Expanding the proteome: disordered and alternatively folded proteins

Proteins provide much of the scaffolding for life, as well as undertaking a variety of essential catalytic reactions. These characteristic functions have led us to presuppose that proteins are in general functional only when well structured and correctly folded. As we begin to explore the repertoire of possible protein sequences inherent in the human and other genomes, two stark facts that belie this supposition become clear: firstly, the number of apparent open reading frames in the human genome is significantly smaller than appears to be necessary to code for all of the diverse proteins in higher organisms, and secondly that a significant proportion of the protein sequences that would be coded by the genome would not be expected to form stable three-dimensional (3D) structures. Clearly the genome must include coding for a multitude of alternative forms of proteins, some of which may be partly or fully disordered or incompletely structured in their functional states. At the same time as this likelihood was recognized, experimental studies also began to uncover examples of important protein molecules and domains that were incompletely structured or completely disordered in solution, yet remained perfectly functional. In the ensuing years, we have seen an explosion of experimental and genome-annotation studies that have mapped the extent of the intrinsic disorder phenomenon and explored the possible biological rationales for its widespread occurrence. Answers to the question 'why would a particular domain need to be unstructured?' are as varied as the systems where such domains are found. This review provides a survey of recent new directions in this field, and includes an evaluation of the role not only of intrinsically disordered proteins but also of partially structured and highly dynamic members of the disorder-order continuum.     

Quantitative plasma proteome analysis reveals aberrant level of blood coagulation-related proteins in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC), one of the most common cancers in Southeast Asia, is not easily diagnosed until advanced stages. To discover potential biomarkers for improving NPC diagnosis, we herein identified the aberrant plasma proteins in NPC patients. We first removed the top-seven abundant proteins from plasma samples of healthy controls and NPC patients, and then labeled the samples with different fluorescent cyanine dyes. The labeled samples were then mixed equally and fractionated with ion-exchange chromatography followed by SDS-PAGE. Proteins showing altered levels in NPC patients were identified by in-gel tryptic digestion and LC-MS/MS. When the biological roles of the 45 identified proteins were assessed via MetaCore? analysis, the blood coagulation pathway emerged as the most significantly altered pathway in NPC plasma. Plasma kallikrein (KLKB1) and thrombin-antithrombin III complex (TAT) were chosen for evaluation as the candidate NPC biomarkers because of their involvement in blood coagulation. ELISAs confirmed the elevation of their plasma levels in NPC patients versus healthy controls. Western blot and activity assays further showed that the KLKB1 active form was significantly increased in NPC plasma. Collectively, our results identified the significant alteration of blood coagulation pathway in NPC patients, and KLKB1 and TAT may represent the potential NPC biomarkers.     

Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors

Background  

The tear film is a thin layer of fluid that covers the ocular surface and is involved in lubrication and protection of the eye. Little is known about the protein composition of tear fluid but its deregulation is associated with disease states, such as diabetic dry eyes. This makes this body fluid an interesting candidate for in-depth proteomic analysis.     

On the number of protein-protein interactions in the yeast proteome

Using two different approaches, we estimated that on average there are about five interacting partners per protein in the proteome of the yeast Saccharomyces cerevisiae. In the first approach, we used a novel method to model sampling overlap by a Bernoulli process, compared the results of two independent yeast two-hybrid interaction screens and tested the robustness of the estimate. The most stable estimate of five interactors per protein was obtained when the three most highly connected nodes in the protein interaction network were removed from the analysis (eight interactors per protein if those nodes were kept). In the second approach, we analysed a published high-confidence subset of putative interaction data obtained from multiple sources, including large-scale two-hybrid screens, complex purifications, synthetic lethals, correlated gene expression, computational predictions and previous annotations. Strikingly, the estimate was again five interactors per protein. These estimates suggest a range of ~16 000–26 000 different interaction pairs in the yeast, excluding homotypic interactions. We also discuss the approaches to estimating the rate of homotypic interactions.     

