Quantitative proteomics analysis of mitochondrial proteins in lung adenocarcinomas and normal lung tissue using iTRAQ and tandem mass spectrometry

Abstract

Lung adenocarcinoma is the most common type of lung cancer. Unfortunately, lung adenocarcinoma has a poor prognosis and the pathogenesis remains unclear. Mitochondria are important mediators of tumorigenesis. However, the proteomics profile of lung adenocarcinoma mitochondrial proteins has not been elucidated. In this study, we investigated differences in the mitochondrial protein profiles between lung adenocarcinomas and normal tissue. Laser capture microdissection (LCM) was used to isolate the target cells from lung adenocarcinomas and normal tissue. The differential expression of mitochondrial proteins was determined using isobaric tags for relative and absolute quantitation (iTRAQ) combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS). Bioinformatics analysis was performed using Gene Ontology and KEGG databases. As a result, 510 differentially expressed proteins were identified, 315 of which were upregulated and 195 that were downregulated. Of these proteins, 35.5% were mitochondrial or mitochondrial-related and were involved in binding, catalysis, molecular transduction, transport, and molecular structure. Based on the differentially expressed proteins, 63 pathways were significantly enriched through KEGG. The overexpression and cellular distribution of the mitochondrial protein C1QBP in the lung cancer samples was confirmed and verified by Western blotting. The relationship between C1QBP expression and clinicopathological features in lung cancer patients was likewise evaluated using immunohistochemistry, which revealed that the upregulation of C1QBP was associated with lymph node metastasis, pathological grade and clinical stage of TNM. The results indicate that the iTRAQ 2D-LC-MS/MS technique is a potential method for comparing mitochondrial protein profiles between tumor and normal tissue and could aid in identifying novel biomarkers and the mechanisms underlying carcinogenesis.

Keywords: Lung adenocarcinoma; iTRAQ; mitochondrial protein; proteomic profile.

Figure 1

Laser capture microdissection of hematoxylin and eosin stained lung adenocarcinoma and paired normal lung tissue slides.

Figure 2

Western blot of purified mitochondrial fractions with cytochrome c oxidase (COX). Note: Na⁺/K⁺ATPase is a mitochondrial protein marker and COX is a membrane protein marker; T-M represents the mitochondrial proteins in lung cancer tissues; T-C represents the lung cancer tissue cytosolic solutions; N-M represents the normal lung tissue mitochondrial proteins; N-C represents the normal lung tissue cytosolic solutions.

Figure 3

MS/MS spectra of four identified peptides: (A) the MS/MS spectra of aFVDFLSDEIk, with m/z of 946.53412 Da (z = +2), MH+: 1892.06096 Da. (B) The MS spectra of sequence MLPLLR with m/z of 371.73535 Da (z = +2), MH+: 742.46343 Da; (C) The MS spectra of sequence aFVDFLSDEIkEER with m/z of 1153.62549 Da (z = +2), MH+: 2951.60300 Da; (D) The MS spectra of sequence VEEQEPELTSTPNFVVEVIk with m/z of 864.46277 Da (z = +3), MH+: 2591.37375 Da.

Figure 4

The molecular weight (MV) distribution of the identified proteins.

Figure 5

The isoelectric point (PI) distribution of the identified proteins.

Figure 6

Significant Gene Ontology analysis of differentially expressed mitochondrial proteins (CC, Cellular component; BP, biological process; MF, molecular function).

Figure 7

Significant pathway analysis of differentially expressed mitochondrial proteins.

Figure 8

Expression of C1QBP in lung adenocarcinomas and paired normal lung tissues. β-actin was used as a loading control.

Figure 9

A. Weak staining of C1QBP in lung adenocarcinoma cell (LAC). B. Moderate staining of C1QBP in LAC. C. High staining of C1QBP in LAC. D. Negative staining in normal lung tissue. E. Weak staining of C1QBP in lung squamous cell (LSC). F. High staining of C1QBP in LSC.