A new methodology, affinity capillary electrophoresis-mass spectrometry (ACE-MS), is introduced as a solution-based approach for screening combinatorial libraries for drug leads. The method allows on-line, one-step selection and structural identification of candidate ligands. ACE-MS is demonstrated using the binding of vancomycin to libraries of all-D-tri-and tetrapeptides as a model system. Peptide libraries of different forms of Fmoc-DDXX and Fmoc-EXX containing up to 361 compounds were successfully employed to determine interacting structural motifs. A consensus structure of the strongest interacting peptides consisted of D-Ala at the C-terminus and an aromatic amino acid in the penultimate position. Ligands with this structure bound more strongly to the receptor than the known ligand, D-Ala-D-Ala. A 1000 peptide library was also screened directly by ACE-MS. It was found that, for this and potentially larger libraries, incorporating an affinity solid phase extraction step prior to ACE-MS was effective in both removing a large number of non-interacting species as well as preconcentrating sample components for sequence determination by MS.

Polyhydroxyalkanoate synthase (PHA) from Chromatium Vinosum catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) and CoA. The synthase is composed of a ∼1:1 mixture of two subunits, PhaC and PhaE. Size-exclusion chromatography indicates that in solution PhaC and PhaE exist as large molecular weight aggregates. The holo-enzyme, PhaEC, has a specific activity of 150 units/mg. Each subunit was cloned, expressed, and purified as a (His) 6 -tagged construct. The PhaC-(His) 6 protein catalyzed polymerization with a specific activity of 0.9 unit/mg; the PhaE-(His) 6 protein was inactive (specific activity <0.001 unit/mg). Addition of PhaE-(His) 6 to PhaC-(His) 6 increased the activity several 100-fold. To investigate the priming step of the polymerization process, the PhaEC was incubated with a trimer of HB-CoA in which the terminal hydroxyl was replaced with tritium ( Sequencing by ion trap mass spectrometry showed that they were identical and that they each contained an altered cysteine (C149). One peptide contained the [ 3 H]-sT while the other two contained, in addition to the [ 3 H]-sT, one and two additional monomeric HBs, respectively. Mutation of C149 to alanine gave inactive synthase. The remaining two cysteines of PhaC, 292 and 130, were also mutated to alanine. The former had wild-type (wt) activity, while the latter had 0.004 wt % activity and was capable of making polymer. A mechanism is proposed in which PhaC contains all the elements essential for catalysis and the polymerization proceeds by covalent catalysis using C149 and potentially C130.Polyhydroxyalkanoates (PHAs 1 ) are polyoxoesters with properties that range from elastomers to thermoplastics (1-4). They are produced by a wide range of bacteria when they are placed in an environment of nutrient limitation (5). Copolymers of polyhydroxybutyrate (PHB) and polyhydroxyvalerate in the correct ratio have properties similar to the petrochemically based polypropylenes, the major component of bulk plastics (6). In 1997, the US market for thermoplastics was on the order of 40 million tons per year (7). PHAs have recently received much attention because they are biodegradable and can be generated from biorenewable sources: bacteria and plants (8). The major focus of many investigators is to make their production economically competitive with the polypropylenes. To achieve this goal, the requirements for the polymerization process need to be established. This paper focuses on the PHA synthase from Chromatium Vinosum which catalyzes the formation of PHB from 3-hydroxybutyryl-CoA (HB-CoA). Evidence is presented that two cysteines and covalent catalysis play an important role in the initiation and elongation of the polymerization process.PHA synthases from 20 organisms have now been identified. They have been divided into three classes based on their substrate specificity and subunit composition (9). The class I synthases, with the Ralstonia eutropha synthase as a prototype, are composed of a single polypeptide (∼65 k...

All stakeholders in cultural heritage share an interest in fabrication methods and material technology. Until now methods for analysis of organic materials, particularly proteins, have not been widely available to researchers at cultural institutions. This paper will describe an analytical method for the identification of collagen-based materials from soft tissue sources and show examples of its application to diverse museum objects. The method, peptide mass fingerprinting (PMF), uses enzymatic digestion of extracted proteins to produce a mixture of peptides. The mass spectrum of the mixture contains characteristic marker ions-a peptide mass fingerprint-which are compared to species-specific markers from references as the basis of identification. Preliminary results indicate that analysis of materials from aged samples, several different tissue types, and tanned or untanned materials yields comparable PMF results. Significantly, PMF is simple, rapid, sensitive and specific, has been implemented in a museum laboratory, and is being practiced successfully by non-specialists.

No abstract

Over the past two decades, centrifugal microfluidic systems have successfully demonstrated their capability for robust, high-performance liquid handling to enable modular, multi-purpose lab-on-a-chip platforms for a wide range of life-science applications. Beyond the handling of homogeneous liquids, the unique, rotationally controlled centrifugal actuation has proven to be specifically advantageous for performing cell and particle handling and assays. In this review we discuss technologies to implement two important steps for cell handling, namely separation and capturing/counting.