[Mirror cutting-assisted orthogonal digestion enabling large-scale and accurate protein complex characterization]

Area of Science:

• Proteomics and Structural Biology
• Biochemistry and Molecular Biology

Background:

• Protein complexes are vital for biological functions, necessitating accurate structural and interaction characterization.
• Chemical cross-linking coupled with mass spectrometry (CXMS) is a powerful technique for analyzing protein complexes.
• Current CXMS methods face limitations in cross-linked site identification accuracy due to poor spectrum quality.

Purpose of the Study:

• To develop an improved orthogonal digestion strategy for enhanced cross-linked peptide identification in CXMS.
• To increase the accuracy, coverage, and quality of cross-linked site identification in protein complex analysis.
• To provide a more robust method for large-scale and highly accurate characterization of protein complexes.

Main Methods:

• Developed an orthogonal digestion strategy combining LysargiNase and trypsin, enzymes with complementary cleavage specificities.
• Analyzed both simple (bovine serum albumin) and complex (Escherichia coli) protein samples using the new digestion method.
• Utilized LC-MS/MS and database searching to identify and analyze cross-linked peptides and sites.

Main Results:

• The orthogonal digestion strategy significantly increased the number of identified cross-linked sites (e.g., 35% increase for BSA, 16% increase for E. coli).
• Improved spectrum quality was observed for commonly identified cross-linked peptides, with more b- and y-type fragment ions and better sequence matching.
• The method yielded substantial structural constraints and identified protein-protein interactions, demonstrating its effectiveness in complex samples.

Conclusions:

• The mirror-cutting-assisted orthogonal digestion strategy substantially enhances the accuracy and coverage of cross-linked peptide identification in CXMS.
• This improved method offers superior performance in identifying cross-linked peptides and increasing the number of identified cross-linked sites.
• The developed strategy is expected to advance large-scale, highly accurate protein complex characterization, offering new insights into their structures and functions.