Development of novel multi-dimensional separation methods for analysis of the proteome using iso-electric focusing, reversed phase HPLC, MALDI TOF/MS and ESI TOF/MS.

Abstract

This work is directed towards the development of novel liquid phase multi-dimensional protein separations designed for efficient interfacing to mass spectrometry based detection and identification methods. The initial work focused on development of RP HPLC methods capable of analyzing a wide mass range of proteins with high efficiency. This work was performed using lysates from <italic>E. coli </italic> that were either induced or uninduced to express proteins involved in the metabolism of L-arabinose. The work was performed using non-porous RP HPLC columns that were optimal for protein analysis due to the absence of pores in the packing material thus allowing elimination of intra-particle resistance to mass transfer problems. Having established a viable RP HPLC method for intact protein collection and identification an orthoganol separation method was needed to increase the resolving power of the method to better match the immense complexity of the typical proteomic sample. For this purpose a liquid phase isoelectric focusing method was developed that was capable of producing 20 discrete pH fractions that contained isoelectrically focused proteins. The detergents and chaotropes used to keep the proteins soluble during the IEF separation were designed to be compatible with the next stage separation, this being the RP HPLC. Individual pH fractions were then analyzed by nonporous silica RP HPLC and a 2D protein map was generated to represent all the protein features detected by the UV detector. The final stage of the work was to identify the eluting proteins from the IEF - nonporous silica (NPS) RP HPLC method using both MALDI-MS peptide mass mapping and ESI TOF/MS of intact proteins. The results of these separations and mass spectrometric detection methods were displayed in 2D and 3D images representing the protein's pI, MW and hydrophobicity. A strong correlation was discovered between the protein's elution time from the NPS RP HPLC column and its hydrophobicity as represented by the ratio of the nonpolar to polar amino acids. This correlation allows for more efficient protein identification and mapping methods as well as the ability to predict the time of elution of a known target protein and thus facilitate its collection and analysis. The IEF - NPS RP HPLC - ESI TOF/MS method was capable of detecting protein MW values from 5 to 85 kDa within 150 ppm mass accuracy, pI values from 3.5 to 9.0 within +/- 0.5 pI units and hydrophobicity values (ratio of nonpolar to polar amino acids) to within a precision of 0.035 units. The method was able to detect over 200 unique mass features and to immediately determine the isoform of identified proteins by associating the highly accurate protein MW with the MW as determined from the annotated Swiss-prot database.