Chemo-Selection Strategy for Limited Proteolysis Experiments on the Proteomic Scale.

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Limited proteolysis coupled with mass spectrometry is valuable for proteome-wide studies.
• Isolating semitryptic peptides, which have one non-tryptic cleavage site, is challenging.
• Existing methods lack efficiency in enriching these specific peptides.

Purpose of the Study:

• To develop a chemo-selective enrichment strategy for semitryptic peptides.
• To improve proteomic coverage and structural insights from limited proteolysis.
• To enable quantitative determination of ligand binding affinities.

Main Methods:

• Developed the semitryptic peptide enrichment strategy for proteolysis procedures (STEPP).
• STEPP involves chemo-selective reaction of N-termini and lysine side chains with isobaric mass tags.
• Utilized N-hydroxysuccinimide (NHS)-activated agarose resin to remove tryptic peptides, isolating semitryptic peptides.
• Interfaced STEPP with pulse proteolysis (PP) and limited proteolysis (LiP) workflows.

Main Results:

• STEPP increased semitryptic peptide detection by 5- to 10-fold in LiP and PP experiments.
• Demonstrated STEPP-PP workflow with yeast cell lysate and drug proteins (cyclosporin A, geldanamycin).
• Validated STEPP-LiP workflow in MCF-7 and MCF-10A breast cancer cell lines.
• Enhanced proteomic coverage and structural information obtained from limited proteolysis.

Conclusions:

• STEPP is an effective strategy for enriching semitryptic peptides in mass spectrometry-based proteomic applications.
• The method significantly improves proteomic coverage and structural analysis.
• STEPP facilitates quantitative analysis of ligand binding affinities, offering new insights into protein interactions.