Establishing a Fast-Releasing Proteolytic Peptide Library to Enhance the Accuracy of Targeted Protein Quantification by Using Mass Spectrometry

The use of mass spectrometry to quantify the target protein has become a key method to validate biomarker molecules in clinical samples. However, the accurate protein quantification depends on whether the peptide can be effectively hydrolyzed, be stably stored in the hydrolysate, and be efficiently extracted. In addition, the absolute content of the target protein is also highly related to the efficiency of the representative peptides successfully detected. Therefore, the introduction of an appropriate target peptide enrichment technique to increase the injection amount of the starting sample and to overcome the problem of the detecting sensitivity of the device provides an opportunity to improve the performance of mass spectrometry-based protein quantification. The purpose of this study is to develop an effective analytical platform for the representative peptide enrichment and detection for rapid verification of target proteins in a large number of samples. The specific aims of this study are listed as follows: 1. To combine chemical modification and cation exchange chromatography to establish a high magnification peptide enrichment method to improve the sensitivity of sample analysis. 2. To evaluate whether the strategy for generating peptides by hydrolyzing proteins in non-reduced status and then fractionated by the cation exchange enrichment method can improve the sensitivity of sample analysis. 3. To establish a database containing the information of releasing speed of peptides during protein digestion. 4. To establish a representative peptide library with the optimized data acquisition parameters for the multiple reaction monitoring mass spectrometric analysis. Finally, these identified peptides will be further evaluated in bio-fluid samples to ensure its practical value. We believe that the database established by this project will be the most straightforward one for the work of biomarker verification.

Project ID:PC10907-1565
External Project ID:MOST109-2320-B182-039