Enhancing Proteoform Sequence Coverage Using Top-Down Mass Spectrometry with In-Source Fragmentation and Middle-Down...

Area of Science:

• Proteomics
• Mass Spectrometry
• Biochemistry

Background:

• Characterizing complex proteoforms with mutations and post-translational modifications is crucial.
• Mass spectrometry (MS)-based techniques are advancing the study of proteoforms.
• High proteoform sequence coverage is essential for accurate characterization.

Purpose of the Study:

• To evaluate top-down and middle-down MS approaches for enhancing proteoform sequence coverage.
• To assess the effectiveness of in-source fragmentation (ISF) and partial enzymatic digestion.
• To determine if these methods can achieve high sequence coverage for complex proteins.

Main Methods:

• Applied top-down MS with in-source fragmentation (ISF) to generate pseudo-MS3 spectra.
• Utilized middle-down MS with short-duration enzymatic digestions to produce longer peptides.
• Tested approaches on three model proteins: ubiquitin, myoglobin, and carbonic anhydrase II.

Main Results:

• In-source fragmentation (ISF) in top-down MS improved sequence coverage.
• Partial enzymatic digestion in middle-down MS yielded longer peptides, preserving proteoform information.
• Both strategies significantly increased protein sequence coverage, exceeding 90%.

Conclusions:

• Top-down and middle-down MS strategies effectively enhance proteoform sequence coverage.
• ISF and partial digestion are valuable techniques for deep proteoform analysis.
• Achieving >90% sequence coverage is feasible for complex proteoforms using these methods.