Protein signatures of molecular pathways in non-small cell lung carcinoma (NSCLC): comparison of glycoproteomics and global proteomics

Shuang Yang, Lijun Chen, Daniel W. Chan, Qing Kay Li, Hui Zhang

Non-small cell lung carcinoma (NSCLC) remains the leading cause of cancer deaths in the United States. More than half of NSCLC patients have clinical presentations with locally advanced or metastatic disease at the time of diagnosis. The large-scale genomic analysis of NSCLC has demonstrated that molecular alterations are substantially different between adenocarcinoma (ADC) and squamous cell carcinoma (SqCC). However, a comprehensive analysis of proteins and glycoproteins in different subtypes of NSCLC using advanced proteomic approaches has not yet been conducted. We applied mass spectrometry (MS) technology featuring proteomics and glycoproteomics to analyze six primary lung SqCCs and eleven ADCs, and we compared the expression level of proteins and glycoproteins in tumors using quantitative proteomics. Glycoproteins were analyzed by enrichment using a chemoenzymatic method, solid-phase extraction of glycopeptides, and quantified by iTRAQ-LC-MS/MS. Protein quantitation was further annotated via Ingenuity Pathway Analysis. Over 6000 global proteins and 480 glycoproteins were quantitatively identified in both SqCC and ADC. ADC proteins (8337) consisted of enzymes (22.11%), kinases (5.11%), transcription factors (6.85%), transporters (6.79%), and peptidases (3.30%). SqCC proteins (6967) had a very similar distribution. The identified glycoproteins, in order of relative abundance, included membrane (42%) and extracellular matrix (>33%) glycoproteins. Oncogene-coded proteins (82) increased 1.5-fold among 1047 oncogenes identified in ADC, while 124 proteins from SqCC were up-regulated in tumor tissues among a total of 827 proteins. We identified 680 and 563 tumor suppressor genes from ADC and SqCC, respectively. Our systematic analysis of proteins and glycoproteins demonstrates changes of protein and glycoprotein relative abundance in SqCC (TP53, U2AF1, and RXR) and in ADC (SMARCA4, NOTCH1, PTEN, and MST1). Among them, eleven glycoproteins were upregulated in both ADC and SqCC. Two glycoproteins (ELANE and IGFBP3) were only increased in SqCC, and six glycoproteins (ACAN, LAMC2, THBS1, LTBP1, PSAP and COL1A2) were increased in ADC. Ingenuity Pathway Analysis (IPA) showed that several crucial pathways were activated in SqCC and ADC tumor tissues.