Quantifying Positional Isomers (QPI) by Top-Down Mass Spectrometry.

Area of Science:

• Proteomics
• Mass Spectrometry
• Biochemistry

Background:

• Proteomics has identified numerous posttranslational modifications, but their functional relevance is often unclear.
• Characterizing the amount, site, and order of modifications on intact proteins is challenging.
• Shotgun proteomics and previous top-down methods struggle with positional isomers and manual interpretation.

Purpose of the Study:

• To develop and validate a top-down proteomics approach for characterizing and quantifying multiple posttranslational modifications on intact proteins.
• To address the challenge of distinguishing and quantifying positional isomers.
• To improve the localization and quantification of modifications using advanced bioinformatics.

Main Methods:

• High-resolution Orbitrap Fusion top-down mass spectrometry.
• Bioinformatics approaches including automated covalent fragment ion type definition.
• Extensive use of replicate spectra for increased sequence coverage and reduced false fragment assignments.

Main Results:

• Significantly reduced false fragment assignments from 10% to 1.5%.
• Accurate quantification of Ubiquitin positional isomers with low standard error (<5%).
• Estimation of phosphosite stoichiometry and discovery of new sites in multiphosphorylated Bora.

Conclusions:

• The developed top-down proteomics method enables precise characterization and quantification of complex protein modifications and positional isomers.
• This approach overcomes limitations of previous methods, offering higher accuracy and reduced manual interpretation.
• The method has significant implications for understanding protein function through detailed modification analysis.