Expanding Proteome Coverage with CHarge Ordered Parallel Ion aNalysis (CHOPIN) Combined with Broad Specificity...

Area of Science:

• Proteomics
• Mass Spectrometry
• Cancer Biology

Background:

• The "deep" proteome, while accessible via mass spectrometry, has shown plateaued protein identification numbers (∼8,000-10,000) without reference data.
• Limited protein sequence coverage using standard proteases like trypsin hinders the accurate discrimination of protein isoforms.

Purpose of the Study:

• To expand proteome and protein sequence coverage in MCF-7 breast cancer cells to an unmatched depth.
• To overcome limitations in current deep proteome analysis workflows.

Main Methods:

• Employed a multi-stage workflow including gel-aided sample preparation (GASP) with combined trypsin/elastase digests for increased peptide orthogonality.
• Utilized concatenated high-pH prefractionation.
• Implemented CHarge Ordered Parallel Ion aNalysis (CHOPIN) on an Orbitrap Fusion (Lumos) mass spectrometer for optimized parallel ion processing.

Main Results:

• Achieved 57% median protein sequence coverage in 13,728 protein groups (8,949 Unigene IDs) within a single cell line.
• Identified a total of 179,549 unique peptides, providing deep proteome coverage in unprecedented detail.
• CHOPIN enabled the use of both Orbitrap detectors on predefined precursor types for enhanced ion processing.

Conclusions:

• The developed workflow significantly enhances deep proteome analysis capabilities.
• This approach offers improved protein sequence coverage and isoform discrimination for cancer cell line research.
• The findings provide a more detailed understanding of the MCF-7 breast cancer cell proteome.