Shashikant B. Joshi

Proteomic Analysis of Colorectal Cancer Reveals Alterations in Metabolic Pathways Mechanism of Tumorigenesis

Colorectal cancer is the second leading killer cancer worldwide and presently the most common cancer among males in Singapore. The study aimed to detect changes of protein profiles associated with the process of colorectal tumorigenesis to identify specific protein markers for early colorectal cancer detection and diagnosis or as potential therapeutic targets. Seven pairs of colorectal cancer tissues and adjacent normal mucosa were examined by two-dimensional gel electrophoresis at basic pH range (pH 7–10). Intensity changes of 34 spots were detected with statistical significance. 16 of the 34 spots were identified by MALDI-TOF/TOF tandem mass spectrometry. Changes in protein expression levels revealed a significantly enhanced glycolytic pathway (Warburg effect), a decreased gluconeogenesis, a suppressed glucuronic acid pathway, and an impaired tricarboxylic acid cycle. Observed changes in protein abundance were verified by two-dimensional DIGE. These changes reveal an underlying mechanism of colorectal tumorigenesis in which the roles of impaired tricarboxylic acid cycle and the Warburg effect may be critical.

Comparative Proteomic Analysis of Extracellular Proteins of Edwardsiella tarda

ABSTRACT A comparison of extracellular proteins of virulent and avirulent Edwardsiella tarda strains revealed several major, virulent-strain-specific proteins. Proteomic analysis identified two of the proteins in the virulent strain PPD130/91 as flagellin and SseB, which are virulence factors in bacterial pathogens. PCR amplification and DNA sequencing confirmed the presence of the genes that encode these proteins. Our results clearly demonstrated the potency of the proteomic approach in identifying virulence factors.

Antifreeze polypeptides from the Newfoundland ocean pout,Macrozoarces americanus: presence of multiple and compositionally diverse components

SummaryEight major antifreeze polypeptides (AFP) were purified from the sera of Newfoundland ocean pout. Except for their approximately identical size (6,000 Dalton), these components were shown to be separate entities by their behaviour on polyacrylamide gel electrophoresis, ion exchange chromatography, gel permeation and reverse phase high performance liquid chromatography. They could also be divided into two cross-reactive, yet distinct, immunological groups. Amino acid analysis demonstrated that ocean pout AFP are different from all of the other antifreezes studied to date. The ocean pout AFP do not contain the abundance of alanine (60 mol%) found in winter flounder and shorthorn sculpin AFP nor the high half-cystine residues (8 mol%) observed in sea raven AFP. It is suggested that ocean pout AFP represent a new type of macromolecular antifreeze.

Proteomic Studies of the Singapore Grouper Iridovirus

The Singapore grouper iridovirus (SGIV) genome consists of a double-stranded circular DNA of 140,131 base pairs with 162 predicted open reading frames. Our earlier study using peptide mass fingerprints generated from MALDI-TOF MS led to the identification of 26 viral proteins. The present investigation aimed to achieve a more comprehensive and precise identification of the SGIV viral proteome by two workflows: one-dimensional gel electrophoresis (1-DE) separation followed by protein identification by MALDI-TOF/TOF MS/MS (1-DE-MALDI workflow) and shotgun proteomics in which the whole virus was digested by trypsin and the resulting peptides were separated by nano-LC and analyzed by MALDI-TOF/TOF MS/MS (LC-MALDI workflow). In total, 44 viral proteins were identified, 25 of which were reported for the first time. Fourteen proteins were uniquely identified by the 1-DE-MALDI workflow, whereas another 10 proteins were only identified by the LC-MALDI workflow with 20 proteins found by both approaches. Moreover 13 proteins were found to have acetylated N termini. Twenty-three proteins identified contain predicted transmembrane domains, accounting for 52.3% of the total proteins identified. RT-PCR confirmed the transcription products of all the identified viral proteins. A large number of proteins identified by both the 1-DE-MALDI and the LC-MALDI workflows from this study have significantly enhanced the coverage of the SGIV proteome. The SGIV proteome is at present the only iridoviral proteome that has been extensively characterized. Our results should provide further insights into the biology of SGIV and other iridoviruses.

Membrane proteins of human fetal primitive nucleated red blood cells.

In humans, primitive fetal nucleated red blood cells (FNRBCs) are thought to be as vital for embryonic life as their counterpart, adult red blood cells (adult RBCs) are in later-gestation fetuses and adults. Unlike adult RBCs, the identity and functions of FNRBC proteins are poorly understood owing to a scarcity of FNRBCs for proteomic investigations. The study aimed to investigate membrane proteins of this unique cell type. We present here, the first report on the membrane proteome of human primitive FNRBCs investigated by two-dimensional liquid chromatography coupled with mass-spectrometry (2D-LCMS/MS) and bioinformatics analysis. A total of 273 proteins were identified, of which 133 (48.7%) were membrane proteins. We compared our data with membrane proteins of adult RBCs to identify common, and unique, surface membrane proteins. Twelve plasma membrane proteins with transmembrane domains and eight proteins with transmembrane domains but without known sub-cellular location were identified as unique-to-FNRBCs. Except for the transferrin receptor, all other 19 unique-to-FNRBC membrane proteins have never been described in RBCs. Reverse-transcriptase PCR (RT-PCR) and immunocytochemistry validated the 2D-LCMS/MS data. Our findings provide potential surface antigens for separation of primitive FNRBCs from maternal blood for noninvasive prenatal diagnosis, and to understand the biology of these rare cells.