Rational Design and Identification of a Non‐Peptidic Aggregation Inhibitor of Amyloid‐β Based on a Pharmacophore Motif Obtained from cyclo[‐Lys‐Leu‐Val‐Phe‐Phe‐]

Inhibition of pathogenic protein aggregation may be an important and straightforward therapeutic strategy for curing amyloid diseases. Small‐molecule aggregation inhibitors of Alzheimer’s amyloid‐β (Aβ) are extremely scarce, however, and are mainly restricted to dye‐ and polyphenol‐type compounds that lack drug‐likeness. Based on the structure‐activity relationship of cyclic Aβ16–20 (cyclo‐[KLVFF]), we identified unique pharmacophore motifs comprising side‐chains of Leu², Val³, Phe⁴, and Phe⁵ residues without involvement of the backbone amide bonds to inhibit Aβ aggregation. This finding allowed us to design non‐peptidic, small‐molecule aggregation inhibitors that possess potent activity. These molecules are the first successful non‐peptidic, small‐molecule aggregation inhibitors of amyloids based on rational molecular design.     

The impact of blood on liver metabolite profiling – a combined metabolomic and proteomic approach

Metabolomics has entered the well‐established omic sciences as it is an indispensable information resource to achieve a global picture of biological systems. The aim of the present study was to estimate the influence of blood removal from mice liver as part of sample preparation for metabolomic and proteomic studies. For this purpose, perfused mice liver tissue (i.e. with blood removed) and unperfused mice liver tissue (i.e. containing blood) were compared by two‐dimensional gas chromatography time of flight mass spectrometry (GC × GC‐TOFMS) for the metabolomic part, and by liquid chromatography tandem mass spectrometry (LC‐MS/MS) for the proteomic part. Our data showed significant differences between the unperfused and perfused liver tissue samples. Furthermore, we also observed an overlap of blood and tissue metabolite profiles in our data, suggesting that the perfusion of liver tissue prior to analysis is beneficial for an accurate metabolic profile of this organ. Copyright © 2013 John Wiley & Sons, Ltd.     

Polymer physics inspired approaches for the study of the mechanical properties of amyloid fibrils

Amyloid structures constitute a class of highly ordered nanomaterials formed by insoluble protein aggregates. These aggregates are characterized by a cross‐β structural motif in which β‐sheets are oriented perpendicular to the fibril axis and bound together by a dense hydrogen bonding network. Although they have been associated with several neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases, amyloid fibrils have also been found in many physiologically beneficial roles, for instance in adhesives and hormone storage. Inspired by this natural occurrence of functional amyloid, the hierarchal self‐assembly of these structures has recently been used to develop artificial biomaterials for applications in medicine and nanotechnology. In order to realize the full potential of amyloids as functional materials, it is important to understand their fundamental mechanical properties. This review explores a range of experimental strategies to determine the mechanical properties of amyloid fibrils and discusses the results in the context of polymer physics concepts. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2014 , 52, 281–292     

Improvement of a sample preparation method assisted by sodium deoxycholate for mass‐spectrometry‐based shotgun membrane proteomics†

In current shotgun‐proteomics‐based biological discovery, the identification of membrane proteins is a challenge. This is especially true for integral membrane proteins due to their highly hydrophobic nature and low abundance. Thus, much effort has been directed at sample preparation strategies such as use of detergents, chaotropes, and organic solvents. We previously described a sample preparation method for shotgun membrane proteomics, the sodium deoxycholate assisted method, which cleverly circumvents many of the challenges associated with traditional sample preparation methods. However, the method is associated with significant sample loss due to the slightly weaker extraction/solubilization ability of sodium deoxycholate when it is used at relatively low concentrations such as 1%. Hence, we present an enhanced sodium deoxycholate sample preparation strategy that first uses a high concentration of sodium deoxycholate (5%) to lyse membranes and extract/solubilize hydrophobic membrane proteins, and then dilutes the detergent to 1% for a more efficient digestion. We then applied the improved method to shotgun analysis of proteins from rat liver membrane enriched fraction. Compared with other representative sample preparation strategies including our previous sodium deoxycholate assisted method, the enhanced sodium deoxycholate method exhibited superior sensitivity, coverage, and reliability for the identification of membrane proteins particularly those with high hydrophobicity and/or multiple transmembrane domains.     

