Abstract
Protein tyrosine kinases (PTKs) play key roles in cellular signal transduction, cell cycle regulation, cell division, and cell differentiation. Dysregulation of PTK-activated pathways, often by receptor overexpression, gene amplification, or genetic mutation, is a causal factor underlying numerous cancers. In this study, we have developed a parallel reaction monitoring-based assay for quantitative profiling of 83 PTKs. The assay detects 308 proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single run. Quantitative comparisons were based on the labeled reference peptide method. We implemented the assay in four cell models: 1) a comparison of proliferating versus epidermal growth factor-stimulated A431 cells, 2) a comparison of SW480Null (mutant APC) and SW480APC (APC restored) colon tumor cell lines, and 3) a comparison of 10 colorectal cancer cell lines with different genomic abnormalities, and 4) lung cancer cell lines with either susceptibility (11-18) or acquired resistance (11-18R) to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. We observed distinct PTK expression changes that were induced by stimuli, genomic features or drug resistance, which were consistent with previous reports. However, most of the measured expression differences were novel observations. For example, acquired resistance to erlotinib in the 11-18 cell model was associated not only with previously reported up-regulation of MET, but also with up-regulation of FLK2 and down-regulation of LYN and PTK7. Immunoblot analyses and shotgun proteomics data were highly consistent with parallel reaction monitoring data. Multiplexed parallel reaction monitoring assays provide a targeted, systems-level profiling approach to evaluate cancer-related proteotypes and adaptations. Data are available through Proteome eXchange Accession PXD002706.
Highlights
Expression of PTKs is measured by enzymelinked immunosorbent assay, fluorescence activated cell sorting and immunoblotting, which provide information for a limited number of proteins in a single assay
Quantitative analyses were done by the labeled reference peptide (LRP) method we described previously [24] using U-13C6, U-15N4-arginine-labeled alkaline phosphatase (AP) peptide (AAQGDITAPGGA*R), -galactosidase (BG) peptide (APLDNDIGVSEAT*R), and -actin (ACTB) peptide (GYSFTTTAE*R) as the reference standard mixture (New England Peptide, Gardner, MA)
Development of PTK parallel reaction monitoring (PRM) Assay Panel—We developed a PRM assay for quantitation of 83 PTKs, which measures proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single run
Summary
Expression of PTKs is measured by enzymelinked immunosorbent assay, fluorescence activated cell sorting and immunoblotting, which provide information for a limited number of proteins in a single assay. Multiplexed Kinase Quantitation ity of MRM measurements, the two-stage mass filtering using a low-resolution MS instrument does not completely avoid interfering ions, which can hamper precise and specific protein quantification [12]. High resolution and accurate mass peptide analysis can be achieved with new generation mass spectrometers, such as the Q Exactive (ThermoFisher Scientific). These instruments combine the quadrupole precursor ion selection with the high resolution and high accuracy of an Orbitrap mass analyzer. A useful approach to targeted proteome analysis is to configure multiplexed assay panels for proteins and their modified forms involved in specific pathways or networks. Multiplexed MRM assay panels have been used to quantify phosphotyrosine sites in tyrosine kinase signaling networks [18] and to monitor the protein expression status of cellular metabolic pathways [19]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
