitation and 1.5 vvm aeration rate, which resulted in an enzymatic activity of 1,317 ± 65 U/ml.     

Domain repertoires as a tool to derive protein recognition rules

Several approaches, some of which are described in this issue, have been proposed to assemble a complete protein interaction map. These are often based on high throughput methods that explore the ability of each gene product to bind any other element of the proteome of the organism. Here we propose that a large number of interactions can be inferred by revealing the rules underlying recognition specificity of a small number (a few hundreds) of families of protein recognition modules. This can be achieved through the construction and characterization of domain repertoires. A domain repertoire is assembled in a combinatorial fashion by allowing each amino acid position in the binding site of a given protein recognition domain to vary to include all the residues allowed at that position in the domain family. The repertoire is then searched by phage display techniques with any target of interest and from the primary structure of the binding site of the selected domains one derives rules that are used to infer the formation of complexes between natural proteins in the cell.     

Mathematical tools to optimize the design of oligonucleotide probes and primers

The identification and quantification of specific organisms in mixed microbial communities often relies on the ability to design oligonucleotide probes and primers with high specificity and sensitivity. The design of these oligonucleotides (or “oligos” for short) shares many of the same principles in spite of their widely divergent applications. Three common molecular biology technologies that require oligonucleotide design are polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH), and DNA microarrays. This article reviews techniques and software available for the design and optimization of oligos with the goal of targeting a specific group of organisms within mixed microbial communities. Strategies for enhancing specificity without compromising sensitivity are described, as well as design tools well suited for this purpose.     

Signaling through cAMP and cAMP-dependent protein kinase: diverse strategies for drug design

The catalytic subunit of cAMP-dependent protein kinase has served as a prototype for the protein kinase superfamily for many years while structures of the cAMP-bound regulatory subunits have defined the conserved cyclic nucleotide binding (CNB) motif. It is only structures of the holoenzymes, however, that enable us to appreciate the molecular features of inhibition by the regulatory subunits as well as activation by cAMP. These structures reveal for the first time the remarkable malleability of the regulatory subunits and the CNB domains. At the same time, they allow us to appreciate that the catalytic subunit is not only a catalyst but also a scaffold that mediates a wide variety of protein:protein interactions. The holoenzyme structures also provide a new paradigm for designing isoform-specific activators and inhibitors of PKA. In addition to binding to the catalytic subunits, the regulatory subunits also use their N-terminal dimerization/docking domain to bind with high affinity to A Kinase Anchoring Proteins using an amphipathic helical motif. This targeting mechanism, which localizes PKA near to its protein substrates, is also a target for therapeutic intervention of PKA signaling.     

青檀SRAP-PCR体系优化设计方案

以青檀(Pteroceltis tatarinowii Maxim.)叶片为材料,采用正交设计和均匀设计两种方法对SRAP-PCR反应体系进行优化,并对这两种设计方案优化出的最佳反应体系进行比较,结果表明:两种设计均可用于青檀SRAP-PCR体系的优化,但与正交设计相比,均匀设计在多因素多水平条件下,得到的条带更清晰、稳定性更好。通过实验比较筛选出的青檀SRAP-PCR最佳反应体系为:2.5μL 10×PCR buffer,20 ng模板DNA,Mg2+2.5 mmol/L,dNTP150μmol/L,引物0.2μmol/L,Taq DNA聚合酶1.0 U,总体积25μL。     

Increased protein identification capabilities through novel tandem MS calibration strategies

High mass measurement accuracy is critical for confident protein identification and characterization in proteomics research. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry is a unique technique which can provide unparalleled mass accuracy and resolving power. However, the mass measurement accuracy of FTICR-MS can be affected by space charge effects. Here, we present a novel internal calibrant-free calibration method that corrects for space charge-induced frequency shifts in FTICR fragment spectra called Calibration Optimization on Fragment Ions (COFI). This new strategy utilizes the information from fixed mass differences between two neighboring peptide fragment ions (such as y(1) and y(2)) to correct the frequency shift after data collection. COFI has been successfully applied to LC-FTICR fragmentation data. Mascot MS/MS ion search data demonstrate that most of the fragments from BSA tryptic digested peptides can be identified using a much lower mass tolerance window after applying COFI to LC-FTICR-MS/MS of BSA tryptic digest. Furthermore, COFI has been used for multiplexed LC-CID-FTICR-MS which is an attractive technique because of its increased duty cycle and dynamic range. After the application of COFI to a multiplexed LC-CID-FTICR-MS of BSA tryptic digest, we achieved an average measured mass accuracy of 2.49 ppm for all the identified BSA fragments.     

Comparison of pretreatment strategies of sugarcane baggase: experimental design for citric acid production

Solid state fermentation was carried out to compare efficiency of acid, alkaline and urea pretreatment of sugarcane bagasse for production of citric acid using Aspergillus niger ATCC 9142. Plackett-Burman statistical design was used to evaluate significance of variables. Pretreatment of bagasse by urea was known as the most influential treatment to increase citric acid production (137.6g/kg of dry sugarcane bagasse and citric acid yield of 96% based on sugar consumed). Finally, up scaling was achieved to a 20L solid state fermentor in which humidity was constant in gas phase and urea-treated sugarcane bagasse. The produced acid concentration and yield in fermentor was 82.38g/kg of dry substrate and 26.45g/kgday, respectively.     

第二遗传密码与蛋白质结构优化算法

核苷酸通过“三联密码”决定氨基酸顺序 ,这就是第一遗传密码。多肽链中氨基酸的一定顺序就是蛋白质的一级结构。 2 0世纪 5 0年代Ａｎｆｉｎｓｅｎ提出假说 ,认为蛋白质特定的三维空间结构是由氨基酸排列顺序所决定的 ,现在已被广泛接受。从无结构的氨基酸序列到有特定功能的蛋白质的信息传递 ,即蛋白质中的氨基酸序列与其空间结构的对应关系 ,被称为第二遗传密码。收稿日期 :2 0 0 2 - 0 6 - 1 1 ;修回日期 :2 0 0 2 - 0 9- 1 8作者简介 :王华伟 (1 978- ) ,男 ,湖北孝感人 ,硕士生 ,从事生物信息学、ＤＮＡ分子生物计算研究。许进 (1 959…     

Comparison of two optimization methods to derive energy parameters for protein folding: perceptron and Z score

Two methods were proposed recently to derive energy parameters from known native protein conformations and corresponding sets of decoys. One is based on finding, by means of a perceptron learning scheme, energy parameters such that the native conformations have lower energies than the decoys. The second method maximizes the difference between the native energy and the average energy of the decoys, measured in terms of the width of the decoys' energy distribution (Z-score). Whereas the perceptron method is sensitive mainly to "outlier" (i.e., extremal) decoys, the Z-score optimization is governed by the high density regions in decoy-space. We compare the two methods by deriving contact energies for two very different sets of decoys: the first obtained for model lattice proteins and the second by threading. We find that the potentials derived by the two methods are of similar quality and fairly closely related. This finding indicates that standard, naturally occurring sets of decoys are distributed in a way that yields robust energy parameters (that are quite insensitive to the particular method used to derive them). The main practical implication of this finding is that it is not necessary to fine-tune the potential search method to the particular set of decoys used.