Identification of Tyrosine-Phosphorylated Proteins Associated with Lung Cancer Metastasis using Label-Free Quantitative Analyses

Abstract

Lung cancer is a lethal disease, and early metastasis is the major cause of treatment failure and cancer-related death. Tyrosine phosphorylated (P-Tyr) proteins are involved in the invasive and metastatic behavior of lung cancer; however, only a limited number of targets were identified. We attempt to characterize P-Tyr proteins and events involved in the metastatic process. In a previous work, we have developed a strategy for identification of protein phosphorylation. Here, this strategy was used to characterize the tyrosine phosphoproteome of lung cancer cells that have different invasive abilities (CL1-0 vs. CL1-5). Using our analytical strategy, we report the identification of 335 P-Tyr sites from 276 phosphoproteins. Label-free quantitative analysis revealed that 36 P-Tyr peptides showed altered levels between CL1-0 and CL1-5 cells. From this list of sites, we extracted two novel consensus sequences and four known motifs for specific kinases and phosphatases including EGFR, Src, JAK2, and TC-PTP. Protein-protein interaction network analysis of the altered P-Tyr proteins illustrated that 11 proteins were linked to a network containing EGFR, c-Src, c-Myc, and STAT, which is known to be related to lung cancer metastasis. Among these 11 proteins, 7 P-Tyr proteins have not been previously reported to be associated with lung cancer metastasis and are of greatest interest for further study. The characterized tyrosine phosphoproteome and altered P-Tyr targets may provide a better comprehensive understanding of the mechanisms of lung cancer invasion/metastasis and discover potential therapies.

Characterization of the Phosphoproteome in SLE Patients

“…Reversible protein phosphorylation, in which phosphate groups are enzymatically added by protein kinases and removed from proteins by phosphatases, often serves as a molecular switch in signaling pathways. Disruptions in phosphorylation-mediated cell signaling events are connected with numerous diseases [1], [2], [3], [4], [5]. Furthermore, the abnormal expression of protein kinases is an important cause or component of many pathologies.…”

Optimization of titanium dioxide and immunoaffinity-based enrichment procedures for tyrosine phosphopeptide using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

“…However, previous literature indicated that the residual DHB interferes with peptide detection and easily contaminates source regions of the LC-MS-MS [37]. In this study, the loading buffer for the TiO 2 microcolumn was prepared following a procedure described elsewhere, and glutamic acid was added as an acidic additive [22]. By using a comparative strategy for loading and elution buffers with different pH values and increasing the phosphopeptide signals of MALDI-TOF MS, we revealed that the enrichment factors of standard phosphopeptides were 10,305, 206, and 406 for tyrosine, serine, and threoninephosphopeptide enrichment, respectively.…”

“…immobilized-metal affinity chromatography (IMAC), in which metal ions (Fe 3+ , Zr 4+ ) are chelated to stationary beads and bind with negatively charged phosphopeptides in a mobile phase [19][20][21]; 2. metal-oxide affinity chromatography, in which TiO 2 enrichment is used to enrich phosphopeptides [22][23][24]; 3. antibody-based enrichment, which includes several phosphopeptide-specific antibodies (for instance 4G10, pY100, and pY99) in phosphoproteome studies [25][26][27][28]; 4. strong cation-anion ion-exchange chromatography (SCX-SAX) [29,30]; and 5. a chemical-derivatization strategy that has been used for tagging phosphate groups in phosphopeptide enrichment.…”

“…MALDI-TOF MS and LC-MS have provided techniques and extensive applications for different biological proteome analyses [22,24,[31][32][33]. Recently, phosphopeptides were enriched on zirconium-oxide plates; the enrichment factors were evaluated using the ratio of all signal intensities for each peptide before and after enrichment.…”

Proteomics in investigation of cancer metastasis: Functional and clinical consequences and methodological challenges

“…in NF-B, TGF-␤, and Wnt metastasis-associated pathways), isolation of secreted glycoproteins captures potential noninvasive biomarkers. Panel of seven tyrosine-phosphorylated proteins was newly identified as associated with lung cancer metastasis [74]. Cadherin-5 was identified as a novel biomarker of metastatic breast cancer using glycoproteomic approach [75], the same approach also revealed the panel of ␣1,6-fucosylated glycoproteins related to HCC metastatic ability [76] and metastatic pathways in osteosarcomas [77].…”