A Streamlined Sequential Enrichment Strategy for Multi-PTM Profiling from Low Micrograms of Samples.

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Post-translational modifications (PTMs) regulate protein function and are implicated in human diseases.
• Analyzing multiple PTM types simultaneously from limited biological samples is challenging due to low stoichiometry and sample loss.

Purpose of the Study:

• To develop a streamlined, sequential enrichment strategy for simultaneous multi-PTM profiling from microscale samples.
• To enable comprehensive analysis of the N-glycoproteome, phosphoproteome, ubiquitinome, and global proteome.

Main Methods:

• Integrated HILIC beads for N-glycopeptides, Ti4+-IMAC beads for phosphopeptides, K-ε-GG antibody for ubiquitinated peptides, and C18 membrane for global proteome analysis.
• Optimized the process to minimize sample loss, simplify operations, and reduce time.

Main Results:

• Successfully identified thousands of sites for N-glycosites, phosphosites, and K-ε-GG sites from microgram-scale human embryonic kidney 293T tryptic peptides.
• Demonstrated high sensitivity, identifying numerous sites even with 1 μg of peptide input.
• Validated the strategy on complex matrices like mouse tissue peptides and human plasma.

Conclusions:

• The developed multi-PTM profiling strategy is sensitive and efficient for simultaneous analysis of diverse PTMs.
• This approach holds promise for PTM crosstalk studies and biomarker discovery, especially with precious clinical samples.