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Controlling nonspecific trypsin cleavages in LC-MS/MS-based shotgun proteomics using optimized experimental
Pan Fang1, Mingqi Liu1, Yu Xue2
1Minhang Hospital and Institutes of Biomedical Sciences, Fudan University, Shanghai, 201199, P. R. China. shenhuali@fudan.edu.cn pyyang@fudan.edu.cn.
The Analyst
|September 30, 2015
Summary
Nonspecific trypsin cleavages are common in proteomics sample preparation. Optimizing digestion parameters significantly reduced these errors, improving protein identification accuracy in shotgun and glycoproteomics.
Area of Science:
- Proteomics
- Biochemistry
- Analytical Chemistry
Background:
- Trypsin is a key enzyme for protein digestion in shotgun proteomics.
- Nonspecific trypsin cleavages can compromise protein identification and quantification accuracy.
- These cleavages are frequently observed across diverse biological samples.
Purpose of the Study:
- To investigate the prevalence of nonspecific trypsin cleavages in various biological systems.
- To optimize sample preparation parameters to minimize these unwanted cleavages.
- To enhance the reliability of shotgun and glycoproteomics workflows.
Main Methods:
- Examined multiple biological systems (protein mixtures, yeast, serum, cell lines, mouse brain).
- Systematically optimized digestion parameters including denaturants, storage time, trypsin type, enzyme ratio, and protein concentration.
- Applied optimized protocol to N-glycoproteomics for validation.
Main Results:
- Nonspecific trypsin cleavages were found to be frequent in analyzed samples.
- Optimized conditions reduced partially tryptic peptides from 28.4% to 2.8%.
- Protocol successfully decreased partially tryptic peptides in N-glycoproteomics samples.
Conclusions:
- Controlling nonspecific trypsin cleavages is crucial for accurate proteomics.
- Optimized sample preparation enhances data quality in shotgun and glycoproteomics.
- Standardized protocols improve routine proteomics sample treatment and understanding.