Estimation of membrane proteins in the human proteome

Genomics and proteomics have added valuable information to our knowledgebase of the human biological system including the discovery of therapeutic targets and disease biomarkers. However, molecular profiling studies commonly result in the identification of novel proteins of unknown localization. A class of proteins of special interest is membrane proteins, in particular plasma membrane proteins. Despite their biological and medical significance, the 3-dimensional structures of less than 1% of plasma membrane proteins have been determined. In order to aid in identification of membrane proteins, a number of computational methods have been developed. These tools operate by predicting the presence of transmembrane segments. Here, we utilized five topology prediction methods (TMHMM, SOSUI, waveTM, HMMTOP, and TopPred II) in order to estimate the ratio of integral membrane proteins in the human proteome. These methods employ different algorithms and include a newly-developed method (waveTM) that has yet to be tested on a large proteome database. Since these tools are prone for error mainly as a result of falsely predicting signal peptides as transmembrane segments, we have utilized an additional method, SignalP. Based on our analyses, the ratio of human proteins with transmembrane segments is estimated to fall between 15% and 39% with a consensus of 13%. Agreement among the programs is reduced further when both a positive identification of a membrane protein and the number of transmembrane segments per protein are considered. Such a broad range of prediction depends on the selectivity of the individual method in predicting integral membrane proteins. These methods can play a critical role in determining protein structure and, hence, identifying suitable drug targets in humans.     

Isolation and characterizations of oxalate-binding proteins in the kidney

Oxalate-binding proteins are thought to serve as potential modulators of kidney stone formation. However, only few oxalate-binding proteins have been identified from previous studies. Our present study, therefore, aimed for large-scale identification of oxalate-binding proteins in porcine kidney using an oxalate-affinity column containing oxalate-conjugated EAH Sepharose 4B beads for purification followed by two-dimensional gel electrophoresis (2-DE) to resolve the recovered proteins. Comparing with those obtained from the controlled column containing uncoupled EAH-Sepharose 4B (to subtract the background of non-specific bindings), a total of 38 protein spots were defined as oxalate-binding proteins. These protein spots were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS) as 26 unique proteins, including several nuclear proteins, mitochondrial proteins, oxidative stress regulatory proteins, metabolic enzymes and others. Identification of oxalate-binding domain using the PRATT tool revealed "L-x(3,5)-R-x(2)-[AGILPV]" as a functional domain responsible for oxalate-binding in 25 of 26 (96%) unique identified proteins. We report herein, for the first time, large-scale identification and characterizations of oxalate-binding proteins in the kidney. The presence of positively charged arginine residue in the middle of this functional domain suggested its significance for binding to the negatively charged oxalate. These data will enhance future stone research, particularly on stone modulators.     

The abundance of short proteins in the mammalian proteome

Short proteins play key roles in cell signalling and other processes, but their abundance in the mammalian proteome is unknown. Current catalogues of mammalian proteins exhibit an artefactual discontinuity at a length of 100 aa, so that protein abundance peaks just above this length and falls off sharply below it. To clarify the abundance of short proteins, we identify proteins in the FANTOM collection of mouse cDNAs by analysing synonymous and non-synonymous substitutions with the computer program CRITICA. This analysis confirms that there is no real discontinuity at length 100. Roughly 10% of mouse proteins are shorter than 100 aa, although the majority of these are variants of proteins longer than 100 aa. We identify many novel short proteins, including a “dark matter” subset containing ones that lack detectable homology to other known proteins. Translation assays confirm that some of these novel proteins can be translated and localised to the secretory pathway.     

Characterization of proteins in the human serum proteome.

