Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteinsfrom human breast cancer whole cell lysates: a novel two-dimensional liquid chromatography/mass spectrometry method

A novel two-dimensional two-column liquid chromatography/mass spectrometry (LC/MS) technique is described in this work, where chromatofocusing (CF) ha s been coupled to nonporous reversed-phase (NPS-RP) HPLC to separate protei ns from human breast epithelial whole cell lysates. The liquid fractions fr om NPS-RP-HPLC are readily amenable to direct on-line analysis using electr ospray ionization orthogonal acceleration time-of-flight mass spectrometry (ESI-TOFMS). A key advantage of this technique is that proteins fan be 'pee led off' in the liquid phase from the CF column according to their isoelect ric points (pI) in the first chromatographic separation dimension. The NPS- RP-HPLC column further separates these pi-focused fractions based upon prot ein hydrophobicity as the second chromatographic dimension. The third dimen sion 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 analo gous to a 2-D gel image. Thus, this technique may provide a means to study differential expression of proteins from whole cell lysates. Copyright (C) 2001 John Wiley & Sons, Ltd.