Differential protein analysis of spasomolytic polypeptide expressing metaplasia using laser capture microdissection and two-dimensional difference gel electrophoresis.
Full analysis of cellular protein constituents is a valuable tool in the evaluation of tissues. Traditional methods of evaluation, however, are time-consuming and difficult to reproduce. Two-dimensional difference gel electrophoresis (2D-DIGE), a recently developed proteomic system, affords the ability to compare and evaluate protein extracts from multiple sources. Coupled with laser capture microdissection (LCM), this technology is a powerful tool in comparing the protein profiles of separate pure cell populations. Proteins are labeled in vitro with reactive cyanine dyes that fluoresce at differential wavelengths, and after comigration on two-dimensional gels, differing protein populations become apparent. The unique aspect of this technology is the ability to identify and quantify proteins from separate preparations without issues of gel-to-gel differences. These techniques coupled with the systems for robotic acquisition of specific spots on the gel, tryptic digestion, and MALDI mass spectrometry permit identification of proteins differentially expressed in two pure cell populations. The authors used these new technologies to analyze the protein constituents of spasmolytic polypeptide expressing metaplasia (SPEM), a gastric mucosal metaplasia (fundic antralization or pseudopyloric metaplasia) that develops in the atrophic fundus mucosa of mice infected with Helicobacter felis and in humans infected with Helicobacter pylori. In addition, SPEM has been identified in the atrophic mucosa surrounding a high percentage of gastric adenocarcinomas and may represent a precursor lineage of malignancy. This technology recognized 28 differentially expressed proteins between SPEM and surface cells. Identification of novel SPEM-related proteins would allow the development of new immunohistochemical antibodies to further study this important metaplasia.
Lee
Jeffrey R
JR
Department of Pathology and Laboratory Medicine, Veterans Affairs Medical Center, Augusta, Georgia, 30904, USA. JLEE@mail.mcg.edu
Baxter
Tammy M
TM
Yamaguchi
Hirokazu
H
Wang
Timothy C
TC
Goldenring
James R
JR
Anderson
Mark G
MG
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D005748:Gastric Fundus / Q000473:pathology; D005753:Gastric Mucosa / Q000473:pathology; D016481:Helicobacter Infections / Q000150:complications; D044922:Helicobacter felis; D007150:Immunohistochemistry / Q000379:methods; D036341:Intercellular Signaling Peptides and Proteins; D007834:Lasers; D008679:Metaplasia / Q000473:pathology* / Q000821:virology; D051379:Mice; D008810:Mice, Inbred C57BL; D042282:Microdissection; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results
D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011506:Proteins; C451060:spasmolytic polypeptide