Efficient extraction of proteins from recalcitrant plant tissue for subsequent analysis by two‐dimensional gel electrophoresis

Protein extraction for two‐dimensional electrophoresis from tissues of recalcitrant species is quite problematic and challenging due to the low protein content and high abundance of contaminants. Proteomics in Shorea robusta is scarcely conducted due to the lack of a suitable protein preparation procedure. To establish an effective protein extraction protocol suitable for two‐dimensional electrophoresis in Shorea robusta, four procedures (borate buffer/trichloroacetic acid extraction, organic solvent/trichloroacetic acid precipitation, sucrose/Tris/phenol, and organic solvent/phenol/sodium dodecyl sulfate) were evaluated. Following these, proteins were isolated from mature leaves and were analyzed for proteomics, and also for potential contaminants, widely reported to hinder proteomics. The borate buffer/trichloroacetic acid extraction had the lowest protein yield and did not result in any banding even in one‐dimensional electrophoresis. In contrast, organic solvent/phenol/sodium dodecyl sulfate extraction allowed the highest protein yield. Moreover, during proteomics, organic solvent/phenol/sodium dodecyl sulfate extracted protein resolved the maximum number (144) of spots. Further, when proteins were evaluated for contaminants, significant (77–95%) reductions in the nucleic acids, phenol, and sugars were discernible with refinement in extraction procedure. Accumulated data suggested that the organic solvent/phenol/sodium dodecyl sulfate extraction was the most effective protocol for protein isolation for proteomics of Shorea robusta and can be used for plants that have a similar set of contaminants.     

Reduced dimensionality (3,2)D NMR experiments and their automated analysis: implications to high‐throughput structural studies on proteins

Protein NMR spectroscopy has expanded dramatically over the last decade into a powerful tool for the study of their structure, dynamics, and interactions. The primary requirement for all such investigations is sequence‐specific resonance assignment. The demand now is to obtain this information as rapidly as possible and in all types of protein systems, stable/unstable, soluble/insoluble, small/big, structured/unstructured, and so on. In this context, we introduce here two reduced dimensionality experiments – (3,2)D‐hNCO canH and (3,2)D‐hN coCA nH – which enhance the previously described 2D NMR‐based assignment methods quite significantly. Both the experiments can be recorded in just about 2–3 h each and hence would be of immense value for high‐throughput structural proteomics and drug discovery research. The applicability of the method has been demonstrated using alpha‐helical bovine apo calbindin‐D9k P43M mutant (75 aa) protein. Automated assignment of this data using AUTOBA has been presented, which enhances the utility of these experiments. The backbone resonance assignments so derived are utilized to estimate secondary structures and the backbone fold using Web‐based algorithms. Taken together, we believe that the method and the protocol proposed here can be used for routine high‐throughput structural studies of proteins. Copyright © 2014 John Wiley & Sons, Ltd.     

亲水作用色谱固定相的发展及应用

亲水作用色谱作为一种高效液相色谱技术,在强极性和离子型化合物如氨基酸、碳水化合物和多肽等的分离分析中发挥着重要作用。作为色谱技术的核心,亲水作用色谱材料的发展直接影响着色谱分离的选择性和分离效率,制约着亲水作用色谱的应用和推广。目前商品化和学术报道的亲水作用色谱材料种类繁多,键合相结构丰富。本文从色谱材料结构出发,综述了近5年内基于硅胶基质的亲水作用色谱固定相,包括纯硅胶、氨基、氰基、二醇基、酰胺型、聚（琥珀酰亚胺）型、糖型和两性离子型键合相的发展及其在极性药物、蛋白质组学、代谢组学等方面的典型应用。同时简要介绍了近年来亲水作用色谱的色谱评价研究。     

Amorphous Aggregation of Amyloid Beta 1‐40 Peptide in Confined Space

The amorphous aggregation of Aβ1‐40 peptide is addressed by using micromolding in capillaries. Both the morphology and the size of the aggregates are modulated by changing the contact angle of the sub‐micrometric channel walls. Upon decreasing the hydrophilicity of the channels, the aggregates change their morphology from small aligned drops to discontinuous lines, thereby keeping their amorphous structure. Aβ1‐40 fibrils are observed at high contact angles.     

Chronological protein synthesis in regenerating rat liver

Liver regeneration has been studied for decades; however, its regulation remains unclear. In this study, we report a dynamic tracing of protein synthesis in rat regenerating liver with a new proteomic technique, ³⁵S in vivo labeling analysis for dynamic proteomics (SiLAD). Conventional proteomic techniques typically measure protein alteration in accumulated amounts. The SiLAD technique specifically detects protein synthesis velocity instead of accumulated amounts of protein through ³⁵S pulse labeling of newly synthesized proteins, providing a direct way for analyzing protein synthesis variations. Consequently, protein synthesis within short as 30 min was visualized and protein regulations in the first 8 h of regenerating liver were dynamically traced. Further, the 3.5–5 h post partial hepatectomy (PHx) was shown to be an important regulatory turning point by acute regulation of many proteins in the initiation of liver regeneration.