PubMed Health⌕ Search

Biomedical subjects

J X Yan

Publications and source records attributed to J X Yan.

29 records · Page 2Linked to original sources
PubMed1995

Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium.

A protein map of the smallest known self-replicating organism, Mycoplasma genitalium (Class: Mollicutes), revealed a high proportion of acidic proteins. Amino acid composition was used to putatively identify, or provide unique parameters, for 50 gene products separated by two-dimensional gel electrophoresis. A further 19 proteins were subjected to peptide-mass fingerprinting using matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry and 4 were subjected to N-terminal Edman degradation. The majority of M. genitalium proteins remain uncharacterised. However, the combined approach of amino acid analysis and peptide-mass fingerprinting allowed gene products to be linked to homologous genes in a variety of organisms. This has allowed proteins to be identified prior to detection of their respective genes via the M. genitalium sequencing initiative. The principle of 'hierarchical' analysis for the mass screening of proteins and the analysis of microbial genomes via their protein complement or 'proteome' is detailed. Here, characterisation of gene products depends upon the quickest and most economical technologies being employed initially, so as to determine if a large number of proteins are already present in both homologous and heterologous species databases. Initial screening, which lends itself to automation and robotics, can then be followed by more time and cost intensive procedures, when necessary.

Amino Acids↗
PubMed1990

Characterization of alpha and beta crystalline forms of nicardipine hydrochloride.

The aim of this work was the characterization of alpha and beta crystalline forms of nicardipine hydrochloride (coronary vasodilator drug) by means of DSC analysis, X-ray diffraction patterns, TLC analysis and IR spectroscopy. The results obtained show that it is possible to distinguish the two forms by means of these techniques.

Chromatography, Thin Layer↗
PubMed1986

The use of micropeptide maps and monoclonal antibodies for antigenic variation analysis of influenza A viruses isolated in China from 1972 to 1983.

125I-labelled haemagglutinin (HA) micropeptide mapping and haemagglutinin inhibition (HAI) tests with monoclonal antibodies were used in analysing antigenic variation of six strains of influenza A virus isolated from 1972 to 1983 in China. The results from micropeptide mapping generally coincided with those of sequence analysis. 125I-labelled HA micropeptide mapping is a simple, reproducible and practical method for surveillance of influenza, especially in combination with HAI test using monoclonal antibodies.

Amino Acid Sequence↗
PubMedDate not supplied

Modified immobilized pH gradient gel strip equilibration procedure in SWISS-2DPAGE protocols.

In the present paper we report a revised protocol for immobilized pH gradient (IPG) gel strip equilibration involving a procedural modification between the first- and second-dimensional separation in both analytical and preparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). By changing the pH of the equilibration buffer (pH 8.0), the concentration of alkylating reagent (125 mM iodoacetamide) and the time of incubation (15 min), it has been possible to achieve increased cysteine (Cys) alkylation to completion with only one adduct of carboxyamidomethyl-Cys formed. Importantly, the modification does not alter the 2-D proteome patterns and therefore maintains the integrity of the existing SWISS-2DPAGE entries. Results are presented for comparative analyses using human plasma, and for Cys analysis of human albumin to illustrate the advantages of the improved protein reduction and Cys alkylation. The modified step of IPG gel strip equilibration will assist protein digestion for matrix-assisted laser desorption/ionisation - time-of-flight - mass spectrometry analysis, and make Cys quantitation possible without further in-gel or on-blot alkylation.

Blood Proteins↗
PubMedDate not supplied

Studies of quantitative analysis of protein expression in Saccharomyces cerevisiae.

In the present study amino acid analysis is applied to quantitation of Saccharomyces cerevisiae proteome expression. The quantitation levels obtained are compared to data using densitometric analysis of silver or amido black staining and to the theoretical expression level (codon bias) of the identified proteins determined from their amino acid analysis (AAA). The results show that relative volume ratio (%vol) using Melanie II is a better parameter for spot quantitation than relative optical density ratio (%OD), and amino black staining provides good linearity within the range 1-100 pmol protein. However, AAA shows that theoretical expression levels are not well correlated with actual protein expression level, although there is better correlation when isoforms of the expressed protein are identified and included. It is concluded that amino acid analysis provides accurate protein quantitation and has a continuing role in proteome studies in terms of the rapid and inexpensive quantitation of proteins displayed on proteome maps. We do however recognize that in the context of future clinical applications and large-scale proteome discovery projects, quantitation and post-translational modification need to be analyzed by 'proteomatic' (i.e., proteome automatic bioinformatic analysis directly from the gel) techniques.

Electrophoresis, Gel, Two-Dimensional↗
PubMedDate not supplied

Method for identification and quantitative analysis of protein lysine methylation using matrix-assisted laser desorption/ionization--time-of-flight mass spectrometry and amino acid analysis.

