Current Proteomics - Volume 5, Issue 3, 2008

Many biological functions are regulated by tyrosine phosphorylation: cell proliferation, migration, differentiation, and tumorgenesis among them. Hence, aspects of cellular behavior may be illustrated by monitoring the global dynamics of cellular tyrosine phosphorylation in response to stimuli. In this review, we describe an approach that combines phosphoprotein/peptide enrichment with stable isotope labeling which can quantify unambiguously those changes in phosphorylation status that occur specifically in response to stimulation by growth factor, autocrines, drug treatment or cellular physiology. Current technologies for enrichment of phosphoproteins and peptides including phosphotyrosine or phosphospecific antibodies, SH2 domains, metal oxide and IMAC chemistries coupled to magnetic particles are discussed. Phosphopeptides from cultures supplemented with either light or heavy lysine and arginine appear in mass spectra as sequence-matched isotopomers, but are chemically identical and co-migrate in any other separation. Hence, the quantification of stimulated phosphorylation (dephosphorylation) at specific sites of a protein may be accomplished with MScoupled chromatographic separation by comparing the relative peak area response of sequence matched phosphopeptides arising from two (or more) cell states. Furthermore, we illustrate a crossover methodology that may be used to disentangle true protein binding partners from nonspecific interactions, which is a common problem in affinity enrichment endeavors.

“Packing defects” which result in the presence of a cavity in the protein continue to be a subject of great interest. Naturally occurring or engineered cavities in the protein structure are empty or hydrated may depend on several factors (size, hydrophobicity, etc.) and is currently the subject of active debate. The role of protein cavities is considerable controversy and still needs further investigation. The presence of internal cavities appears to be crucial for conferring the conformational flexibility needed for their biological function. In several studies, stabilities of the protein could be improved by cavity filling mutations but concomitant loss of functionality, indicating their role in stability-function tradeoff. In this review, we hope to bring together the experimental studies available on protein cavities.

Proteome analysis plays a key role in the elucidation of the functions and applications for numerous proteins. For proteome analyses, various microplate- and microarray-based techniques have been developed by a number of researchers. Their intent was to immobilize proteins on the surface of a solid substrate in a site-directed manner while retaining structure and native biological function. In this review, we focus on recent advances in immobilization methodology for proteins/enzymes on a surface, including those using the affinity peptides screened by random peptide library systems. We also discuss applications of the affinity peptide-mediated immobilization method in fields related to proteome analysis, particularly our recent work concerning immunoassay and protein-protein interaction analysis.

The sulfur compound metabolizing sox operon of microbes consists of two transcriptional units. Recently a new orf, which code for protein named ORF1, was identified in Pseudaminobacter salicylatoxidans which is one of the most prominent sulfur oxidizers. Sequence analyses reveal ORF1 has the signature sequence of the dsr family of sulfate ion binding proteins. It is present in an analogoues position as the ORF1 of Starkeya novella. There are no studies regarding the structural biology ORF1 of Pseudaminobacter salicylatoxidans. In order to elucidate the structural aspects of the mechanistic details of of the protein, homology modeling technique has been employed to construct the three-dimensional structure of ORF1. ORF1 from Starkeya novella is known to interact with the transcriptional activator SigE which is known to bind to its adjacent promoter DNA sequence and thereby regulate the sox gene cluster in Starkeya novella. Pseudaminobacter salicylatoxidans does not contain the SigE protein but instead possesses another transcriptional activator SoxR which performs similar function as SigE. In order to verify whether ORF1 of Pseudaminobacter salicylatoxidans serves the similar function as that of Starkeya novella by molecular docking analyses have been performed using the models of ORF1 and SoxR to predict the favourable binding interactions of the proteins. The putative sulfate ion binding residues of ORF1 has also been identified by docking sulfate ion on to it. This study provides a rational framework for understanding of the structural as well as the molecular basis of the mechanism of the regulation of sulfur oxidation reactions ORF1 proteins via the sox operon.

Elucidating protein structure and function relationships from sequence data, and predicting protein structure and function by an automatic, high-throughput means pose important and imminent challenges for structural proteomics. In recent years, there has been growing interest in applying network concepts and theory to meet this challenge. The network approach transforms a protein structure into a network by representing each amino acid residue as a node and each residue-residue interaction with an edge connecting two residues. Through this transformation, insights into various aspects of protein structure and function are explored from the perspective of networks. Current applications of the network approach include: understanding protein stability, studying protein folding, developing scoring functions for structure discrimination, predicting protein functional sites and analyzing protein-protein and protein-DNA complexes. The network approach is computationally efficient and proves to be a useful tool in structural proteomics. This review covers the applications of the network approach in the field of structural proteomics.

Proteomics and the technology supporting this science are proving to be invaluable in elucidating the tremendous complexity of biological processes. Among proteomic techniques, two-dimensional electrophoresis (2-DE) has been applied to resolve thousands of proteins and 2-DE has been especially useful for comparative studies between paired samples or populations. Improved protein extraction and purification protocols for recalcitrant fruits and vegetables have resulted in 2-DE techniques appearing in almost all fruit and vegetable proteomic studies currently being published. Numerous proteins involving various metabolic pathways have already been reported for tomato, pepper, strawberry, grape, banana, apple and pear. Significant improvements have been made to both gel and non-gel based proteomic research platforms. Better quantitative analyses and higher throughput proteomic technologies will further improve fruit and vegetable proteomic research and will have a significant impact on future breeding and post-harvest handling technologies to improve nutritional and eating quality.

Proteomics research typically starts by reducing the sample complexity through multidimensional separation methods based on the unique characteristics of the proteins or their peptides followed by identification and quantification. Protein-carbohydrate bindings play very important roles in cell-cell communication, cell proliferation and differentiation as well as microbial adhesion. Therefore, carbohydrates can serve as protein capturing ligand for protein purification and identification and protein-carbohydrate binding research provides important insights for basic biological research, medicine and the biotechnology as well as drug development. In the past decades, a diverse carbohydrate-based technology, termed glycotechnology, such as glyco-arrays and glyco-affinity chromatography, and glyco-affinity probe, have been developed. A guiding principle of these glycotechnologies is the display of carbohydrates onto a pertinent support or carrier such as glass slide, microplate, and microbeads as well as functional polymers and probe molecules. This review highlights recent progresses of carbohydrate-containing intelligent polymers for rapid and efficient glyco-affinity precipitation purification of carbohydrate-binding protein and the enhanced proteomics research. Specifically, two innovation methods, inverse reversible thermal-responsive glyco-affinity precipitation and magnetic assistant glyco-affinity precipitation purification of carbohydrate-binding protein for enhanced proteomics application are summarized.