Optimization of mass spectrometry-compatible surfactants for shotgun proteomics

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Shotgun proteomics relies on efficient protein digestion for comprehensive peptide and protein identification.
• Traditional trypsin digestion methods can be limited by protein solubility and proteolytic efficiency, especially in complex biological samples.

Purpose of the Study:

• To optimize and compare different trypsin digestion strategies for enhanced peptide and protein identification using micro liquid chromatography-tandem mass spectrometry (microLC-MS/MS).
• To evaluate the impact of MS-compatible detergents on protein digestion efficiency in various solvent systems.

Main Methods:

• Comparison of trypsin digestion protocols with and without MS-compatible detergents.
• Analysis of peptide and protein identifications using microLC-MS/MS.
• Investigation of mixed organic-aqueous and aqueous solvent systems.

Main Results:

• MS-compatible detergents significantly increased peptide and protein identifications in complex protein mixtures.
• A modified protocol with MS-compatible detergents identified over 300 proteins from 5 µg of pancreatic cell lysates, outperforming urea-based digestion.
• Combining trypsin digestion with three different MS-compatible detergents led to the identification of over 700 proteins.
• Solvent systems influenced protein identifications in conjunction with MS-compatible detergents, affecting peptide hydrophobicity.

Conclusions:

• Incorporating MS-compatible detergents into trypsin digestion protocols substantially enhances both qualitative and quantitative peptide identifications.
• These optimized digestion schemes lead to a greater overall number of protein identifications, including potential improvements in identifying low-abundance proteins.