The discovery of polypeptides and proteins with relevance to a particular biological state is complicated by their vast number and concentration range in most biological mixtures. Depletion methodologies are frequently used to remove the most abundant species; however, this removal not only fails significantly to enrich trace proteins, it may also nonspecifically deplete them due to their interactions with the removed high-abundance proteins. Here we report a simple-to-use methodology that reduces the protein concentration range of a complex mixture like whole serum through the simultaneous dilution of high-abundance proteins and the concentration of low-abundance proteins. This methodology utilizes solid-phase ligand libraries of immense diversity, generated by "split, couple, recombine" combinatorial chemistry, that are used for affinity-based binding to the proteins of a given mixture. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that many peptides or proteins that are undetectable by classical analytical methods become easily accessible. The reduction in the dynamic range of unfractionated serum is specifically described along with treatment of other proteomes such as extracts from Escherichia coli, chicken egg white and cell culture supernatant. Mono- and bi-dimensional electrophoresis (1-DE and 2-DE respectively) and surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-TOF-MS) technology demonstrate the reduction in protein concentration range. Combining this approach with additional fractionation methods further increased the number of detectable species.
The mechanisms by which inhibition of the epidermal growth factor receptor (EGFR) sensitizes non-small cell lung cancer (NSCLC) cells to ionizing radiation remain poorly understood. We set out to characterize the radiosensitizing effects of the tyrosine kinase inhibitor erlotinib and the monoclonal antibody cetuximab in NSCLC cells that contain wild-type p53. Unexpectedly, EGFR inhibition led to pronounced cellular senescence but not apoptosis of irradiated cells both in-vitro and in-vivo. Senescence was completely dependent on wild-type p53 and associated with a reduction in cell number as well as impaired clonogenic radiation survival. Study of ten additional NSCLC cell lines revealed that senescence is a prominent mechanism of radiosensitization in 45% of cell lines and occurs not only in cells with wild-type p53 but also in cells with mutant p53 where it is associated with an induction of p16. Interestingly, senescence and radiosensitization were linked to an increase in residual radiation-induced DNA double-strand breaks irrespective of p53/p16 status. This effect of EGFR inhibition was at least partially mediated by disruption of the MEK-ERK pathway. Thus, our data indicate a common mechanism of radiosensitization by erlotinib or cetuximab across diverse genetic backgrounds. Our findings also suggest that assays that are able to capture the initial proliferative delay that is associated with senescence should be useful for screening large cell line panels to identify genomic biomarkers of EGFR inhibitor-mediated radiosensitization.
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