Isoelectric focusing nonporous silica 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.
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 silica (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, heat shock 90 beta, HINT and alpha-enolase. Sequence modifications or conflicts were observed for beta-and gamma-actin, ATP beta-synthase and heat shock 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 % acetonitrile elution to generate a 3-D pI-MS-%B plot where each protein can be tagged according to three parameters.
Wall
D B
DB
Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109-1055, USA.
Kachman
M T
MT
Gong
S S
SS
Parus
S J
SJ
Long
M W
MW
Lubman
D M
DM
D000595:Amino Acid Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000737:chemistry; D004586:Electrophoresis; D006801:Humans; D006868:Hydrolysis; D007525:Isoelectric Focusing; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000302:isolation & purification; D010449:Peptide Mapping; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D014357:Trypsin