An Informatics-assisted Label-free Quantitation Strategy that Depicts Phosphoproteomic Profiles in Lung Cancer Cell Invasion

Abstract

Aberrant protein phosphorylation plays important roles in cancer-related cell signaling. With the goal of achieving multiplexed, comprehensive, and fully automated relative quantitation of site-specific phosphorylation, we present a simple label-free strategy combining an automated pH/acid-controlled IMAC procedure and informatics-assisted SEMI (sequence, elution time, mass-to-charge, and internal standard) algorithm. The SEMI strategy effectively increased the number of quantifiable peptides more than 4-fold in replicate experiments (from 262 to 1171, p < 0.05, false discovery rate = 0.46%) by using a fragmental regression algorithm for elution time alignment followed by peptide cross-assignment in all LC-MS/MS runs. In addition, the strategy demonstrated good quantitation accuracy (10-12%) for standard phosphoprotein and variation less than 1.9 fold (within 99% confidence range) in proteome scale and reliable linear quantitation correlation (R(2) = 0.99) with 4000-fold dynamic concentrations, which was attributed to our reproducible experimental procedure and informatics-assisted peptide alignment tool to minimize system variations. In an attempt to explore metastasis-associated phosphoproteomic alterations in lung cancer, this approach was used to delineate differential phosphoproteomic profiles of a lung cancer metastasis model. Without sample fractionation, the SEMI algorithm enabled quantification of 1796 unique phosphopeptides (false discovery rate = 0.56%) corresponding to 854 phosphoproteins from a series of non-small cell lung cancer lines with varying degrees of in vivo invasiveness. Nearly 40% of the phosphopeptides showed >2-fold change in highly invasive cells; validation of phosphoprotein subsets by Western blotting not only demonstrated the consistency of data obtained by our SEMI strategy but also revealed that such dramatic changes in the phosphoproteome result mostly from translational or post-translational regulation. Mapping of these differentially expressed phosphoproteins in multiple cellular pathways related to cancer invasion and metastasis suggests that the site and degree of phosphorylation might have distinct patterns or functions in the complex process of cancer progression.

Global Analysis of Cdc14 Dephosphorylation Sites Reveals Essential Regulatory Role in Mitosis and Cytokinesis

“…Quantitative Analysis by IDEAL-Q-The quantitative analysis of phosphopeptides was performed with the SEMI label-free algorithm (34) and IDEAL-Q software (35). The raw data files acquired from the LTQ-Orbitrap were converted into files of mzXML format by the program ReAdW (XCalibur, Thermo Finnigan), and the search results in MASCOT were exported in Extensible Markup Language data (.xml) format.…”

“…It also demonstrated that Cdc14 is a major phosphatase in the cell cycle. To identify potential Cdc14 substrates, we filtered the cdc14-1/CDC14 ratio with a threshold of 2-fold determined from the technical replicate (34). A total of 835 dephosphorylation sites on 455 potential Cdc14 substrates showed a Ͼ2-fold increase in cdc14-1 cells in vivo (Fig.…”

DcR3 suppresses influenza virus-induced macrophage activation and attenuates pulmonary inflammation and lethality

“…The enriched samples were subjected to liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) analysis using a NanoAcquity system (Waters) connected to an LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific) equipped with a nanospray interface (Proxeon). The quantitative analysis was as described previously with minor modifications [25,26]. Raw MS/MS data were converted into peak lists (MGF file) using Raw2MSM [27] with default parameters.…”

“…The phosphopeptides enriched from non-, IgG1-, and DcR3-treated cell lysates were analyzed by LC-MS/MS, and a total of 1887 phosphopeptides derived from 826 proteins were identified. Based on the standard deviation of our label-free quantitation strategy [25], phosphopeptides showing more than twofold changes in abundance compared to control were considered as the altered phosphorylation sites with either upregulation or downregulation. Among all the identified phosphopeptides (n=1887), 532 phosphopeptides from 319 proteins were modulated in DcR3-Mϕ [i.e., with at least twofold of up-or downregulation of expression ratio (DcR3 versus IgG1-treatment)], while ratio in the control group (IgG1 versus non-treated group) did not show any statistically significant change before IAV infection.…”

Phosphorylation of Crm1 by CDK1-cyclin B promotes Ran-dependent mitotic spindle assembly

“…One site, serine 391, which has been identified in proteomic analyses of mitotic phosphoproteins (Daub et al, 2008;Dephoure et al, 2008;Olsen et al, 2010;Wang et al, 2010), is followed by Fig. 2.…”