This paper presents a multidimensional profile of the human serum proteome, produced by a two-dimensional protein fractionation system based on liquid chromatography followed by characterization with capillary electrophoresis (CE). The first-dimension separation was done by chromatofocusing over a pH range from 8.5 to 4.0, where proteins were separated by their isoelectric points (pI). In this dimension, fractions were collected based on pH. The first-dimension pI fractions were then resolved in the second dimension by high-resolution, reversed-phase chromatography with a gradient of trifluoroacetic acid (TFA) in acetonitrile and TFA in water. A selected protein fraction collected from the second dimension by time was characterized by CE for molecular-weight estimation and for presence of isoforms. Molecular-weight estimation was done by sodium dodecyl sulfate capillary gel electrophoresis, where proteins were separated in the range of 10,000-225,000 Da. Detection of isoforms was done by capillary isoelectric focusing over a pH range of 3-10. A selected second-dimension fraction that contained the putative serum iron-binding protein transferrin was analyzed by these two CE techniques for molecular-weight determination and the presence of isoforms. The combination of two-dimensional protein fractionation and CE characterization represents an advanced tool for proteomics.     

Quantitative whole-cell proteome analysis of pseudorabies virus-infected cells

A quantitative proteome study using the stable isotope labeling with amino acids in cell culture technique was performed on bovine kidney cells after infection with the alphaherpesvirus pseudorabies virus (PrV), the etiological agent of Aujeszky's disease. To enhance yields of proteins to be identified, raw extracts were fractionated by affinity solid-phase extraction with a combination of a cibacron blue F3G-A and a heparin matrix and with a phosphoprotein-specific matrix. After two-dimensional gel electrophoresis in different pH ranges between pH 3 and pH 10, 2,600 proteins representing 565 genes were identified by mass spectrometry and screened for virus-induced changes in relative protein levels. Four hours after infection, significant quantitative variations were found for constituents of the nuclear lamina, representatives of the heterogeneous nuclear ribonucleoproteins, proteins involved in membrane trafficking and intracellular transport, a ribosomal protein, and heat shock protein 27. Several proteins were present in multiple charge variants that were differentially affected by infection with PrV. As a common pattern for all these proteins, a mass shift in favor of the more acidic isoforms was observed, suggesting the involvement of viral or cellular kinases.     

Quantitative proteome profiling of normal human circulating microparticles

Circulating microparticles (MPs) are produced as part of normal physiology. Their numbers, origin, and composition change in pathology. Despite this, the normal MP proteome has not yet been characterized with standardized high-resolution methods. We here quantitatively profile the normal MP proteome using nano-LC-MS/MS on an LTQ-Orbitrap with optimized sample collection, preparation, and analysis of 12 different normal samples. Analytical and procedural variation were estimated in triply processed samples analyzed in triplicate from two different donors. Label-free quantitation was validated by the correlation of cytoskeletal protein intensities with MP numbers obtained by flow cytometry. Finally, the validity of using pooled samples was evaluated using overlap protein identification numbers and multivariate data analysis. Using conservative parameters, 536 different unique proteins were quantitated. Of these, 334 (63%) were present in all samples and represent an MP core proteome. Technical triplicates showed <10% variation in intensity within a dynamic range of almost 5 decades. Differences due to variable MP numbers and losses during preparative steps could be normalized using cytoskeletal MP protein intensities. Our results establish a reproducible LC-MS/MS procedure, provide a simple and robust MP preparation method, and yield a baseline MP proteome for future studies of MPs in health and disease.     

Quantitative profiling of the membrane proteome in a halophilic archaeon

We present a large scale quantitation study of the membrane proteome from Halobacterium salinarum. To overcome problems generally encountered with membrane proteins, we established a membrane preparation protocol that allows the application of most proteomic techniques originally developed for soluble proteins. Proteins were quantified using two complementary approaches. For gel-based quantitation, DIGE labeling was combined with two-dimensional gel electrophoresis on an improved 16-benzyldimethyl-n-hexadecylammonium chloride/SDS system. MS-based quantitation was carried out by combining gel-free separation with the recently developed isotope-coded protein labeling technique. Good correlations between these two independent quantitation strategies were obtained. From computational analysis we conclude that labeling of free amino groups by isotope-coded protein labeling (Lys and free N termini) is better suited for membrane proteins than Cys-based labeling strategies but that quantitation of integral membrane proteins remains cumbersome compared with soluble proteins. Nevertheless we could quantify 155 membrane proteins; 101 of these had transmembrane domains. We compared two growth states that strongly affect the energy supply of the cells: aerobic versus anaerobic/phototrophic conditions. The photosynthetic protein bacteriorhodopsin is the most highly regulated protein. As expected, several other membrane proteins involved in aerobic or anaerobic energy metabolism were found to be regulated, but in total, however, the number of regulated proteins is rather small.     

