| Summary: | 碩士 === 國立中正大學 === 化學暨生物化學研究所 === 102 === Protein phosphorylation is an important post-translational modification since it plays a key process in many cellular processes. Although MS technology has been commonly used for phosphorylation site identification in a complex phosphoproteome, achieving comprehensive coverage of the human phosphorylation network remains a challenge. Among the serine, threonine and tyrosine phosphorylation in human, tyrosine phosphorylation is most well-known therapeutic target, yet its infrequent occurrence (1.8%) and low substiochmetric levels of modified form low abundance cause difficulty for the currently under-represented tyrosine phosphorproteome. To address this challenge, in this thesis, we have developed an integrated subcellular and chemical fractionation strategy to enhance the number of the phosphotyrosine component of the phosphoproteome.
Taking advantage of the distinct binding affinities of Ga3+ and Fe3+ for phosphopeptides, in the first part, we designed a metal-directed immobilized metal ion affinity chromatography (IMAC) for the sequential enrichment of phosphopeptides. To further reduce the sample complexity, subcellular fractionation was performed on cell lysate to separate into membrane, nuclear and cytosol components. 5288 phosphopeptides and 599 phosphotyrosine peptides from the model cell line (Raji B) were identified using sequential Ga3+-Fe3+-IMAC, which is higher than identifications obtained using only a single Fe3+-IMAC enrichment, with only 4553 phosphopeptides and 421 phosphotyrosine peptides identified. This corresponds to an increase of 1.4 folds in the identification of tyrosine phosphopeptides. With incorporation of subcellular fractionation before Ga3+-Fe3+-IMAC phosphopeptide enrichment, we identified 5205 phosphopeptides and 1388 phosphotyrosine peptides, including 274 previously unidentified tyrosine phosphorylation sites. We also found that 1088 phosphotyrosine peptides (78%) can be effectively purified in 1st Ga3+-IMAC fraction. The increase in the numbers of identified phosphotyrosine peptides is due to the increase in the ratio of phosphotyrosine peptides obtained from all the detected phosphopeptides. 26% phosphotyrosine peptides was obtained with subcellular-Ga3+-Fe3+-IMAC method while only 11% with Ga3+-Fe3+-IMAC method. In addition, subcellular-Ga3+-Fe3+-IMAC method also effectively increased the BCR pathway (B cell receptor signaling pathway) identification coverage in Raji B cell.
For deep phosphoproteome analysis with high sensitivity, on the second part of thesis, a small-scale high-pH RP (reverse phase) stage tip was developed for further phosphopeptides fractionation after IMAC enrichment. The number of phosphopeptides can be increased from 5205 to 7962 (15 % tyrosine sites) in 400 ug HeLa cell lysate. Comparing the two integrated fractionation approaches, Subcellular-Ga3+-Fe3+-IMAC and Fe3+-IMAC-RP from HeLa cell line, more tyrosine phosphopeptide can be detected by Fe3+-IMAC-RP (n=1225) compared to Subcellular-Ga3+-Fe3+-IMAC (n=545) and only 20% overlap of identified tyrosine phosphopeptides were observed between these two approaches. We demonstrated that this strategy can be applied for more comprehensive characterization of the tyrosine phosphoproteome.
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