Abstract
Esophageal adenocarcinoma, currently the seventh leading cause of cancer-related death, has been associated with the presence of Barrett metaplasia. The malignant potential of Barrett metaplasia is evidenced by ultimate progression of this condition to invasive adenocarcinoma. We utilized liquid phase separation of proteins with chromatofocusing in the first dimension and nonporous reverse phase HPLC in the second dimension followed by ESI-TOF mass spectrometry to identify proteins differentially expressed in six Barrett metaplasia samples as compared with six esophageal adenocarcinoma samples; all six Barrett samples were obtained from the identical six patients from whom we obtained the esophageal adenocarcinoma tissue. Approximately 300 protein bands were detected by mass mappings, and 38 differentially expressed proteins were identified by microLC-MS/MS. The false positive rates of the peptide identifications were evaluated by reversed database searching. Among the proteins that were identified, Rho GDP dissociation inhibitor 2, alpha-enolase, Lamin A/C, and nucleoside-diphosphate kinase A were demonstrated to be up-regulated in both mRNA and protein expression in esophageal adenocarcinomas relative to Barrett metaplasia. Candidate proteins were examined at the mRNA level using high density oligonucleotide microarrays. The cellular expression patterns were verified in both esophageal adenocarcinomas and in Barrett metaplasia by immunohistochemistry. These differentially expressed proteins may have utility as useful candidate markers of esophageal adenocarcinoma.
Highlights
Esophageal adenocarcinoma, currently the seventh leading cause of cancer-related death, has been associated with the presence of Barrett metaplasia
Liquid Phase 2-D Separation and On-line ESI-TOF MS Analysis—Tissue lysates containing 4.5 mg of protein each from the six premalignant Barrett metaplasia tissues and six esophageal adenocarcinomas were resolved individually in the first dimension using CF, which combines the high capacity of ion-exchange chromatography with the high resolution of IEF
0.42 a ϩ, weak; ϩϩ, medium; ϩϩϩ, high expression; Ϫ, not found. b Comparison based on six pairs of samples; differences evaluated by 2 test. c Value indicates the number of tumors demonstrating increased or decreased mRNA expression compared with mean expression Ϯ 1 S.D. observed in Barrett metaplasia. d Indicates the probability for significant difference between mean mRNA expression values of Barrett metaplasia and esophageal adenocarcinoma by Student’s t test, assuming unequal sample variance
Summary
2-D, two-dimensional; NPS-RP, nonporous reverse phase; CF, chromatofocusing; OG, n-octyl -D-glucopyranoside; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; RhoGDI, Rho GDP dissociation inhibitor. Protein lysates were prepared from both high grade dysplasia and esophageal adenocarcinoma tissue, both obtained from the same six patients. These lysates were resolved by 2-D LC using chromatofocusing in the first dimension and nonporous silica reverse phase (NPS-RP) HPLC in the second dimension. Proteins were quantified based on individual molecular weight intensity and assembly of a protein mass map. Hierarchical clustering of the mass maps correctly segregated each of the 12 samples by histological type into Barrett metaplasia or esophageal adenocarcinoma. The 2-D mass maps and the corresponding protein identification have utility for analysis of cellular protein expression changes that are associated with progression of Barrett metaplasia to esophageal adenocarcinoma
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