Current Analytical Chemistry - Volume 3, Issue 1, 2007

With over 500 members catalyzing an estimated 100,000 phosphorylation reactions, the kinases are among the largest and most complex families of enzymes. As phosphorylation represents the pivotal mechanism for regulation of biological processes, they are also one of the most biologically significant. Development of analytical techniques for the characterization of phosphorylation events, in particular from a global vantage point, is therefore a central priority for proteomic and cell biology researchers. There are two differing philosophies on the most appropriate perspective for global phosphorylation analysis, either through characterization of the phosphoproteome, the sub-population of proteins that undergo phosphorylation, or the kinome, the activities of the cellular protein kinases that catalyze phosphorylation. While each of these approaches strives to describe the same biological event, they employ unique experimental approaches to provide distinct, yet complimentary information. Here we review recent advances in each area, highlight their relative strengths and weaknesses, and discuss their complimentary nature for global phosphorylation analysis.

Sensitive yet selective techniques for characterizing protein unfolding are important for assessing the conformational stability of wild-type and recombinant protein. This review presents a summary of recent developments in protein unfolding by capillary electrophoresis (CE). Relative to conventional spectroscopic methods, CE offers a versatile microseparation format for performing unfolding studies using small amounts of protein mixtures based on thermal or chemical denaturation. The fundamental theory of protein unfolding in CE, as well as correction factors required to normalize non-specific changes in apparent mobility are examined in this review. The integration of in-capillary ligand stripping with dynamic protein unfolding by CE provides a convenient way to characterize holoproteins without sample pretreatment. CE is also a useful format for resolving protein conformational intermediates involving multimeric proteins or oligomers that is relevant to understanding the dynamics of misfolded proteins. This review covers protein unfolding and conformational studies from 1991-2006 with emphasis on recent developments in CE that highlight its significance as a complementary biophysical tool for protein characterization.

Since ancient times many natural organic materials have been used in artistic and historic works as binders, adhesives, fillers and coatings. The identification of these materials is important not only for a proof of authenticity; for restorers and conservators it is essential to recognize the materials and technologies employed by artists and craftsmen. For identification and characterisation of the different natural organic binders spectrometric and chromatographic methods are well established. Recently, capillary electrophoresis has been introduced as an alternative technique to receive analytical information about the kind and composition of the binding media used in artefacts. These materials are waxes, resins, drying oils, animal glues and plant gums. In the present review the application of capillary electrophoresis in this area is discussed against the background of a general survey of the more common analytical techniques.

A simple, rapid, selective and sensitive electrochemical method for the direct determination of prednisolone in spiked human serum and tablets was developed. The electrochemical oxidation and determination of prednisolone has been carried out at glasy carbon electrode (GCE) in various aquaeous solution in the pH range of 0.56-12.30 by cyclic (CV) and Osteryoung square wave voltammetry (OSWV). The best results were obtained for the determination of prednisolone by OSWV method in 0.5 mol L-1 sulphuric acid at about pH 0.56. The peak current and peak potential depends on pH, so its influence and also scan rate were studied. The diffusion controlled by nature of the peak was established. This electroanalytical procedure enabled to determine prednisolone in the concentration range 1.0 x10-6- 2.0x10-5 mol L-1. This limit of detection (LOD) and limit of quantification (LOQ) were obtained as 3.4x10-7 and 4.5x10-7 mol L-1 respectively. Precision and accuracy of the developed method were checked by recovery studies in spiked tablets and human serum.

Heteronuclear coherence transfer in liquid-state NMR applications has been traditionally performed using pulse-interrupted delay schemes such as INEPT-type pulse trains. So far, the alternative use of heteronuclear crosspolarization (HCP) has only been limited to a few cases involving exclusively in-phase to in-phase transfers. In this revision work a theoretical description on the effect and the characteristic anisotropic features of HCP is introduced in terms of product operator formalism. A very intuitive and simple graphical black-box approach based on a pictorial nonclassical vector representation that only consider the available input/output magnetization is also presented to understand the general transformations that are undergoing under such rather complex HCP processes. The appropriate manipulation of magnetization components during the HCP block offers novel concepts in pulse sequence design. In this way, new liquid- state multidimensional NMR methods incorporating HCP-driven processes have recently been developed and successfully applied for both small-to-medium-sized molecules and large labeled bio-molecules, as will be discussed in this review.

One of the most important ecological problems is mutagenic pollution in the environment, and the determination of mutagenic compounds in environmental samples is of special interest. In the current article, a review of the liquid chromatography-mass spectrometry (LC/MS) based methods published so far for the determination of mutagens in the environment is presented. Mutagens included in this review are polycyclic aromatic compounds, heterocyclic aromatic amine compounds, azo dyes, aldehydes and pesticides. Advanced aspects of current analysis of mutation research using LC/MS, including analysis of urinary metabolites of mutagens and DNA modification, are also discussed.

Early studies in to lipids that comprised cellular membranes noted an asymmetrical distribution of lipids between different membranes which contrasted with the contemporary concept of the plasma membrane as a homogeneous mixture of lipid in which both lipids and membrane-associated proteins exhibit unfettered mobility. The ability of lipids to form inhomogeneities capable of restricting lateral mobility has since been demonstrated in both model and cell membranes. It has further been shown that lipids possess the ability to preferentially associate and co-exist in domains rich in particular classes of lipid, most notably cholesterol, sphingolipids, and glycolipids. In cells, ‘rafts’ further appear to be enriched in raft-targeted proteins. These observations have led to the hypothesis that phase-separated domains may act as signaling platforms capable of recruiting proteins thus facilitating their interaction for transduction of cellular signals. Experimental evidence largely based upon detergent isolation of raft domains has since been provided implicating laterally organized lipid domains in a litany of both physiological and pathophysiological processes including T-cell activation, B-cell antigen signaling, thromboregulation, Alzheimers disease and atherosclerosis. However, the field is often criticized for interpretations which may lack physiological relevance due to artifact arising from isolation procedures. Thus, investigators are increasingly looking to spectroscopic techniques to obtain information about phase-separated domains. This review seeks to summarize the current knowledge and understanding of raft structure, and provide a first foray in to the fundamentals and application of spectroscopic and microscopic techniques to characterize attributes and dynamics of cellular lipid microdomains.