Biomedical subjects

G E Reid

Publications and source records attributed to G E Reid.

40 records · Page 3Linked to original sources

PubMedDate not supplied

Two-dimensional electrophoretic analysis of proteins expressed by normal and cancerous human crypts: application of mass spectrometry to peptide-mass fingerprinting.

Protein patterns of normal human colonic crypts, isolated from different regions of the large intestine, and several colorectal cancer cell lines were compared using two-dimensional electrophoresis gels (2-DE). As detected by intrinsic radiolabeling and Coomassie Brilliant Blue staining, the protein patterns for normal crypts isolated from the ascending, and descending, regions of the colon and the rectum, were almost (> 95%) identical. While 75-80% of the protein spots from normal crypts and the colorectal cancer cell line (LIM 1863), a cell line that grows as organoids and differentiates spontaneously into crypt-like structures in vitro, can be matched, the relative expression levels of a large number of proteins differ. At least two protein spots (undetectable in the protein pattern from normal cells), proteins a (M(r) approximately 18,000, pI 6.7-6.9) and b (M(r) approximately 24,000, pI 5.9-6.0), were detected in the 2-DE gel protein pattern in the three cell lines LIM 1863, LIM 1215 and LIM 1899. The identity of these proteins is not yet known and further studies are required before they can be considered as potential colon tumor markers. Approximately 60% of the cellular proteins from LIM 1215 cells, a colon carcinoma cell line that exhibits many properties associated with columnar cells, can be matched with LIM 1863 cells. The results presented here represent an initial phase in our efforts to develop a comprehensive protein database for normal human colon cells and several colorectal cancer cell lines. While our initial protein identification relied on microsequencing methodologies, we are presently evaluating peptide-mass fingerprinting, utilizing capillary reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray mass spectrometry, as a means for rapid identification of proteins at subpicomole levels. Using this approach, protein #3 (M(r) approximately 66,000, pI 6.2) was identified as heat shock protein 60 from as few as seven tryptic peptide masses when they were screened against the molecular weight search (MOWSE) peptide-mass database.

Amino Acid Sequence↗

PubMedDate not supplied

Two-dimensional electrophoretic analysis of human breast carcinoma proteins: mapping of proteins that bind to the SH3 domain of mixed lineage kinase MLK2.

MLK2, a member of the mixed lineage kinase (MLK) family of protein kinases, first reported by Dorow et al. (Eur. J. Biochem. 1993, 213, 701-710), comprises several distinct structural domains including an src homology-3 (SH3) domain, a kinase catalytic domain, a unique domain containing two leucine zipper motifs, a polybasic sequence, and a cdc42/rac interactive binding motif. Each of these domains has been shown in other systems to be associated with a specific type of protein interaction in the regulation of cellular signal transduction. To study the role of MLK2 in recruiting specific substrates, we constructed a recombinant cDNA encoding the N-terminal 100 amino acids of MLK2 (MLK2N), including the SH3 domain (residues 23-77), fused to glutathione S-transferase. This fusion protein was expressed in Escherichia coli, purified using gluthathione-Sepharose affinity chromatography and employed in an affinity approach to isolate MLK2-SH3 domain binding proteins from lysates of 35S-labelled MDA-MB231 human breast tumour cells. Electrophoretic analysis of bound proteins revealed that two low-abundance proteins with a molecular weights (Mr) of approximately 31,500 and approximately 34,000, bound consistently to the MLK2N protein. To establish accurately the Mt / isoelectric point (pI) loci of these MLK2-SH3 domain binding proteins, a number of abundant proteins in a two-dimensional electrophoresis (2-DE) master gel were identified to serve as triangulation marker points. Proteins were identified by (i) direct Edman degradation following electroblotting onto polyvinylidene difluoride (PVDF) membranes, (ii) Edman degradation of peptides generated by in-gel proteolysis and fractionation by rapid (approximately 12 min) microbore column (2.1 mm ID) reversed-phase high performance liquid chromatography (HPLC), (iii) mass spectrometric methods including peptide-mass fingerprinting and electrospray (ESI)-mass spectrometry (MS)-MS utilizing capillary (0.2-0.3 mm ID) column chromatography, or (iv) immunoblot analysis. Using this information, a preliminary 2-DE protein database for the human breast carcinoma cell line MDA-MB231, comprising 21 identified proteins, has been constructed and can be accessed via the World Wide Web (URL address: http:(/)/ www.ludwig.edu.au/www/jpsl/jpslhome.htm l).

