Biomarker discovery from the plasma proteome using multidimensional fractionation proteomics

Because biomarkers are typically low in abundance, the crucial step of biomarker discovery is to efficiently separate clinically relevant sets of proteins that might define disease stages and/or predict disease development. It is anticipated that a multi-dimensional fractionation system (MDFS) will provide an efficient means of separating low abundance proteins from plasma proteins, resulting in the extension of the detection limit. However, when using an MDFS to analyze the plasma proteome it is important to consider how sample processing, yield, resolution and throughput potential may influence the detection limit. This review evaluates the recent advances in MDFS research with respect to '4RS criterion' (4R: resolution, reproducibility, recovery, and robustness; 4S: simplicity, speed, selectivity and sensitivity) and discusses perspectives for future plasma-derived biomarker discovery.     

Molecular dynamics simulations of a cyclic-beta-(1-->2) glucan containing an alpha-(1-->6) linkage as a 'molecular alleviator' for the macrocyclic conformational strain

The conformational preferences of a cyclic osmoregulated periplasmic glucan of Ralstonia solanacearum (OPGR), which is composed of 13 glucose units and linked entirely via beta-(1-->2) linkages excluding one alpha-(1-->6) linkage, were characterized by molecular dynamics simulations. Of the three force fields modified for carbohydrates that were applied to select a suitable one for the cyclic glucan, the carbohydrate solution force field (CSFF) was found to most accurately simulate the cyclic molecule. To determine the conformational characteristics of OPGR, we investigated the glycosidic dihedral angle distribution, fluctuation, and the potential energy of the glucan and constructed hypothetical cyclic (CYS13) and linear (LINEAR) glucans. All beta-(1-->2)-glycosidic linkages of OPGR adopted stable conformations, and the dihedral angles fluctuated in this energy region with some flexibility. However, despite the inherent flexibility of the alpha-(1-->6) linkage, the dihedral angles have no transition and are more rigid than that in a linear glucan. CYS13, which consists of only beta-(1-->2) linkages, is somewhat less flexible than other glycans, and one of its linkages adopts a higher energy conformation. In addition, the root-mean-square fluctuation of this linkage is lower than that of other linkages. Furthermore, the potential energy of glucans increases in the order of LINEAR, OPGR, and CYS13. These results provide evidence of the existence of conformational constraints in the cyclic glucan. The alpha-(1-->6)-glycosidic linkage can relieve this constraint more efficiently than the beta-(1-->2) linkage. The conformation of OPGR can reconcile the tendency for individual glycosidic bonds to adopt energetically favorable conformations with the requirement for closure of the macrocyclic ring by losing the inherent flexibility of the alpha-(1-->6)-glycosidic linkage.     

κ-Casein-Based Hierarchical Suprastructures and Their Use for Selective Temporal and Spatial Control over Neuronal Differentiation

Functions are diversified by producing hierarchical structures from a single raw material. Biologically compatible milk protein of κ-casein has been employed to fabricate higher-order suprastructures. In the presence of dithiothreitol and heat treatment, κ-casein transforms into amyloid fibrils with distinctive morphology attributable to mechanism-based fibrillar polymorphism. As the fibrils elongate to yield high aspect ratio during high-temperature incubation, the resulting fibrils laterally associate into the liquid crystalline state by forming a two-dimensional fibrillar array. Following a desalting process, the fibrillar arrays turn into a three-dimensional matrix of hydrogel that could be selectively disintegrated by subsequent salt treatment. The hydrogel was demonstrated to be a matrix capable of exhibiting controlled release of bioactive substances like retinoic acid, which led to temporal and spatial control over the differentiation of neuronal cells. Therefore, the hierarchical suprastructure formation derived from the single protein of κ-casein producing one-dimensional protein nanofibrils, a two-dimensional liquid crystalline state and a three-dimensional hydrogel could be widely appreciated in various areas of nanobiotechnology including drug delivery and tissue engineering.     

