A liquid-phase three-dimensional protein separation method has been developed that is used to separate the cytosolic fraction of a HEL cell lysate via isoelectric focusing (IEF), nonporous @entity3440 (NPS) reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS), respectively. Several hundred unique protein molecular weights were observed in a pI range from 4.8 to 8.5 and a mass range from 5 to 85 kDa. Proteins were positively identified by analysis of the pI (+/-0.5 pI units), an intact protein molecular weight (+/-150 ppm), and peptide mass mapping results. Using the molecular weight (MW) and peptide mapping results of identified proteins it was possible to characterize their posttranslational (PTMs) and/or sequence modifications. PTMs were detected on both forms of cytosolic actin, @entity1457 90 beta, @entity5387 and alpha-enolase. Sequence modifications or conflicts were observed for @entity217 , @entity855 beta-synthase and @entity1457 90 beta. IEF-NPS-RP-HPLC/ESI-TOFMS was used to determine experimental pI, MW and relative hydrophobicity values for each protein detected. This data was used to generate a 2-D pI-MS protein map, where proteins are displayed according to their pI and molecular weight. Protein molecular weight peaks are represented as bands in the 2-D pI-MS image where the gray scale of each band is proportional to the intensity of the protein molecular weight peak. In addition, a third hydrophobicity dimension (%B) was added as the % @entity1626 elution to generate a 3-D pI-MS-%B plot where each protein can be tagged according to three parameters.

Isoelectric focusing nonporous XXXX reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry: a three-dimensional liquid-phase protein separation method as applied to the human erythroleukemia cell-line.

@entity217 :: ('728378', 'Gene') :: ['beta-and gamma-actin'] @entity1457 :: ('MESH:D012769', 'Disease') :: ['heat shock'] @entity1626 :: ('MESH:C032159', 'Chemical') :: ['acetonitrile'] @entity3440 :: ('MESH:D012822', 'Chemical') :: ['silica'] @entity5387 :: ('3094', 'Gene') :: ['HINT'] @entity855 :: ('MESH:D000255', 'Chemical') :: ['ATP']
@entity3440 :: (MESH:D012822,Chemical) :: ['silica']
