SHOTGUN CHARACTERIZATION OF NUCLEAR AND WC PROTEINS IN ENCODE CELL LINES

University Of Washington, Seattle WA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The previous project using chromatographic intensities from the FT-ICR and crawdad to identify proteins in nuclei that were changing between cell lines identified several hundred proteins. However, only 10 of these were transcription factors. Consequently, we sought to utilize the increased sampling speed of the LTQ-VELOS in order to better identify low abundant nuclear proteins, such as transcription factors. Whole cell extracts and nuclear extracts from BJ, SKNSH, HepG2 and K562 cell lines were run in multiple replicates and analyzed using a data dependent acquisition (DDA) approach. This experiment was able to identify several dozen more transcription factors (TFs) in the nuclear samples of each of these cell lines. Additionally, these experiments were able to identify several transcription factors that were unique to the whole cell extract. These transcription factors correspond to proteins that are known to be predominately cytoplasmic under the growth conditions. One startling finding was the discrepancy between the cell-lines in total number of TFs identified. BJ nuclear extracts showed only a fraction of the number of TFs found in K562 nuclear extracts. This finding is thought to be due to the higher dynamic range of proteins present in BJ versus K562 nuclear extracts. As shown in the previous experiment. BJ nuclei contain an elevated amount of structural proteins that are most likely "hogging" the exclusion list in a DDA run.