Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: an alternative approach to high-resolution peptide separation for shotgun proteome analysis

A two-dimensional separation scheme for shotgun proteome analysis employing high-pH reversed-phase HPLC in the first and low-pH ion-pair reversed-phase HPLC in the second dimension (RP x IP-RP-HPLC) has been developed and evaluated. Compared to the classical strong cation exchange x ion-pair reversed-phase (SCX x IP-RP-HPLC) approach, the RP x IP-RP-HPLC system was characterized by a lower degree of orthogonality, which was, however, more than counterbalanced by higher separation efficiency, more homogeneous distribution of peptide elution, and easier experimental handling. Peptide fragment fingerprinting by electrospray-ionization tandem mass spectrometry (ESI-MS/MS) was employed for peptide detection and identification. About 13% more peptides and 7% more proteins could be identified with the alternative approach in 30% less analysis time, enabling the analysis of the proteome of Corynebacterium glutamicum with a coverage of 24.9% (745 proteins). Combining the identification results both of the SCX- x IP-RP-HPLC-ESI-MS/MS and RP- x IP-RP-HPLC-ESI-MS/MS methods, a total of 871 proteins were identified in a cytosolic protein preparation, which represented 29.1% of all proteins annotated in the genome of C. glutamicum.     

GTP-Ras disrupts the intramolecular complex of C1 and RA domains of Nore1

The novel Ras effector mNore1, capable of inducing apoptosis, is a multidomain protein. It comprises a C1 domain homologous to PKC and an RA domain similar to the Ras effectors AF-6 and RalGDS. Here, we determine the affinity of these two domains to the active forms of Ras and Rap1 using isothermal calorimetric titration. The interaction of Ras/Rap1-GTP with the RA domain of mNore1 is weakened significantly by direct binding of the C1 domain to the RA domain. In order to analyze this observation in atomic detail, we solved the C1 solution structure by NMR. By determining chemical shifts and relaxation rates, we can show an intramolecular complex of C1-RA. GTP-Ras titration and binding to RA disrupts this complex and displaces the C1 domain. Once the C1 domain tumbles freely in solution, a lipid binding interface becomes accessible. Furthermore, we provide evidence of phosphatidylinositol 3-phosphate binding of the free C1 domain.     

Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures

Aptamers that target a specific cell subpopulation within composite mixtures represent invaluable tools in biomedical research and in the development of cell-specific therapeutics. Here we describe a detailed protocol for a modular and generally applicable scheme to select aptamers that target the subpopulations of cells in which you are interested. A fluorescence-activated cell-sorting device is used to simultaneously differentiate and separate those subpopulations of cells having bound and unbound aptamers. There are fewer false positives when using this approach in comparison with other cell-selection approaches in which unspecific binding of nucleic acids to cells with reduced membrane integrity or their unselective uptake by dead cells occurs more often. The protocol provides a state-of-the-art approach for identifying aptamers that selectively target virtually any cell type under investigation. As an example, we provide the step-by-step protocol targeting CD19(+) Burkitt's lymphoma cells, starting from the pre-SELEX (systematic evolution of ligands by exponential amplification) measurements to establish suitable SELEX conditions and ending at completion of the SELEX procedure, which reveals the enriched single-stranded DNA library.     

Structure-activity relationships of diastereomeric lysine ring size analogs of the antimicrobial peptide gramicidin S: mechanism of action and discrimination between bacterial and animal cell membranes

Structure-activity relationships were examined in seven gramicidin S analogs in which the ring-expanded analog GS14 [cyclo-(VKLKVdYPLKVKLdYP)] is modified by enantiomeric inversions of its lysine residues. The conformation, amphiphilicity, and self-association propensity of these peptides were investigated by circular dichroism spectroscopy and reversed phase high performance liquid chromatography. (31)P nuclear magnetic resonance spectroscopic and dye leakage experiments were performed to evaluate the capacity of these peptides to induce inverse nonlamellar phases in, and to permeabilize phospholipid bilayers; their growth inhibitory activity against the cell wall-less mollicute Acholeplasma laidlawii B was also examined. The amount and stability of beta-sheet structure, effective hydrophobicity, propensity for self-association in water, ability to disrupt the organization of phospholipid bilayers, and ability to inhibit A. laidlawii B growth are strongly correlated with the facial amphiphilicity of these GS14 analogs. Also, the magnitude of the parameters segregate these peptides into three groups, consisting of GS14, the four single inversion analogs, and the two multiple inversion analogs. The capacity of these peptides to differentiate between bacterial and animal cell membranes exhibits a biphasic relationship with peptide amphiphilicity, suggesting that there may only be a narrow range of peptide amphiphilicity within which it is possible to achieve the dual therapeutic requirements of high antibiotic effectiveness and low hemolytic activity. These results were rationalized by considering how the physiochemical properties of these GS14 analogs are likely to be reflected in their partitioning into lipid bilayer membranes.     

Reconstitution of the recombinant human γ-tubulin ring complex

Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.     

Potato-legume intercropping on a sloping terrain and its effects on soil physico-chemical properties

Plant and Soil - The objective of this paper was to develop a method based on infrared spectroscopy to compare mineral content in soils and apply it to evaluate soil mineralogical variations in...     

Preparative enzymatic solid phase synthesis of cis(+)-12-oxo-phytodienoic acid - physical interaction of AOS and AOC is not necessary

The pathway of jasmonic acid (JA) biosynthesis was established in the 1980s by Vick and Zimmerman but, until now, the preparative biosynthesis of the jasmonic acid precursors 12-oxo-phytodienoic acid (OPDA) and 3-oxo-2-[2'-pentenyl]-cyclopentan-1-octanoic acid (OPC-8:0) in their endogenous and biologically relevant cis(+)-configuration was only possible in small amounts and had to put up with high costs. This was mainly due to the lack of high amounts of pure and enzymatically active allene oxide cyclase (AOC), which is a key enzyme in the biosynthesis of jasmonates in that it releases, in a coupled reaction with allene oxide synthase (AOS), the first cyclic and biological active metabolite - OPDA. We describe here the expression and purification of AOS and AOC and their subsequent coupling to solid matrices to produce an enantioselective, reusable bioreactor for octadecanoid production. With the method described here it is possible to produce optically pure enantiomers of octadecanoids in high amounts in a cost- and time-efficient manner. Furthermore, it could be demonstrated that a physical interaction of AOS and AOC, hitherto postulated to be required for substrate channeling from AOS to AOC, is not necessary for the in vitro cyclization of the unstable epoxide generated by the AOS reaction.     

Towards multidimensional liquid chromatography separation of proteins using fluorescence and isotope-coded protein labelling for quantitative proteomics

HPLC has emerged as a valuable tool for separating proteins. To address the analysis of complex proteomes and quantitative changes of proteins therein, we developed a multidimensional LC (MDLC)-based approach followed by large gel 1-D SDS-PAGE. Here we present a novel strategy that allows for simultaneously identifying and quantifying differentially regulated proteins following three separation and fractionation steps. This MDLC platform integrates advantages of dual protein labelling using both fluorescence and isotope-coded tags for subsequent detection and quantification of abundance ratios of proteins by MS.