PMC

Validation of Prosaposin using immunohistochemical labeling. Representative sections from tissue microarrays stained with anti-prosaposin is shown (A) expression of prosaposin in representative normal esophageal squamous mucosa and (B) expression of prosaposin in ESCC.

Classification of proteins by gene ontology based on their cellular localization and biological process. (A) Distribution of proteins based on their cellular component using gene ontology classifier. (B) Distribution of proteins based on their biological process using gene ontology classifier.

Validation of Plectin 1 using immunohistochemical labeling. Representative sections from tissue microarrays stained with anti-Plectin is shown (A) expression of Plectin 1 in representative normal esophageal squamous mucosa and (B) expression of plectin 1 in ESCC.

Validation of protein disulfide isomerase A4 using immunohistochemical labeling. Representative sections from tissue microarrays stained with anti-protein disulfide isomerase A4 as shown (A) expression of protein disulfide isomerase A4 in representative normal esophageal squamous mucosa and (B) expression of protein disulfide isomerase A4 in ESCC.

MS and MS/MS spectra of known and novel upregulated proteins in ESCC tissues as compared with the adjacent normal epithelia. MS and MS/MS spectra of peptide from representative differentially expressed proteins identified in this study. (A) Periostin (POSTN); (B) Plectin 1 (PLEC1); (C) Prosaposin (PSAP); and (D) Protein disulfide isomerase A4 (PDIA4).

Work flow for quantitative tissue proteomics using iTRAQ labeling and validation of biomarkers for esophageal squamous cell carcinoma. For iTRAQ labeling, proteins were isolated from ten tumor and adjacent normal tissues. Proteins were subjected to trypsin digestion followed by iTRAQ labeling of peptides. Post labeling of peptides the tumor and adjacent normal derived peptide mixture was pooled and fractionated using strong cation exchange (SCX) chromatography, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on a QTOF mass spectrometer. The data was searched using Mascot and Spectrum Mill search engines. Some of the overexpressed proteins that were not previously described (e.g., PLEC1) were validated using IHC labeling using tissue microarrays.