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Chromatofocusing nonporous reversed‐phase high‐performance liquid chromatography/electrospray ionization time‐of‐flight mass spectrometry of proteins from human breast cancer whole cell lysates: a novel two‐dimensional liquid chromatography/mass spectrometry method
Author(s) -
Chong Bathsheba E.,
Yan Fang,
Lubman David M.,
Miller Fred R.
Publication year - 2001
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.227
Subject(s) - chemistry , chromatography , mass spectrometry , chromatofocusing , electrospray ionization , high performance liquid chromatography , time of flight mass spectrometry , electrospray , protein mass spectrometry , reversed phase chromatography , hydrophilic interaction chromatography , sample preparation in mass spectrometry , analytical chemistry (journal) , isoelectric point , ionization , biochemistry , ion , organic chemistry , enzyme
A novel two‐dimensional two‐column liquid chromatography/mass spectrometry (LC/MS) technique is described in this work, where chromatofocusing (CF) has been coupled to nonporous reversed‐phase (NPS‐RP) HPLC to separate proteins from human breast epithelial whole cell lysates. The liquid fractions from NPS‐RP‐HPLC are readily amenable to direct on‐line analysis using electrospray ionization orthogonal acceleration time‐of‐flight mass spectrometry (ESI‐TOFMS). A key advantage of this technique is that proteins can be ‘peeled off’ in the liquid phase from the CF column according to their isoelectric points ( pI ) in the first chromatographic separation dimension. The NPS‐RP‐HPLC column further separates these pI ‐focused fractions based upon protein hydrophobicity as the second chromatographic dimension. The third dimension involves on‐line molecular weight determination using ESI‐TOFMS. As a result, this method has the potential to be fully automated. In addition, a 2‐D protein map of pI versus molecular weight is generated, which is analogous to a 2‐D gel image. Thus, this technique may provide a means to study differential expression of proteins from whole cell lysates. Copyright © 2001 John Wiley & Sons, Ltd.