Expanding Proteoform Identifications in Top-Down Proteomic Analyses by Constructing Proteoform Families

Area of Science:

• Proteomics
• Biochemistry
• Computational Biology

Background:

• Top-down proteomics identifies protein variants (proteoforms) using mass spectrometry.
• Many proteoforms detected in MS1 spectra remain unidentified due to lack of fragmentation data.

Purpose of the Study:

• To adapt Proteoform Suite software for analyzing MS1-only intact proteoform data.
• To increase proteoform identification in top-down proteomics workflows.

Main Methods:

• Utilized open-source Proteoform Suite software.
• Adapted software to analyze MS1 spectra for intact proteoform mass identification.
• Performed mass calibration using high-scoring top-down identifications.
• Constructed and visualized proteoform families from combined MS1 and MS2 data.

Main Results:

• Identified 1861 proteoforms in yeast lysate.
• Achieved an approximate 40% increase in proteoform identifications compared to traditional top-down analysis alone.
• Successfully supplemented MS2-based identifications with MS1-based intact mass data.

Conclusions:

• The adapted Proteoform Suite enhances proteoform discovery in top-down proteomics.
• Integrating MS1-only data significantly expands the number of identified proteoforms.
• This strategy provides a more comprehensive view of the proteome.