Amino Acid Sequence↗

PubMedDate not supplied

A two-dimensional gel database of human colon carcinoma proteins.

The master two-dimensional gel database of human colon carcinoma cells currently lists cellular proteins from normal crypts and the colorectal cancer cell lines LIM 1863, LIM 1215 and LIM 1899 (Ward et al., Electrophoresis 1990, 11, 883-891; Ji et al., Electrophoresis 1994, 15, 391-405). Updated two-dimensional electrophoretic (2-DE) maps of cellular proteins from LIM 1215 cells, acquired under both nonreducing and reducing conditions, are presented. Fifteen cellular proteins are identified in the reducing 2-DE gel map, and seven in the nonreducing gel map, along with a tabular listing of their M(r)/pI loci and mode of identification. We also include our mass spectrometric based procedures for identifying 2-DE resolved proteins. This procedure relies on a combination of capillary column (0.10-0.32 mm internal diameter) reversed-phase HPLC peptide mapping of in-gel digested proteins, peptide mass fingerprinting, sequence analysis by either collision-induced dissociation or post-source-decay fragmentation, and protein identification using available database search algorithms. These data, and descriptions of the micro-techniques employed in this laboratory for identifying 2-DE resolved proteins can be accessed via the internet URL: http:(/)/www.ludwig.edu.au.

Amino Acid Sequence↗

PubMedDate not supplied

Electrophoretic analysis of the novel antigen for the gastrointestinal-specific monoclonal antibody, A33.

The murine monoclonal antibody A33 (mAbA33) recognises a human cell membrane-associated antigen selectively expressed in epithelial cells of the lower gastrointestinal tract and > 90% of colonic cancers, but is not detected in a wide range of other normal tissues by immunohistochemical analysis. In phase I/II clinical triasl, mAbA33 has been shown to target advanced colon cancers and the humanized version is currently being evaluated in therapy studies. Although the mAbA33 has been well characterised by immunohistochemical and clinical studies, until recently, the target antigen has remained poorly defined. This was largely attributable to the antigenic determinant recognised by mAbA33 being dependent on the native spatial conformation of the A33 antigen which impeded its identification by conventional two-dimensional electrophoresis (2-DE) and immunoblot analysis. We have developed an immunoblot method, based on nonreducing/non-urea precast 2-DE gels, that has facilitated the purification of the detergent (0.3% Triton X-100) solubilised A33 antigen from the human colon cancer cell lines LIM1215 and SW1222. Under these 2-DE conditions, the A33 antigen electrophoreses with an apparent M(r) approximately 41000 and pI 5.0-6.0. Attempts to isolate the A33 antigen from 2-DE gels for direct structural analysis were unsuccessful, due to its co-electrophoresis with actin and cytokeratin proteins. However, using Western blot and biosensor detection the A33 antigen has been purified chromatographically and N-terminal sequence analysis was possible. Using polyclonal antibodies raised against a synthetic peptide corresponding to the N-terminal region of the A33 antigen we have used Western blot analysis to localise the molecule in our master 2-DE protein database for normal human colon crypts and several colon carcinoma cell lines (URL address: http:(/)/www.ludwig.edu.au). Under reducing 2-DE conditions, the A33 antigen electrophoresis as 6 differentially charged isoforms (pI 4.6-4.8) with a single molecular weight species at M(r) approximately 55000.

3T3 Cells↗