CAPILLARY ISOELECTRIC FOCUSING-BASED MULTIDIMENSIONAL PEPTIDE/PROTEIN SEPARATIONS FOR PROTEOMICS ANALYSIS
Lee, Cheng S
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With the completion of the human genome project, the proteomics has become the focus of research interest for better understanding the complex biological processes. The mass spectrometry (MS) detection of vast number and broad dynamic range of the proteins requires that sample fractionation and separation be performed prior to MS analysis. Realizing the limitations of gel-based proteomic techniques, capillary-based and gel free separation technologies are presented as attractive alternatives holding the promises of high separation efficiency and resolution, broad dynamic range, easy system automation as well as high throughput. Coupled with laser free microdissection technology, the combination of CIEF with nano-RPLC in an automated and integrated platform was employed for comprehensive and sensitive proteome studies of limited protein quantities obtained from tissue samples. The peptides were first separated and concentrated by CIEF and were sequentially fractionated. All the CIEF fractions were further resolved by nano-RPLC, followed by tandem MS analysis. A total of 6,866 fully tryptic peptides were detected, leading to the identification of 1,820 distinct proteins. Due to limited peptide sequence coverage of identified proteins, the bottom-up approaches provide very limited molecular information about the intact proteins, particularly towards the detection of post-translational modifications. In contrast, top-down methods are advantageous for the detection of protein modifications. To improve separation efficiency and resolution of nano-RPLC separations for intact proteins, various chromatography conditions, including the chain length of the stationary phase, the column temperature, and the ion-pairing agent utilized in the mobile phase, were optimized using model proteins. Building upon the experience in the development of automated and integrated multidimensional peptide separation platform and the optimization of protein chromatography separation, a top-down proteome characterization of yeast cell lysates was further evaluated. An overall system capacity of 4,320-7,200 was achieved and a total of 534 distinct yeast protein masses were measured, yet required a protein loading of only 9.6 ug. This protein loading is two to three orders of magnitude less than those used in current top-down proteome techniques, illustrating the potential usage of this proteome technology for the analysis of protein profiles within small cell populations or limited tissue samples.