Optimizing the strategy of combining TiO2- and immunoaffinity- based enrichment methods for tyrosine phosphoproteome
碩士 === 國立成功大學 === 環境醫學研究所 === 102 === Protein phosphorylation is one of the crucial post-translational modifications and regulates many biological functions in eukaryote cells. Several studies revealed that phosphorylation of protein tyrosine residues are involved in carcinogenesis. Thus, the identi...
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| Format: | Others |
| Language: | en_US |
| Published: |
2014
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| Online Access: | http://ndltd.ncl.edu.tw/handle/gay232 |
| Summary: | 碩士 === 國立成功大學 === 環境醫學研究所 === 102 === Protein phosphorylation is one of the crucial post-translational modifications and regulates many biological functions in eukaryote cells. Several studies revealed that phosphorylation of protein tyrosine residues are involved in carcinogenesis. Thus, the identification of tyrosine phosphorylated proteins require an efficient method from a complex sample matrix since the relative abundance of phosphotyrosine (P-Tyr) proteins only around 1% of the total human phosphoproteome. There are many procedures published for phosphopeptide enrichment, but only a few focusing on tyrosine phosphopeptides. So far there is no standard protocol to be followed for selective enrichment of tyrosine phosphopeptides. Here, we propose an analytical strategy of combining the optimized TiO2- enrichment experimental conditions and immunoaffinity- based enrichment methods for phosphotyrosine-specific phosphoproteomics analysis using MALDI-TOF MS analysis. We applied an enrichment factor to evaluate enrichment efficiency for pTyr peptides in many different parameters. The results showed that the optimized TiO2 enrichment is in loading solution pH 1.5 and elution solution pH 12.0 with enrichment factor 10305. The optimized immunoaffinity enrichment in Tris incubation buffer system pH9.0 with enrichment factor 1450 is higher than MOPS buffer. A combination of sequential TiO2 and immunoaffinity is the most effective and selective enrichment method for phosphotyrosine peptides. Additionally, enrichment factors evaluated by LC-MS yielded similar results as those obtained by MALDI-TOF MS analysis.
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