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An approach to proteomic analysis of human tumors
Author(s) -
EmmertBuck Michael R.,
Gillespie John W.,
Paweletz Cloud P.,
Ornstein David K.,
Basrur Venkatesha,
Appella Ettore,
Wang QuanHong,
Huang Jing,
Hu Nan,
Taylor Phil,
Petricoin Emanuel F.
Publication year - 2000
Publication title -
molecular carcinogenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.254
H-Index - 97
eISSN - 1098-2744
pISSN - 0899-1987
DOI - 10.1002/(sici)1098-2744(200003)27:3<158::aid-mc2>3.0.co;2-2
Subject(s) - biology , laser capture microdissection , cytokeratin , proteomics , microdissection , microbiology and biotechnology , isoelectric focusing , annexin a2 , gel electrophoresis , epithelium , isoelectric point , annexin , biochemistry , immunohistochemistry , genetics , enzyme , gene expression , gene , immunology , flow cytometry
A strategy for proteomic analysis of microdissected cells derived from human tumor specimens is described and demonstrated by using esophageal cancer as an example. Normal squamous epithelium and corresponding tumor cells from two patients were procured by laser‐capture microdissection and studied by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE). Fifty thousand cells resolved approximately 675 distinct proteins (or isoforms) with molecular weights ranging between 10 and 200 kDa and isoelectric points of pH 3–10. Comparison of the microdissected protein profiles showed a high degree of similarity between the matched normal‐tumor samples (98% identical). However, 17 proteins showed tumor‐specific alterations, including 10 that were uniquely present in the tumors and seven that were observed only in the normal epithelium. Two of the altered proteins were characterized by mass spectrometry and immunoblot analysis and were identified as cytokeratin 1 and annexin I. Acquisition of 2D‐PAGE protein profiles, visualization of disregulated proteins, and subsequent determination of the identity of selected proteins through high‐sensitivity MS‐MS microsequencing are possible from microdissected cell populations. These separation and analytical techniques are uniquely capable of detecting tumor‐specific alterations. Continued refinement of techniques and methodologies to determine the abundance and status of proteins in vivo holds great promise for future study of normal cells and associated neoplasms. Mol. Carcinog. 27:158–165, 2000. Published by Wiley‐Liss Inc.