Three-dimensional protein map according to pI, hydrophobicity and molecular mass

A three-dimensional method has been developed to map the protein content of cells according to pI, Mw and hydrophobicity. The separation of complex protein mixtures from cells is performed using isoelectric focusing (IEF) in the liquid phase in the first dimension, non-porous silica (NPS) RP-HPLC in the second dimension and on-line electrospray ionization (ESI) time-of-flight mass spectrometry (TOF-MS) detection in the third dimension. The experimentally determined pI, Mw and hydrophobicity can then be used to produce a three-dimensional map of the protein expression of a cell, where now each protein can be tagged by three independent parameters. The ESI-TOF-MS provides an accurate Mw for the intact protein while the hydrophobicity dimension results from the RP-HPLC component of the separation. The elution time, or percent acetonitrile at time of elution, of the protein is related to the hyrophobicity, which is an inherent property of the protein. 3D protein maps can thus be generated showing pI, Mw and % acetonitrile at time of elution as well as pI, Mw and hydrophobicity. The potential of the 3D plot for effective mapping of proteins from cells compared to current 2D methods is discussed.