Proteomic Profiling Of Candidate Biomarkers In Urine Of Non-Small Cell Lung Cancer Patients

ObjectiveIn the present study, we aimed to screen for candidate NSCLC-related biomarkers in urine by researching on the urinary soluble and exosomal proteome of non-small cell lung cancer (NSCLC) patients, and to lay new theoretical basis for the diagnosis, prognosis and personal therapy of NSCLC patients and provide new technological strategy.Methods1. Comparative analysis of soluble urinary proteins of normal individuals and NSCLC patients: Urinary proteomes between 3 normal persons and 3 NSCLC patients were compared by 1D SDS-PAGE. After 1D SDS-PAGE analysis, Agilent nanoHPLC-chip-MS/MS ION TRAP 6330 was used to characterize differentially expressed proteins in the bands. The MS/MS data was then automatically searched against UniProtKB/Swiss-Prot protein database by Spectrum Mill Proteomics Workbench Rev A.03.03.078 software. 2. Validation of putative urinary biomarker by western blot and immunohistochemistry staining : To validate the LC-MS/MS data, we analyzed the levels of AACT in three healthy and NSCLC urine samples by western blot;To verify the expression level of AACT in lung tissues, immunohistochemistry staining was performed on paraffin-embedded tissue sections that contained both tumor tissue and adjacent non-tumor lung tissue from 20 NSCLC patients.3. Comprehensive analysis of low abundance proteome in human urinary exosomes:We employed differential ultracentrifugation to purify urinary exosomes. To verify whether the sediments separated from normal human urine were exosomes, the vesicles were concentrated and visualized by immunogold electron microscopy using antibodies reacting with water channel aquaporin-2 (AQP2). To comprehensively explore the low abundance proteome, combinatorial peptide ligand libraries, combined with peptide OFFGEL electrophoresis were employed for the enrichment and separation of relatively low abundant proteins in urinary exosomes. After peptide OFFGEL electrophoresis, separated peptides were analyzed by nanoHPLC-chip-MS/MS. Gene ontology (GO) analysis was also employed to analyze the biological process, molecular function, and cellular component of the low abundance proteome of urinary exosomes.4. Comparative analysis of urinary exosomal proteins of normal individuals and NSCLC patients: Urinary exosomal proteomes were compared by 1D SDS-PAGE between 10 normal persons and 8 NSCLC patients. After 1D SDS-PAGE analysis, Agilent nanoHPLC-chip-MS/MS ION TRAP 6330 was utilized to characterize differentially expressed proteins in the bands. Peptide and protein identifications were run automatically with the Spectrum Mill Proteomics Workbench Rev A.03.03.078 software.5. Validation of candidate urinary exosomal biomarker by western blot and immunohistochemistry staining:To validate the LC-MS/MS data, we analyzed the levels of LRG1 in six healthy and NSCLC urinary exosomes by Western blot;To verify the expression level of LRG1 in lung tissues, immunohistochemistry staining was performed on paraffin-embedded tissue sections that contained 20 tumor tissue and 10 adjacent non-tumor lung tissue from NSCLC patients.Results1. A total of 40 unique proteins were identified by comparative analysis of urinary soluable proteins from normal individuals and NSCLC patients. Among these proteins, eight proteins were related with NSCLC, they were: CLU, KLK1, Gelsolin, LRG1, Galectin-3-binding protein, ZAG, AACT, and AAT.2. The level of AACT was higher on the whole in the urine from three NSCLC patients compared to samples from normal healthy individuals by western blot analysis. The result of immunohistochemistry staining showed significantly increased AACT level in lung cancer tissues compared with normal lung tissues.3. Purified exosomes are relatively round vesicles ranging from approximately 30–100 nm in diameter. The expression of AQP2 on the vesicles also argues positively for the presence of exosomes, as has been previously reported in human urine samples.4. 1D SDS-PAGE analysis of all fractions under reducing conditions revealed different patterns of proteins. After treatment with CPLL, very different patterns were obtained for the column eluates, which showed a large decrease of the intense bands, and the apparition of many new protein bands.5. The combinatorial peptide ligand library treated urinary exosomes were predigested and fractionated by peptide OFFGEL electrophoresis and characterized by mass detection of HPLC-CHIP-MS/MS analysis. By using this strategy, 512 non-redundant proteins were identified from the human urinary exosomes, and 374 proteins had not been reported. GO analysis showed that 55.36% of identified proteins were soluble proteins, and 44.93% were membrane proteins.6. A total of 18 proteins were identified in the 45kD～35kD band by comparative analysis of urinary exosomal proteins from normal individuals and NSCLC patients. Among them, 4 were only from normal individuals, 11 were only from NSCLC patients, and the other 3 were from both normal individuals and NSCLC patients.7. The result of western blot showed that the level of LRG1 was higher on the whole in the urinary exosomes from NSCLC patients compared to samples from healthy individuals. The result of immunohistochemistry staining showed significantly increased AACT level in lung cancer compared with normal lung tissue.Conclusions1. In the present research, using a comparative proteomic discovery approach combined 1D SDS-PAGE and nano-HPLC-chip-MS/MS, we effectively identified and validated candidate urinary biomarkers for objective and non-invasive diagnosis of NSCLC.2. The higher expression level ofAACT in urine may be associated with lung tissue of NSCLC patients, and the results suggested that LRG1 may be a candidate biomarker for non-invasive diagnosis of NSCLC in urine.3. The CPLL technology can effectively remove the abundant proteins and enrich the relatively low abundance proteome of exosmes.4. The urinary exosomal proteome contains exceedingly rich low abundance proteins, and the research result contributes a lot to our understanding of the urinary exosomal proteome.5. The higher expression level of LRG1 in urinary exosomes may be associated with lung tissue of NSCLC patients, and the results suggested that LRG1 may be a candidate biomarker for non-invasive diagnosis of NSCLC in urine.