Purification of organelles and analysis of their
proteins is an important initial step for
biological proteomics, simplifying the
proteome prior to analysis by established techniques such as two-dimensional liquid chromatography (2-DLC) or two-dimensional gel electrophoresis (2-DE).
Nuclear proteins play a central role in regulating gene expression, but are often under-represented in proteomic studies due to their lower abundance in comparison to cellular 'housekeeping' metabolic
enzymes and structural
proteins. A reliable procedure for separation and proteomic analysis of
nuclear proteins would be useful for investigations of cell proliferation and differentiation during disease processes (e.g., human
cancer). In this study, we have purified nuclei from the human
Burkitt's lymphoma B-cell line, Raji, using
sucrose density gradient centrifugation. The integrity and purity of the nuclei were assessed by light microscopy and
proteins from the nuclear fractions were separated by 2-DE and identified using matrix assisted
laser desorption ionization mass spectrometry (MALDI-MS). A total of 124 unique
proteins were identified, of which 91% (n=110) were predicted to be nuclear using PSORT.
Proteins from the nuclear fraction were subjected to affinity chromatography on
DNA-
agarose to isolate
DNA-binding proteins. From this purified fraction, 131 unique
proteins were identified, of which 69% (n=90) were known or predicted
DNA-binding proteins. Purification of nuclei and subsequent enrichment of
DNA-binding proteins allowed identification of a total of 209 unique
proteins, many involved in transcription and/or correlated with
lymphoma,
leukemia or
cancer in general. The data obtained should be valuable for identification of
biomarkers and targets for
cancer therapy, and for furthering our understanding of the molecular mechanisms underlying
lymphoma development and progression.