Proteomics multidimensional HPLC

With the hnman genome now sequenced (primarily by capillary electrophoresis technology), scientists are turning their attention to proteomics. The term pro-teome has been introdnced to define the full complement of proteins in a cell. It also reqnires a description of the localization, concentration, and multisubunit association of each of these proteins (25). The multidimensional hplc with automated column switching technology can successfully complement the more conventional approaches to protein separation, such as two-dimensional gel electrophoresis, which nsnally involve many manual steps and are not conducive to high throughput applications. 

Human peripheral blood-derived DCs take up therapeutic proteins via macropinocytosis or receptor-mediated endocytosis, degrade them proteolyti-cally, and load the resulting peptide fragments onto HLA class II molecules, provided that the latter contain an appropriate epitope and resist downstream quality control by the peptide editor HLA-DM (Vogt et al., 2005). The second step foresees cell lysis, affinity extraction of HLA-peptide complexes, and acid elution of bound peptides. This procedure provides a mixture of both selfepitopes from the DC proteome and potential epitopes derived from the therapeutic protein. To separate mixtures of 1500-2000 distinct peptides with maximal resolution, the so-called Multidimensional Protein Identihcation Technology is employed (Kropshofer and Spindeldreher, 2005). Peptides leaving the HPLC capillary upon separation are sprayed directly into the orifice of an ion trap mass spectrometer for sequence determination. 

McLean, J. A. Tao, L. Perkins, S. L. Russell, D. H. Multidimensional proteomic analysis of Escherichia Coli whole cell lysates by HPLC—MALDI—ion mobility—MS Extending dynamic range in protein analysis. In the 53rd American Society for Mass Spectrometry Annual Meeting, San Antonio, TX (June 2005). 

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