Proteomic analysis of protein deamidation.

Area of Science:

• Biochemistry
• Proteomics
• Analytical Chemistry

Background:

• