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
dc.contributor.author | Wall, Daniel B. | en_US |
dc.contributor.author | Kachman, Maureen T. | en_US |
dc.contributor.author | Gong, Siyuan S. | en_US |
dc.contributor.author | Parus, Stephan J. | en_US |
dc.contributor.author | Long, Michael W. | en_US |
dc.contributor.author | Lubman, David M. | en_US |
dc.date.accessioned | 2006-04-19T14:08:10Z | |
dc.date.available | 2006-04-19T14:08:10Z | |
dc.date.issued | 2001-09-30 | en_US |
dc.identifier.citation | Wall, Daniel B.; Kachman, Maureen T.; Gong, Siyuan S.; Parus, Stephen J.; Long, Michael W.; Lubman, David M. (2001)."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." Rapid Communications in Mass Spectrometry 15(18): 1649-1661. <http://hdl.handle.net/2027.42/35080> | en_US |
dc.identifier.issn | 0951-4198 | en_US |
dc.identifier.issn | 1097-0231 | en_US |
dc.identifier.uri | https://hdl.handle.net/2027.42/35080 | |
dc.identifier.uri | http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=11555863&dopt=citation | en_US |
dc.description.abstract | 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 14kDa. Proteins were positively identified by analysis of the pI (±0.5 pI units), an intact protein molecular weight (±150 14ppm), 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 Β, HINT and Α-enolase. Sequence modifications or conflicts were observed for Β-and Γ-actin, ATP Β-synthase and heat shock 90 Β. 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. Copyright © 2001 John Wiley & Sons, Ltd. | en_US |
dc.format.extent | 503942 bytes | |
dc.format.extent | 3118 bytes | |
dc.format.mimetype | application/pdf | |
dc.format.mimetype | text/plain | |
dc.language.iso | en_US | |
dc.publisher | John Wiley & Sons, Ltd. | en_US |
dc.subject.other | Chemistry | en_US |
dc.subject.other | Analytical Chemistry and Spectroscopy | en_US |
dc.title | 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 | en_US |
dc.type | Article | en_US |
dc.rights.robots | IndexNoFollow | en_US |
dc.subject.hlbtoplevel | Science | en_US |
dc.description.peerreviewed | Peer Reviewed | en_US |
dc.contributor.affiliationum | Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109-1055, USA | en_US |
dc.contributor.affiliationum | Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109-1055, USA | en_US |
dc.contributor.affiliationum | Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109-1055, USA | en_US |
dc.contributor.affiliationum | Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109-1055, USA | en_US |
dc.contributor.affiliationum | Department of Pediatrics, School of Medicine, The University of Michigan, Ann Arbor, MI 48109, USA | en_US |
dc.contributor.affiliationum | Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109-1055, USA ; Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109-1055, USA. | en_US |
dc.identifier.pmid | 11555863 | en_US |
dc.description.bitstreamurl | http://deepblue.lib.umich.edu/bitstream/2027.42/35080/1/421_ftp.pdf | en_US |
dc.identifier.doi | http://dx.doi.org/10.1002/rcm.421 | en_US |
dc.identifier.source | Rapid Communications in Mass Spectrometry | en_US |
dc.owningcollname | Interdisciplinary and Peer-Reviewed |
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