Study of Burkitt lymphoma cell line proteins by high resolution two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry

We paper describe a mass spectrometric approach generally applicable for th e rapid identification and characterization of proteins isolated by two-dim ensional gel electrophoresis (2-DE). The highly sensitive nanoflow-electros pray mass spectrometry employing a quadrupole-time of flight mass spectrome ter was used for the direct identification of proteins from the peptide mix ture generated from only one high resolution 2-DE get without high performa nce liquid chromatography (HPLC) separation or Edman sequencing. Due to the high sensitivity and high mass accuracy of the instrument employed, this t echnique proved to be a powerful tool for the identification of proteins fr om femtomole amounts of materials. We applied the technique for the investi gation of Burkitt lymphoma BL60 cell proteins. This cell line has been used as a model to assign apoptosis-associated proteins by subtractive analysis of normal and apoptotic cells. From the nuclear fraction of these cells, 3 6 protein spots were examined, from only one micropreparative Coomassie Bri lliant Blue R-250 stained gel,after proteolytic digestion by matrix assiste d laser desorption ionization (MALDI) and nanospray mass spectrometry (MS). In combination with database searches, of 33 proteins were successfully id entified by nanospray-MS/MS-sequencing of up to eight peptides per protein. Three proteins were new proteins not listed in any of the available databa ses. Some of the identified proteins are known to be involved in apoptosis processes, the others were common proteins in the eukaryotic cell. The give n technique and the protein data are the basis for construction of a databa se to compare normal and apoptosis-induced cells and, further, to enable fa st screening of drug impact in apoptosis-associated processes.