Interaction proteome analysis of Xanthomonas Hrp proteins

Because of the importance of the type III protein-secretion system in bacteria-plant interaction, its function in bacterial pathogenesis of plants has been intensively studied. To identify bacterial proteins interacting with Xanthomonas hrp gene products that are involved in pathogenicity, we performed the glutathione-bead binding analysis of Xanthomonas lysates containing GST-tagged Hrp proteins. Analysis of glutathione-bead bound proteins by 1-DE and MALDI-TOF has demonstrated that Avr proteins, RecA, and several components of the type III secretion system interact with HrpB protein. This proteomic approach could provide a powerful tool in finding interaction partners of Hrp proteins whose roles in host-pathogen interaction need further studies.     

Towards the proteome of the marine bacterium Rhodopirellula baltica: mapping the soluble proteins

The marine bacterium Rhodopirellula baltica, a member of the phylum Planctomycetes, has distinct morphological properties and contributes to remineralization of biomass in the natural environment. On the basis of its recently determined complete genome we investigated its proteome by 2-DE and established a reference 2-DE gel for the soluble protein fraction. Approximately 1000 protein spots were excised from a colloidal Coomassie-stained gel (pH 4-7), analyzed by MALDI-MS and identified by PMF. The non-redundant data set contained 626 distinct protein spots, corresponding to 558 different genes. The identified proteins were classified into role categories according to their predicted functions. The experimentally determined and the theoretically predicted proteomes were compared. Proteins, which were most abundant in 2-DE gels and the coding genes of which were also predicted to be highly expressed, could be linked mainly to housekeeping functions in glycolysis, tricarboxic acid cycle, amino acid biosynthesis, protein quality control and translation. Absence of predictable signal peptides indicated a localization of these proteins in the intracellular compartment, the pirellulosome. Among the identified proteins, 146 contained a predicted signal peptide suggesting their translocation. Some proteins were detected in more than one spot on the gel, indicating post-translational modification. In addition to identifying proteins present in the published sequence database for R. baltica, an alternative approach was used, in which the mass spectrometric data was searched against a maximal ORF set, allowing the identification of four previously unpredicted ORFs. The 2-DE reference map presented here will serve as framework for further experiments to study differential gene expression of R. baltica in response to external stimuli or cellular development and compartmentalization.     

Linking proteome and genome: how to identify parasite proteins

Parasite genome projects are generating an avalanche of sequence data. If this resource is to be exploited effectively for drug and vaccine design, there is an urgent need to make the link between these DNA sequences and the functional proteins of the parasite, which they encode. Here, we seek to demystify the revolutionary advances in protein identification based on mass spectrometry.     

New insights into the rat spermatogonial proteome: identification of 156 additional proteins

Despite the essential role played by spermatogonia in testicular function, little is known about these cells. To improve our understanding of their biology, our group recently identified a set of 53 spermatogonial proteins using two-dimensional (2-D) gel electrophoresis and mass spectrometry. To continue this work, we investigated a subset of the spermatogonial proteome using narrow range immobilized pH gradients to favor the detection of less abundant proteins. A 2-D reference map of spermatogonia in the pH range 4-9 was created, and protein entities fractionated in a pH 5-6 2-D gel were further processed for protein identification. A new set of 156 polypeptides was identified by peptide mass fingerprinting and tandem mass spectrometry. These polypeptides corresponded to 102 different proteins, which reflect the complexity of post-translational modifications. Seventy-nine of these proteins were identified for the first time in spermatogonia. All identified proteins were classified into functional groups. This work represents a first step toward the establishment of a systematic spermatogonia protein database.     

