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 matrix-assisted [correction of multi-assisted] laser desorption/ionization mass spectrometry analysis and sequence database searching.
Non-porous reversed phase (NPRP) high-performance liquid chromatography (HPLC) has been used as a rapid method to separate proteins from whole cell lysates 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 method, using a programmed acetonitrile:H2O gradient. The separated proteins were collected as liquid fractions as they eluted, and were further separated on the NPRP column with a different gradient to separate coeluting peaks. The isolated protein fractions were analyzed by matrix-assisted laser desorption/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 extraction 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 exactly one match against the database. This methodology is demonstrated for several proteins isolated from HEL cells and identified via database matching.
Chen
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Y
Department of Chemistry, University of Michigan, Ann Arbor 48109-1055, USA.
Wall
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D
Lubman
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DM
D002147:Calmodulin / Q000737:chemistry / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification; D016208:Databases, Factual; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000302:isolation & purification; D010449:Peptide Mapping; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification; D012261:Ribonucleoproteins / Q000737:chemistry / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013947:Thymosin / Q000737:chemistry / Q000302:isolation & purification; D014407:Tumor Cells, Cultured / Q000737:chemistry; D000099201:Thymosin beta(4)
D002147:Calmodulin; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D012261:Ribonucleoproteins; D013947:Thymosin; D000099201:Thymosin beta(4)