Rapid identification and screening of proteins from whole cell lysates of human erythroleukemia cells in the liquid phase, using non-porous reversed phase high-performance liquid chromatography separations of proteins followed by multi-assisted laser desorption/ionization mass spectrometry analysisand sequence database searching

Non-porous reversed phase (NPRP) high-performance liquid chromatography (HP LC) has been used as a rapid method to separate proteins from whole cell ly sates of human erythroleukemia (HEL) cells. Using phosphate-buffered saline (PBS) as a lysis buffer to extract proteins from HEL cells, more than 100 proteins of molecular weight up to 30 kDa were separated by the NPRP HPLC m ethod, using a programmed acetontirile:H2O gradient. The separated proteins were collected as liquid fractions as they eluted, and were further separa ted on the NPRP column with a different gradient to separate coeluting peak s. The isolated protein fractions were analyzed by matrix-assisted laser de sorption/ionization mass spectrometry (MALDI-MS) to determine the molecular weight of the protein. The proteins were cleaved by chemical or enzymatic digestion to produce peptide maps, which were analyzed by pulsed delayed ex traction MALDI-MS. The peptide maps were matched against a database search to determine the protein identity. In some cases, several enzymes were used in order to find erectly one match against the database. This methodology is demonstrated for several proteins isolated from HEL cells and identified via database matching. (C) 1998 John Wiley & Sons, Ltd.