Current Proteomics - Volume 2, Issue 4, 2005

Two dimensional polyacrylamide gel electrophoresis (2DE) has been a core technology of current proteomics for its high resolution and ability to detect proteins with post-translational modifications. This technology has been widely applied in numerous biomedical research projects including biomarker discovery and drug development. The application of narrow-range immobilized pH gradient strips (IPGs) and advanced detection methodologies have increased the resolution of 2DE technology. However, 2DE still suffers from its limitation in unambiguous detection of proteins of lowabundance. To improve this, sample preparation is the first and key step for successful 2DE analysis. This article summarizes current progresses in sample handling and prefractionation aiming to enrich proteins of interest according to their special physical and chemical properties so that the application of 2DE can be greatly extended.

Efficient sample preparation in the micro and nano-litre range, prior to analysis of proteins and peptides of biological origin by liquid chromatography (LC), liquid chromatography coupled to mass spectrometry (LC-MS), micro- liquid chromatography (μ-LC) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, is the key to success for different applications, such as biomarker discovery. Sample preparation in proteomics mainly comprises purification and preconcentration by solid-phase extraction (SPE) using different polymers (polystyrene, poly acrylate, cellulose, etc.) as a stationary phase following several cleaning-up procedures. These polymers can be derivatized with several functional groups, for example, C18, ion-exchanger or immobilized metal affinity chromatography (IMAC) groups. Based on these "traditional" techniques new methods enabling higher selectivity, sensitivity and speed of separation are required. Capillary coatings, for example, with latex particles has been used successfully. Derivatization of these latex particles, for example, with IMAC-functional groups enables a selective purification of phosphorylated or His-tagged proteins. Alternatively, polymeric disks possessing a thickness of approximately 2 mm can be used even in the case of multidimensional separations. In this review, we summarize currently available sample pretreatment techniques, provide an overview on the technical background and discuss the advantages of the individual methods using recently developed selective stationary phases such as silica based materials, polymers, etc.

Stable isotope labeling coupled with multidimensional liquid chromatography/tandem mass spectrometry is an advanced platform for global proteome-wide quantification. Compared to conventional methods, such as two dimensional polyacrylamide gel or electroblot based quantification methods, stable isotope labeling holds greater promise for accurate and large-scale quantitative analyses. Various labeling strategies have been developed to analyze protein expression, posttranslational modification, and protein/protein interactions, as well as for absolute quantification. There are advantages and disadvantages inherent with each method, but their applicability is dependent on many factors. In addition to the choice of which labeling chemistry to be used, there are several bottleneck issues associated with this approach that are of critical importance. These include finding effective multidimensional separation steps to resolve low abundant proteins present in relatively complicated mixtures, validating the methods, and interpreting the large amount of statistical data generated. This review covers the current progress in solving these concerns and summarizes the various labeling strategies and applications. We believe that a judicious integration of each component of the technique is crucial for the success of such a global systems approach. Based on the current progress, it is clear that the stable isotope labeling/mass spectrometry technique will find tremendous use in many fields, such as drug discovery, clinical diagnostics, disease prevention, basic biological research, and biotechnology.

Over many years, the protein components of pulmonary surfactant have been the subject of a large number of analyses using high resolution methods for protein analysis. In fact, identification of protein biomarkers for lung disorder is extremely important in order to gain insight into the mechanisms underlying lung diseases. For separation of protein components of pulmonary surfactant proteins, two-dimensional gel electrophoresis (2-DE) coupled with Western blot analysis involving electrophoretic transfer of the separated proteins onto a membrane followed by immunodetection of proteins by enhanced chemiluminescence has been used. This method allows very high resolution, sensitivity and specificity for proteomic study of pulmonary surfactant-associated proteins, particularly for water-soluble surfactant-associated protein-A (SP-A). Analysis of SP-A has also been carried out with 2-DE followed by either amino acid sequence analysis using Edman degradation or mass spectrometry. Other pulmonary surfactant-associated proteins, SP-B, SP-C and SP-D were identified using either gel electrophoresis based or non-gel-electrophoresis based methods, such as high performance liquid chromatography. This review summarizes the major achievements in proteomic studies of pulmonary surfactantassociated proteins.

A novel method of structure prediction for membrane-bound proteins is reviewed. The approach is based on a sequence-function analysis, secondary structure prediction and subsequent geometry optimization. The prediction of the structure of ligand-gated P2X4 receptor subunit, which is a membrane-embedded cation channelforming protein with extracellularly occuring ATP binding sites, is shown as an example. The potential Nglycosylation sites of the glycoprotein, location of the five disulfide bridges, and phospholipid-dependent protein kinase C (PKC) phosphorylation attachment sites were determined by sequence-function analysis. Subsequently an attempt was made to predict its conformation using homology-based comparative modeling and threading; however, the modeling could not be accomplished. Because of this, secondary structure prediction of the protein was carried out. The input coordinates of the spatial structure were obtained by a profile-based neural network prediction method. The resulting secondary structure was converted into a three-dimensional geometry. The secondary and tertiary structures were optimized by the quantum chemistry RHF/3-21G minimal basic set and all-atom molecular mechanics AMBER96 force field. The predicted shape is similar to the shape of the experimentally obtained monomeric structure of the classical ion channel, the K+ion channel from Streptomyces lividans (KcsA channel), and agrees with the P2X shape proposed by biological experimenters. The geometry optimized structure of the P2X4 receptor is freely available (Protein Data Bank format) from the authors on e-mail request ([email protected]. de).

Plants need to perceive and process information both from the biotic and abiotic surroundings for their optimum growth and development. Because plants are not motile, they have to be especially responsive to environmental changes, including stress conditions. Subjecting rice seedlings to environmental stresses results in various biochemical changes, many of which are poorly understood. Proteomics approaches to identifying proteins that are regulated in response to different environmental conditions are becoming common in the post-genomic era in rice research. This review describes initial steps toward determining the physiological significance of some proteins identified from rice.