Biomedical subjects

Raghothama Chaerkady

Publications and source records attributed to Raghothama Chaerkady.

4 recordsLinked to original sources

PubMed2005

Plasma Proteome Database as a resource for proteomics research.

Plasma is one of the best studied compartments in the human body and serves as an ideal body fluid for the diagnosis of diseases. This report provides a detailed functional annotation of all the plasma proteins identified to date. In all, gene products encoded by 3778 distinct genes were annotated based on proteins previously published in the literature as plasma proteins and the identification of multiple peptides from proteins under HUPO's Plasma Proteome Project. Our analysis revealed that 51% of these genes encoded more than one protein isoform. All single nucleotide polymorphisms involving protein-coding regions were mapped onto the protein sequences. We found a number of examples of isoform-specific subcellular localization as well as tissue expression. This database is an attempt at comprehensive annotation of a complex subproteome and is available on the web at http://www.plasmaproteomedatabase.org.

Amino Acid Motifs↗

PubMed2005

A functional annotation of subproteomes in human plasma.

The data collected by Human Proteome Organization's Plasma Proteome Pilot project phase was analyzed by members of our working group. Accordingly, a functional annotation of the human plasma proteome was carried out. Here, we report the findings of our analyses. First, bioinformatic analyses were undertaken to determine the likely sources of plasma proteins and to develop a protein interaction network of proteins identified in this project. Second, annotation of these proteins was performed in the context of functional subproteomes involved in the coagulation pathway, the mononuclear phagocytic system, the inflammation pathway, the cardiovascular system, and the liver; as well as the subset of proteins associated with DNA binding activities. Our analyses contributed to the Plasma Proteome Database (http://www.plasmaproteomedatabase.org), an annotated database of plasma proteins identified by HPPP as well as from other published studies. In addition, we address several methodological considerations including the selective enrichment of post-translationally modified proteins by the use of multi-lectin chromatography as well as the use of peptidomic techniques to characterize the low molecular weight proteins in plasma. Furthermore, we have performed additional analyses of peptide identification data to annotate cleavage of signal peptides, sites of intra-membrane proteolysis and post-translational modifications. The HPPP-organized, multi-laboratory effort, as described herein, resulted in much synergy and was essential to the success of this project.

Blood Coagulation↗

PubMed2004

Characterization of a bradykinin-hydrolyzing protease from the bovine lens.

PURPOSE: To isolate and characterize bovine lens endopeptidase activity that cleaves the Phe-Ser bond in peptide substrates. METHODS: The protease activity in young bovine lens homogenate was measured using the Mca-(Ala(7),Lys(Dnp)(9))-bradykinin substrate. Degradation of bradykinin and other unlabeled peptide substrates was monitored by reversed-phase HPLC on a C18 column. The protease was purified by means of several chromatography steps. An in-gel tryptic digest of the purified protease was analyzed by using matrix-assisted desorption ionization-time of flight mass spectrometry (MALDI-ToF-MS and nanospray quadrupole time of flight mass spectrometry (QqToF MS). The specificity of the protease was determined with bradykinin and its analogues. Crystallin fragments isolated from aged bovine lenses were tested for their susceptibility to degradation by a newly identified endopeptidase. RESULTS: Bradykinin hydrolyzing endopeptidase activity was localized mainly in the outer cortex of the lens. A characterization study showed that the purified protease was thiol and metal dependent. Peptide mass fingerprinting and tandem mass spectrometry (MS/MS) analysis of an in-gel tryptic digest matched the protein sequence of thimet oligopeptidase (TOP). The purified protease cleaved bradykinin specifically at Phe(5)-Ser(6) and neurotensin at Arg(8)-Arg(9). Basic or hydrophobic amino acids at P1 and P1' positions in the substrate were preferred over acidic residues. Enzyme activity was also inhibited by physiological levels of adenosine triphosphate (1 mM; ATP) and glutathione (3 mM; GSH). The crystallin fragments obtained from aged bovine lenses were cleaved by the purified enzyme. CONCLUSIONS: This study shows the presence of TOP in the bovine lens. Its unique substrate specificity and regulation of its activity by ATP and GSH suggest that TOP has an important role in peptide hydrolysis in the lens.

Adenosine Triphosphate↗

PubMed2002

Identification and properties of anti-chaperone-like peptides derived from oxidized bovine lens betaL-crystallins.

Thermal aggregation of betaL-crystallin was higher in the presence of peptide fragments generated from oxidized and trypsin-digested betaL-crystallin compared with thermal aggregation of the control proteins without oxidized betaL-crystallin fragments. Increased aggregation of betaL-crystallin was also observed despite the presence of alpha-crystallin (which has anti-aggregating properties) in the system. Self-aggregation of the oxidized betaL-crystallin fragments per se was not observed under the experimental conditions. Reverse-phase HPLC analysis of the precipitate obtained after heating a mixture of betaL-crystallin and oxidized betaL-crystallin fragments revealed that more than one peptide co-precipitates with betaL-crystallin. Electrospray mass spectrometry analysis of the peptides revealed that the molecular weight(s) of the peptides ranged from 1400-1800. Tandem mass spectrometry and a data base search revealed that two of the peptides originated from betaA4-crystallin (LTIFEQENFLGR, residues 121-132) and betaB3-crystallin (AINGTWVGYEFPGYR, residues 153-167) respectively. Oxidized synthetic peptides representing the same sequence were also found to enhance the aggregation of betaL-crystallin in a manner similar to oxidized lens betaL-crystallin peptides. These data suggest that the polypeptides generated after oxidation and proteolysis of betaL-crystallins interact with denaturing proteins and facilitate their aggregation and light scattering, thus behaving like anti-chaperones.

Amino Acid Sequence↗