研究目的
To develop a high-throughput sample preparation method that is amenable to both top-down and bottom-up proteomics and overcome the limitations of the conventional acid cleavable surfactants.
研究成果
The study demonstrates that Azo is fully compatible with bottom-up proteomics and uniquely serves as an “all-in-one” MS-compatible surfactant for both bottom-up and top-down proteomics, greatly facilitating high-throughput sample handling before MS-analysis. Azo enables effective protein extraction, rapid enzymatic digestion, and is amenable to MS analysis without an additional desalting step, improving throughput for a wide range of applications.
研究不足
The study focuses on the use of Azo for proteomics and does not compare it with all existing surfactants. The method's applicability to all types of proteins and modifications is not fully explored.
1：Experimental Design and Method Selection:
The study utilized a newly developed MS-compatible photocleavable surfactant, 4-hexylphenylazosulfonate (Azo), for protein extraction, rapid enzymatic digestion, and subsequent MS-analysis following UV degradation.
2：Sample Selection and Data Sources:
Standard proteins (myoglobin, insulin, and carbonic anhydrase) and complex cell lysates (HEK293T cells) were used.
3：List of Experimental Equipment and Materials:
Azo surfactant, UV-Vis spectroscopy, SDS-PAGE, LC-MS/MS analysis using a Q-TOF mass spectrometer.
4：Experimental Procedures and Operational Workflow:
Proteins were extracted and solubilized using Azo, digested with trypsin, degraded by UV irradiation, and analyzed by LC-MS/MS.
5：Data Analysis Methods:
Data was processed using MaxQuant for protein identification and label-free quantitation.
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