Protein methylation is a post-translational modification that might have important functional roles in cell regulation. We present a new technique with sufficient sensitivity (sub-pmol level) for analysis of methylation of proteins in abundances typically found on proteome maps produced by two-dimensional (2-D) gel electrophoresis. The method involves the identification and quantitation of lysine (Lys) methylation using Fmoc (9-fluorenylmethyl chloroformate)-based amino acid analysis (AAA). Tri- and monomethyl-Lys were baseline-separated from other amino acids using a modified buffer system. Trimethyl-Lys was quantitatively recovered after acid hydrolysis and AAA of two known methylated proteins - yeast cytochome c and human calmodulin. The methylated peptides from tryptic digestion of those two proteins were identified by high sensitivity matrix-assisted laser desorption/ionization - time-of-flight (MALDI-TOF) mass spectrometry (MS). An automated mass-screening approach is proposed for the study of various post-translational modifications to understand the distribution of those protein isoforms separated by two-dimensional polyacrylamide gel electrophoresis. It is concluded that the combination of AAA and MALDI-TOF-MS provides a high sensitivity quantitative tool for the analysis of protein post-translational methylation in the context of proteome studies.

Amino Acid Sequence↗
PubMedDate not supplied

Bovine dilated cardiomyopathy: proteomic analysis of an animal model of human dilated cardiomyopathy.

Bovine hereditary dilated cardiomyopathy (bCMP) is endemic in Switzerland and hearts from diseased animals display important clinical and biochemical similarities to human DCM. Recent research has identified at least one protein (myoglobin) to be significantly reduced in bovine DCM. Using a proteomic approach, we have separated over 1125 protein species from bovine ventricular tissue. Gel analysis and protein characterisation have identified a number of proteins whose abundance is significantly altered in bovine DCM. Twenty-four proteins are of decreased abundance in diseased tissue, whilst 11 proteins are of increased abundance in the diseased state. A combination of amino acid compositional analysis, peptide mass profiling, N-terminal microsequencing and MultiIdent (http://www.expasy.ch/sprot/multiident. html) has been employed in order to elucidate the identities of the differentially expressed proteins. Using these techniques we have currently determined the identity of 12 of the 35 altered proteins. We have also detected three proteins that are differentially expressed in genotypically diseased but phenotypically normal animals, identifying a possible mechanism for the onset of the disease. The possibility that inappropriate ubiquination of proteins plays an important role in the disease is discussed. A database of bovine proteins is currently being established. The identity of the proteins affected, together with a comparison of the human and bovine expression patterns, is displayed.

Animals↗
PubMedDate not supplied

The Dictyostelium discoideum proteome--the SWISS-2DPAGE database of the multicellular aggregate (slug).

The cellular slime mold Dictyostelium discoideum is a eukaryotic microorganism which has developmental life stages attractive to the cell and molecular biologist. By displaying the two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein map of different developmental stages, the key molecules can be identified and characterised, allowing a detailed understanding of the D. discoideum proteome. Here we describe the preparation of reference gel of the D. discoideum multicellular aggregate, the slug. Proteins were separated by 2-D PAGE with immobilised pH gradients (pH 3.5-10) in the first dimension and sodium dodecyl sulfate (SDS)-PAGE in the second dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride (PVDF) membranes and 150 spots were visualised by amido black staining. Protein spots were excised and 31 were putatively identified by matching their amino acid composition, estimated isoelectric point (pI) and molecular weight (M(r)) against the SWISS-PROT database with the ExPASy AAcompID tool (http:// expasy.hcuge.ch/ch2d/aacompi.html). A total of 25 proteins were identified by matching against database entries for D. discoideum, and another six by cross-species matching against database entries for Saccharomyces cerevisiae proteins. This map will be available in the SWISS-2DPAGE database.

Animals↗
PubMedDate not supplied

Characterisation of wool intermediate filament proteins separated by micropreparative two-dimensional electrophoresis.

Wool intermediate filament proteins (IFP) are a subclass of the cytokeratins, a group of structural proteins which form intermediate filaments in many cell types. Post-translational modifications, such as phosphorylation, play an important role in the control of intermediate filament assembly. Two-dimensional electrophoresis has previously been used to study the IFP distribution in wools with different physical characteristics. Charge heterogeneity has been observed in Type I and Type II IFP. In a previous study, two-dimensional electrophoresis of alkaline phosphatase-treated wool protein extracts was used to show that Type II IFP are phosphorylated. To facilitate post-separation analysis, micropreparative two-dimensional electrophoresis was used to separate milligram quantities of wool protein. Direct phosphoamino acid analysis has confirmed the presence of phosphorylation on serine residues on Type II IFP, whose identity was confirmed by amino acid compositional analysis. The isoelectric points of Type I IFP are very similar and they do not separate completely on the commercially available pH 4-7 immobilized pH gradients (IPG) used in this study. In situ tryptic digestion followed by automated Edman sequencing of the high performance liquid chromatography (HPLC)-separated peptides was used to confirm the identity of this group as Type I IFP. To improve the separation of the Type I IFP it will be necessary to use narrow range IPGs such as Immobiline DryPlates which are available from Pharmacia Biotech, in the pH ranges 4.2-4.9, 4.5-5.4, 5.0-6.0 and 5.6-6.6.

Animals↗