Protein LC-MS Using Slip Flow Chromatography
Histones are essential chromosomal proteins with large numbers of variants and post-translational modifications (PTMs). PTM levels of histones are known to correlate with different stages of cancer. Due to the lack of resolution on intact histone separations, conventional methods for histone analysis require time-consuming digestion, which often leads to the loss of PTM information. Slip flow chromatography with orderly packed nonporous silica particles has been shown to greatly increase the efficiency of protein separation by reducing eddy diffusion and resistance to mass transfer. In this work, higher resolution for intact histones separation was achieved by using a 5 cm capillary with 470 nm particle size and C18 bonded phase. The levels of histone phosphorylation and other PTMs were verified by deconvoluted MS spectra. This methodology was also applied to the separation of a complex mixture of protein digests. A peak capacity of 500 was achieved by using a 30 min gradient elution. Other bonded phases such as C4 and TDP were also developed to separate different protein samples. This dissertation also includes 1) separation of intact monoclonal antibodies by RPLC; 2) characterization of reduced monoclonal antibodies by nano-RPLC-MS; 3) analysis of ubiquitin and ubiquitin linker in a new pathway of ubiquitination; 4) separation of RRM2 monomer and aggregates. Above results demonstrated the effectiveness of slip flow capillary for the separation of both protein and peptide separation for proteomic studies.
Wirth, Purdue University.
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