SILAC-based quantitative proteomic analysis of gastric cancer secretome

Abstract

Purpose: Gastric cancer is a commonly occurring cancer in Asia and one of the leading causes of cancer deaths. However, there is no reliable blood-based screening test for this cancer. Identifying proteins secreted from tumor cells could lead to the discovery of clinically useful biomarkers for early detection of gastric cancer.

Experimental design: A SILAC-based quantitative proteomic approach was employed to identify secreted proteins that were differentially expressed between neoplastic and non-neoplastic gastric epithelial cells. Proteins from the secretome were subjected to SDS-PAGE and SCX-based fractionation, followed by mass spectrometric analysis on an LTQ-Orbitrap Velos mass spectrometer. Immunohistochemical labeling was employed to validate a subset of candidates using tissue microarrays.

Results: We identified 2205 proteins in the gastric cancer secretome of which 263 proteins were overexpressed greater than fourfold in gastric cancer-derived cell lines as compared to non-neoplastic gastric epithelial cells. Three candidate proteins, proprotein convertase subtilisin/kexin type 9 (PCSK9), lectin mannose binding 2 (LMAN2), and PDGFA-associated protein 1 (PDAP1) were validated by immunohistochemical labeling.

Conclusions and clinical relevance: We report here the largest cancer secretome described to date. The novel biomarkers identified in the current study are excellent candidates for further testing as early detection biomarkers for gastric adenocarcinoma.

Figure 1. Workflow employed for the analysis of gastric cancer secretome

SILAC was employed to identify the more abundant proteins in the secretome of gastric cancer cells compared to the normal cells. Gastric cancer cell lines (AGS, SNU-1, SNU-1, SNU-5, SNU-16, NCI-N87, AGS and KATO-III) were grown in medium containing normal arginine and lysine. The normal gastric epithelial cell line was grown in a medium containing heavy arginine and lysine. Equal amounts of protein from pooled tumor secretome and from normal secretome were mixed. Proteins were subjected to fractionation by SDS-PAGE and strong cation exchange chromatography. The fractionated proteins were subjected to LC-MS/MS analysis using an LTQ-Orbitrap Velos mass spectrometer. The data was searched and quantitated using the Proteome Discoverer software suite.

Figure 2. Representative MS and MS/MS spectra of a subset of proteins that were found to be differentially expressed in the gastric cancer secretome

Representative MS/MS spectra of identified peptides from A) proprotein convertase subtilisin/kexin type 9 (PCSK9) B) Lectin mannose binding 2 (LMAN2) C) PDGFA associated protein 1 (PDAP1) and D) Follistatin related protein 1 (FSTL1) are shown. The MS spectra in the inset provide the relative abundance of the representative peptides in secretome derived from tumor and normal cells.

Figure 3. Validation of PCSK9 by immunohistochemical labeling

Representative sections from tissue microarrays for normal gastric tissues and tumor tissues stained with anti-PCSK9 antibody are shown.

Figure 4. Validation of LMAN2 by immunohistochemical labeling

Representative sections from tissue microarrays for normal gastric tissues and tumor tissues stained with anti-LMAN2 antibody are shown.

Figure 5. Validation of PDAP1 by immunohistochemical labeling

Representative sections from tissue microarrays for normal gastric tissues and tumor tissues stained with anti-PDAP1 antibody are shown.