Proteomic analysis of human saliva from lung cancer patients using two-dimensional difference gel electrophoresis and mass spectrometry

Abstract

Lung cancer is often asymptomatic or causes only nonspecific symptoms in its early stages. Early detection represents one of the most promising approaches to reduce the growing lung cancer burden. Human saliva is an attractive diagnostic fluid because its collection is less invasive than that of tissue or blood. Profiling of proteins in saliva over the course of disease progression could reveal potential biomarkers indicative of oral or systematic diseases, which may be used extensively in future medical diagnostics. There were 72 subjects enrolled in this study for saliva sample collection according to the approved protocol. Two-dimensional difference gel electrophoresis combined with MS was the platform for salivary proteome separation, quantification, and identification from two pooled samples. Candidate proteomic biomarkers were verified and prevalidated by using immunoassay methods. There were 16 candidate protein biomarkers discovered by two-dimensional difference gel electrophoresis and MS. Three proteins were further verified in the discovery sample set, prevalidation sample set, and lung cancer cell lines. The discriminatory power of these candidate biomarkers in lung cancer patients and healthy control subjects can reach 88.5% sensitivity and 92.3% specificity with AUC = 0.90. This preliminary data report demonstrates that proteomic biomarkers are present in human saliva when people develop lung cancer. The discriminatory power of these candidate biomarkers indicate that a simple saliva test might be established for lung cancer clinical screening and detection.

Fig. 1.

Study design for lung cancer salivary proteomic biomarker discovery.

Fig. 2.

Salivary proteomic biomarker discovery. The merged 2D-DIGE image of proteins from lung cancer patients (Red) and healthy control subjects (Green).

Fig. 3.

The Western blot of HP (A), AZGP1 (B), and β-actin (C) in the saliva of lung cancer patients and healthy control subjects. Equal amount of protein were loaded to each lane.

Fig. 4.

Salivary proteomic biomarker evaluation and confirmation. The dot plots of HP (A), AZGP1 (B), and human calprotectin (C) in the saliva of lung cancer patients and healthy control subjects of prevalidation sample set. D, ROC curve for human calprotectin (ROC = 0.817).

Fig. 5.

Performance of biomarker panel. A, the dot plots of logistic regression combined biomarkers of human calprotectin, HP and AZGP1; B, ROC curve for the combined three biomarkers.

Fig. 6.

Biomarker verification in lung cancer cell lines. A, The Western blot of HP (a) and AZGP1 (b) in different lung cancer cell lines. Their corresponding β-actin is shown in (c); B, The corresponding quantification of the Western blot in (A).

Fig. 7.

The quantification data of HP (A), AZGP1 (B), and human calprotectin (C) in smoking and nonsmoking groups (*: p < 0.05; **: p < 0.01; ***: p < 0.001).