Constructing Human Proteoform Families Using Intact-Mass and Top-Down Proteomics with a Multi-Protease Global...

Area of Science:

• Proteomics
• Molecular Biology
• Biochemistry

Background:

• Human proteoforms, diverse variants from the same gene, are crucial for complex biomolecular processes.
• Identifying proteoforms with high confidence using mass spectrometry remains a significant challenge in proteomics.

Purpose of the Study:

• To enhance the identification of human proteoforms in total cell lysate from the Jurkat T lymphocyte cell line.
• To integrate multiple mass spectrometry strategies for maximizing confident proteoform identifications.

Main Methods:

• Collected and processed three types of proteomics data: NeuCode-labeled intact-mass, label-free top-down, and multi-protease bottom-up.
• Integrated these datasets to improve mass calibration and utilize a global post-translational modification discovery database.

Main Results:

• Identified 5950 unique experimentally observed proteoforms, organized into 848 proteoform families.
• Achieved confident identification of 1207 unique proteoforms (20% of total) from 484 genes at a 3.9% false discovery rate.

Conclusions:

• The integrated multi-omics approach significantly enhances the scope and confidence of proteoform identification.
• This comprehensive proteoform catalog provides a valuable resource for studying human cellular diversity and function.