Prioritization of SNPs for genome-wide association studies using an interaction model of genetic variation,gene expression,and trait variation

The identification of true causal loci to unravel the statistical evidence of genotype-phenotype correlations and the biological relevance of selected single-nucleotide polymorphisms (SNPs) is a challenging issue in genome-wide association studies (GWAS). Here, we introduced a novel method for the prioritization of SNPs based on p-values from GWAS. The method uses functional evidence from populations, including phenotype-associated gene expressions. Based on the concept of genetic interactions, such as perturbation of gene expression by genetic variation, phenotype and gene expression related SNPs were prioritized by adjusting the p-values of SNPs. We applied our method to GWAS data related to drug-induced cytotoxicity. Then, we prioritized loci that potentially play a role in druginduced cytotoxicity. By generating an interaction model, our approach allowed us not only to identify causal loci, but also to find intermediate nodes that regulate the flow of information among causal loci, perturbed gene expression, and resulting phenotypic variation.     

Conjugation of Proteins by Installing BIO-Orthogonally Reactive Groups at Their N-Termini

N-terminal site-specific modification of a protein has many advantages over methods targeting internal positions, but it is not easy to install reactive groups onto a protein in an N-terminal specific manner. We here report a strategy to incorporate amino acid analogues specifically in the N-terminus of a protein in vivo and demonstrate it by preparing green fluorescent protein (GFP) having bio-orthogonally reactive groups at its N-terminus. In the first step, GFP was engineered to be a foldable, internal methionine-free sequence via the semi-rational mutagenesis of five internal methionine residues and the introduction of mutations for GFP folding enhancement. In the second step, the N-terminus of the engineered protein was modified in vivo with bio-orthogonally functional groups by reassigning functional methionine surrogates such as L-homopropargylglycine and L-azidohomoalanine into the first methionine codon of the engineered internal methionine-free GFP. The N-terminal specific incorporation of unnatural amino acids was confirmed by ESI-MS analysis and the incorporation did not affect significantly the specific activity, refolding rate and folding robustness of the protein. The two proteins which have alkyne or azide groups at their N-termini were conjugated each other by bio-orthogonal Cu(I)-catalyzed click chemistry. The strategy used in this study is expected to facilitate bio-conjugation applications of proteins such as N-terminal specific glycosylation, labeling of fluorescent dyes, and immobilization on solid surfaces.     

Characterization of tailoring genes involved in the modification of geldanamycin polyketide in Streptomyces hygroscopicus JCM4427

Geldanamycin and its analogs are important anticancer agents that inhibit the newly targeted, heat-shock protein (Hsp) 90, which is a chaperone protein in eukaryotic cells. To resolve which geldanamycin biosynthetic genes are responsible for particular post-polyketide synthase (PKS) processing steps and in which order the reactions occur, we individually inactivated candidate genes in Streptomyces hygroscopicus subsp. duamyceticus JCM4427, and isolated and elucidated the structures of intermediates from each mutant. The results indicated that gel7 governs at least one of the benzoquinone ring oxidation steps. In addition, gel16 was found to be involved in double-bond formation between C-4 and C-5 of 4,5-dihydrogeldanamycin, which confirmed our previous findings that this double bond reduced during the post-PKS modification of the polyketide assembly. In addition, pro-geldanamycin, which does not possess a double bond at C-4/5, was purified from the gel7 and 8 double-gene-inactivated mutant.     

Application of a peptide-based PF2D platform for quantitative proteomics in disease biomarker discovery

A peptide-based 2-D liquid phase fractionation (PF2D) system was used in a quantitative proteomic analysis of hepatocellular carcinoma. 2-D liquid maps of peptide specimens showed better resolution than those of proteins, leading to the identification of differentially expressed proteins. Peptide-based PF2D gave well-matched theoretical and experimental pI values and was proven to be a very efficient and versatile analytical tool for both large-scale profiling and quantification of phosphoproteins in disease biomarker discovery.