Challenges facing the development and use of protein chips to analyze the phosphoproteome

Recent advances in analytical methods, particularly in the area of protein microarrays, have brought the field of proteomics to the forefront of biological science. Protein arrays have shown to be useful for the multiplexed analysis of several hundreds of proteins in parallel. While much of the effort has focused on developing methods to identify expressed proteins, the identification of post-translational modifications is equally important for comprehensive proteome characterization. Protein phosphorylation constitutes a major type of post-translational modification that mobilizes a high number of genes, is involved in many crucial cell functions and largely contriubutes to the complexity of the proteome. One of the major challenges to analyze phosphoproteins using arrays is the availability of specific antibodies. Thus far, this has hampered the development of highly complex phosphoprotein arrays. This review discusses some of the recent progress made in the development of techniques and reagents to quantitatively determine sites of protein phosphorylation.     

Challenges facing drug development for malaria

Malaria is a significant cause of morbidity and mortality in the developing world. Until recently malaria was winning but with increase in funding particularly from philanthropic groups the ability to control malaria is again possible. There are still many challenges to developing the next generations of anti-malarials. This article will briefly discuss the challenges and the advance that are being made.     

Challenges facing sex preselection of stallion spermatozoa

Since the production of the first live offspring from sex-sorted spermatozoa in 1989, there have been many developments in the fluorescence-activated cell separation (FACS) procedures to preselect X- and Y-chromosome bearing spermatozoa prior to insemination. During this time, FACS technology has been applied to a range of species and has resulted in offspring from rabbits, cattle, sheep, elk and horses. In horses, satisfactory fertility rates have been achieved after hysteroscopic insemination of 20 x 10(6) fresh or stored, sex-sorted spermatozoa. However, many of the sperm processing protocols are still based on the original protocol and components of these procedures may not necessarily be suitable for the stallion. This review examines the details of FACS protocols that have resulted in the production of live offspring and makes comparisons with the published stallion protocols in an attempt to determine how best to improve the fertility of sorted, frozen-thawed stallion spermatozoa.     

Tools to analyze protein farnesylation in cells

Farnesylation of proteins is catalyzed by protein farnesyl transferase (FTase) and is obligatory for the function of the oncoprotein Ras and a variety of other physiologically important proteins. The rapid and selective detection of cellular protein farnesylation status is crucial to understanding both the function of farnesylated proteins and FTase inhibitors. The unnatural FPP analogue 8-anilinogeranyl diphosphate (AGPP, 3b) is an effective alternative substrate for mammalian FTase. Using antibodies specific for the anilinogeranyl moiety, we show that the alcohol precursor (AGOH, 5b) of AGPP is incorporated into cellular proteins in an FTase dependent manner competitive with endogenous pools of FPP. Continuous treatment of HEK-293 cells with 100 microM AGOH for up to 3 days is neither cytotoxic or cytostatic. Antibodies to detect the unnatural anilinogeranyl group were raised against bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) bioconjugates of the activated hapten N-hydroxyphthalimido-succinyl-(S-anilinogeranyl)-L-cysteine methyl ester 9a. Polyclonal antisera containing anti-anilinogeranyl antibodies were generated by immunization of rabbits and a monoclonal anti-anilinogeranyl antibody was raised in mice. ELISA and western blotting of anilinogeranyl modified proteins were used to show the selectivity and measure the titer of the antibodies. The unnatural FPP analogue and corresponding antibodies provide a simple and rapid method for monitoring FTase activity in cells and detection of cellular proteins modified by AGOH 5a.     

The use of antibodies to the polypyrimidine tract binding protein (PTB) to analyze the protein components that assemble on alternatively spliced pre-mRNAs that use distant branch points.

We are using the rat beta-tropomyosin (beta-TM) gene as a model system to study the mechanism of alternative splicing. Previous studies demonstrated that the use of the muscle-specific exon is associated with the use of distant branch points located 147-153 nt upstream of the 3' splice site. In addition, at least one protein, the polypyrimidine tract binding protein (PTB), specifically interacts with critical cis-acting sequences upstream of exon 7 that are involved in blocking the use of this alternative exon in nonmuscle cells. In order to further study the role of PTB, monoclonal antibodies to PTB were prepared. Anti-PTB antibodies did not inhibit the binding of PTB to RNA because they were able to supershift RNA-PTB complexes. To determine if additional proteins interact with sequences within the pre-mRNA, 35S-met-labeled nuclear extracts from HeLa cells were mixed with RNAs and the RNA-protein complexes were recovered by immunoprecipitation using antibodies to PTB. When RNAs containing intron 6 were added to an 35S-met-labeled nuclear extract, precipitation with PTB antibodies showed a novel set of proteins. By contrast, addition of RNAs containing introns 5 or 7 gave the same results as no RNA, indicating that these RNAs are unable to form stable complexes with PTB. These results are in agreement with our previous studies demonstrating that PTB interacts with sequences within intron 6, but not with sequences within introns 5 and 7. When 35S-met-labeled HeLa nuclear extracts were mixed with biotinylated RNA containing intron 6 and the RNA-protein complexes were recovered using streptavidin-agarose beads, an identical pattern of proteins was observed when compared with the immunoprecipitation assay. Analysis of the proteins that assembled on introns 5, 6, or 7 using biotinylated RNA revealed a unique set of proteins that interact with each of these sequences. The composition of proteins interacting with sequences associated with the use of the 3' splice site of intron 6 included proteins of 30, 40, 55, 60, 65, 70, 80, and 100 kDa. Microsequencing identified two of the proteins to be Sam68 and the Far Upstream Element Binding Protein (FBP) from the c-myc gene. In addition, a comparison of the proteins that assemble on introns from the alpha- and beta-TM genes that utilize distant branch points revealed common as well as unique proteins that assemble on these introns. These studies identify a set of proteins, in addition to PTB, that are likely involved in the use of distant branch sites associated with the use of alternatively spliced introns.     

Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome

In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling networks also requires quantitation of these phosphorylation events. In this article, we outline several methods for enrichment of phosphorylated proteins and peptides and discuss various options for their identification and quantitation with special emphasis on mass spectrometry-based techniques.     

Generation of bioreagents for protein chips

Protein microarrays have the potential to dramatically increase the throughput of proteomic analysis. Protein expression profiling chips with distinct spots of immobilized protein capture agents will allow the simultaneous measurement of hundreds to thousands of proteins from one sample. In contrast to DNA chips, for which the capture probes are easily designed and synthesized, the development of content for protein biochips is a long and laborious process. Careful consideration must be given to the specificities desired, the format of the assay, and the requirements of the capture agents, as well as to process optimization to minimize development time and cost. Monoclonal antibodies have been the prime choice as protein capture agents for the majority of protein chips developed to date. New technologies for the production of protein capture agents are more amenable to automation than traditional monoclonal antibody production and therefore carry the promise for industrialization.     

A new computational approach to analyze human protein complexes and predict novel protein interactions

We propose a new approach to identify interacting proteins based on gene expression data. By using hypergeometric distribution and extensive Monte-Carlo simulations, we demonstrate that looking at synchronous expression peaks in a single time interval is a high sensitivity approach to detect co-regulation among interacting proteins. Combining gene expression and Gene Ontology similarity analyses enabled the extraction of novel interactions from microarray datasets. Applying this approach to p21-activated kinase 1, we validated α-tubulin and early endosome antigen 1 as its novel interactors.     

An effort to use human-based exome capture methods to analyze chimpanzee and macaque exomes

Non-human primates have emerged as an important resource for the study of human disease and evolution. The characterization of genomic variation between and within non-human primate species could advance the development of genetically defined non-human primate disease models. However, non-human primate specific reagents that would expedite such research, such as exon-capture tools, are lacking. We evaluated the efficiency of using a human exome capture design for the selective enrichment of exonic regions of non-human primates. We compared the exon sequence recovery in nine chimpanzees, two crab-eating macaques and eight Japanese macaques. Over 91% of the target regions were captured in the non-human primate samples, although the specificity of the capture decreased as evolutionary divergence from humans increased. Both intra-specific and inter-specific DNA variants were identified; Sanger-based resequencing validated 85.4% of 41 randomly selected SNPs. Among the short indels identified, a majority (54.6%-77.3%) of the variants resulted in a change of 3 base pairs, consistent with expectations for a selection against frame shift mutations. Taken together, these findings indicate that use of a human design exon-capture array can provide efficient enrichment of non-human primate gene regions. Accordingly, use of the human exon-capture methods provides an attractive, cost-effective approach for the comparative analysis of non-human primate genomes, including gene-based DNA variant discovery.     

Application of metal-chelate affinity chromatography to the study of the phosphoproteome

Summary. With the increasing importance of proteome analysis, studying the phosphoproteome is a priority for functional studies. Therefore, a rational approach to simplifying the proteome is needed. In this work, we examined the use of immobilized metal affinity chromatography (IMAC) using ferric ions-chelated column for enriching crude cell extracts in phosphoproteins. The adsorption of the proteins on Fe³⁺ was obtained at an acidic pH 5.6, and their elution at a more basic pH in Tris buffer. To evaluate the separation, western blots were performed with either anti-phosphotyrosine or anti-phosphoserine/threonine. The analysis of the eluates demonstrated the selectivity of the separation, particularly for proteins phosphorylated on serine or threonine. In conclusion, the advantages and the limits of this approach are discussed.     

An advantage for use of isotope labeling and NMR chemical shifts to analyze the structure of four homologous IgG-binding domains of staphylococcal protein A

Because of the complexity arising from the large molecular size and the amino acid sequence homologies of IgG-binding domains of Staphylococcal Protein A (SpA), we have introduced, a combination of stable isotope labeling and both qualitative and quantitative investigations of the structural dependence of the NMR chemical shifts for its structure analysis. In order to enable selective isotope labeling with high efficiency, a mutated low molecular weight Protein A (LPA; MWt = 27 kDa) which consists of E, D, A, B and 13 residues of the C-domain was used in this study. Amide proton chemical shifts, measured using uniformly 15N-labeled LPA and LPA labeled selectively with 15N-alanine, show that the turn between helices 1 and 2, and its tertiary interactions with helix 3, are very similar in all domains. This contradicts previous results obtained using independent structure calculations on isolated domains. The close similarity in NH and 15N chemical shifts of alanine residues in the interdomain linker suggests that the linker maintains a similar structure both in isolated domains and in the intact protein. We show that the high-field shifted methyl signal of Ala 48 is affected by the ring-current effect arising from Phe 30, and has a very similar helical environment in all four domains. Thus, helix 3 is present in all domains, as we previously reported [Kikuchi et al., J Biochem Biophys Method, 1999:38:203-208], even though it is not observed in the crystal structure [Deisenhofer J. Biochemistry 1981;20:2361-2370].     

不同经济类型高粱的RAPD分析

对四种不同经济类型高粱采用ＤＮＡ随机扩增多态技术（ＲＡＰＤ）进行了比较研究。１８种引物扩增后共得到５４个ＲＡＰＤ标记,其中２３个标记在进行４种高粱成对比较时呈多态。根据计算出的遗传距离值可以发现虽然不同种高粱之间差异虽然较小,但仍具有丰富的遗传多样性。