Proteomic analysis of gastric cancer and immunoblot validation of potential biomarkers

Abstract

Aim: To search for and validate differentially expressed proteins in patients with gastric adenocarcinoma.

Methods: We used two-dimensional gel electrophoresis and mass spectrometry to search for differentially expressed proteins in patients with gastric adenocarcinoma. A set of proteins was validated with immunoblotting.

Results: We identified 30 different proteins involved in various biological processes: metabolism, development, death, response to stress, cell cycle, cell communication, transport, and cell motility. Eight proteins were chosen for further validation by immunoblotting. Our results show that gastrokine-1, 39S ribosomal protein L12 (mitochondrial precursor), plasma cell-induced resident endoplasmic reticulum protein, and glutathione S-transferase mu 3 were significantly underexpressed in gastric adenocarcinoma relative to adjacent non-tumor tissue samples. On the other hand, septin-2, ubiquitin-conjugating enzyme E2 N, and transaldolase were significantly overexpressed. Translationally controlled tumor protein was shown to be differentially expressed only in patients with cancer of the gastric cardia/esophageal border.

Conclusion: This work presents a set of possible diagnostic biomarkers, validated for the first time. It might contribute to the efforts of understanding gastric cancer carcinogenesis.

Keywords: Biomarkers; Gastric adenocarcinoma; Mass spectrometry; Proteome; Two-dimensional gel electrophoresis.

Figure 1

Results of two-dimensional gel electrophoresis from gastric adenocarcinoma patients. A: Examples of silver-stained two-dimensional gel electrophoresis gels for non-tumor and tumor tissue; B: Examples of differentially expressed spots (circled) for the non-tumor and tumor tissue that were chosen for immunoblot validation. MW: Molecular weight; GKN1: Gastrokine-1 precursor; MRPL12: 39S ribosomal protein L12 (mitochondrial precursor); PACAP: Plasma cell-induced resident endoplasmic reticulum protein; GSTM3: Glutathione S-transferase mu 3; SEPT2: Septin-2; UBE2N: Ubiquitin-conjugating enzyme E2 N; TALDO1: Transaldolase; TPT1: Translationally controlled tumor protein.

Figure 2

An example of mass spectrometry identification results for septin-2.

Figure 3

Distribution of differentially expressed proteins according to the biological processes in which they are involved.

Figure 4

Examples of immunoblotting results on four pairs of non-tumor and tumor tissue for eight differentially expressed proteins (A) and densitometric results from immunoblotting for the same eight proteins (B). The x-axis represents non-tumor (N) and tumor (T) tissues and the y-axis represents relative band density. The P values are from the Wilcoxon signed-rank test. GKN1: Gastrokine-1 precursor; MRPL12: 39S ribosomal protein L12 (mitochondrial precursor); PACAP: Plasma cell-induced resident endoplasmic reticulum protein; GSTM3: Glutathione S-transferase mu 3; SEPT2: Septin-2; UBE2N: Ubiquitin-conjugating enzyme E2 N; TALDO1: Transaldolase; TPT1: Translationally controlled tumor protein.

Figure 5

Densitometric results from immunoblotting for three proteins describing potential associations of differential protein expression and clinical parameters. The x-axis represents different clinical parameters and the y-axis represents relative band density. The P values are from the repeated measures analysis of variance. ^aP < 0.01 and ^bP < 0.05 are from the Bonferroni post-test. MRPL12: 39S ribosomal protein L12 (mitochondrial precursor); TPT1: Translationally controlled tumor protein; TALDO1: Transaldolase; N: Non-tumor; T: Tumor.

Figure 6

Densitometric results from immunoblotting of 10 pairs of non-tumor and tumor tissue from cardia/gastroesophageal border adenocarcinoma patients. The x-axis represents non-tumor (N) and tumor (T) tissues and the y-axis represents relative band density. The P value is from the Wilcoxon signed-rank test. TPT1: Translationally controlled tumor protein.