From genes to proteins: high-throughput expression and purification of the human proteome

The development of high-throughput methods for gene discovery has paved the way for the design of new strategies for genome-scale protein analysis. Lawrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have produced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) collection. This high-throughput protein expression system has been developed for the analysis of the human proteome, the protein equivalent of the human genome, comprising the translated products of all expressed genes. Functional and structural analysis of novel genes identified by EST (Expressed Sequence Tag) sequencing and the Human Genome Project will be greatly advanced by the application of this high-throughput expression system for protein production. A prototype was designed to demonstrate the feasibility of our approach. Using a PCR-based strategy, 72 unique IMAGE cDNA clones have been used to create an array of recombinant baculoviruses in a 96-well microtiter plate format. Forty-two percent of these cDNAs successfully produced soluble, recombinant protein. All of the steps in this process, from PCR to protein production, were performed in 96-well microtiter plates, and are thus amenable to automation. Each recombinant protein was engineered to incorporate an epitope tag at the amino terminal end to allow for immunoaffinity purification. Proteins expressed from this system are currently being analyzed for functional and biochemical properties.     

Most non-canonical proteins uniquely populate the proteome or immunopeptidome

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Quantitative characterizations of the cholesterol-related pathways in the retina and brain of hamsters

The retina and brain are separated from the systemic circulation by the anatomical barriers, which are permeable (the outer blood-retinal barrier) and impermeable (the blood-brain and inner blood-retina barriers) to cholesterol. Herein we investigated whether whole-body cholesterol maintenance affects cholesterol homeostasis in the retina and brain. We used hamsters, whose whole-body cholesterol handling is more similar to those in humans than in mice, and conducted separate administrations of deuterated water and deuterated cholesterol. We assessed the quantitative significance of the retinal and brain pathways of cholesterol input and compared the results with those from our previous studies in mice. The utility of the measurements in the plasma of deuterated 24-hydroxycholesterol, the major cholesterol elimination product from the brain, was investigated as well. We established that despite a sevenfold higher serum LDL to HDL ratio and other cholesterol-related differences, in situ biosynthesis remained the major source of cholesterol for hamster retina, although its quantitative significance was reduced to 53% as compared to 72%–78% in the mouse retina. In the brain, the principal pathway of cholesterol input was also the same, in situ biosynthesis, accounting for 94% of the total brain cholesterol input (96% in mice); the interspecies differences pertained to the absolute rates of the total cholesterol input and turnover. We documented the correlations between deuterium enrichments of the brain 24-hydroxycholesterol, brain cholesterol, and plasma 24-hydroxycholesterol, which suggested that deuterium enrichment of plasma 24-hydroxycholesteol could be an in vivo marker of cholesterol elimination and turnover in the brain.     

Extraction of wheat endosperm proteins for proteome analysis

Total protein extracts of wheat endosperm are widely used for the analysis of the highly abundant gliadins and glutenins. In this review, the most popular total endosperm extraction methods are compared for their effectiveness in proteome coverage. A drawback of total endosperm extracts is that the enormous dynamic range of protein abundance limits the detection, quantification, and identification of low abundance proteins. Protein fractionation is invaluable for improving proteome coverage, because it reduces sample complexity while enriching for specific classes of less abundant proteins. A wide array of techniques is available for isolating protein subpopulations. Sequential extraction is a method particularly suited for subfractionation of wheat endosperm proteins, because it takes advantage of the specific solubility properties of the different classes of endosperm proteins. This method effectively separates the highly abundant gliadins and glutenins from the much less abundant albumins and globulins. Subcellular fractionation of tissue homogenates is a classical technique for isolating membranes and organelles for functional analysis. This approach is suitable for defining the biochemical processes associated with amyloplasts, specialized organelles in the endosperm that function in the synthesis and storage of starch. Subproteome fractionation, when combined with 2-DE and protein identification, provides a powerful approach for defining endosperm protein composition and providing new insights into cellular functions.