Accurate Proteoform Identification and Quantitation Using pTop 2.0.

Area of Science:

• Proteomics
• Mass Spectrometry
• Bioinformatics

Background:

• Top-down proteomics has advanced significantly due to technological progress in separation, mass spectrometry (MS), fragmentation, and bioinformatics.
• Accurate identification and quantification of proteoforms, distinct protein variants from a single gene, remain computationally challenging due to complex mass spectra of intact proteoforms.
• Existing bioinformatics tools struggle to handle large-scale, complex top-down MS data effectively.

Purpose of the Study:

• To address the limitations in analyzing complex top-down mass spectrometry data.
• To introduce pTop 2.0, an enhanced bioinformatics tool for accurate proteoform identification and quantification.
• To improve the analysis of proteoforms with unexpected modifications or terminal truncations.

Main Methods:

• Development of pTop 2.0, an upgraded version of the pTop bioinformatics software.
• Focus on identifying proteoforms with unexpected modifications or terminal truncations.
• Integration of quantitative analysis using isotopic labeling with a user-friendly 'one-key operation'.

Main Results:

• pTop 2.0 demonstrates significantly improved accuracy and running speed compared to its predecessor.
• The software effectively handles complex mass spectra from intact proteoforms.
• New functionalities include identification of proteoforms with unexpected modifications and isotopic labeling-based quantification.

Conclusions:

• pTop 2.0 enhances the identification and quantification of intact proteoforms in top-down proteomics.
• The tool offers a user-friendly interface and improved performance for complex MS data analysis.
• This advancement pushes the field towards higher accuracy in proteoform analysis.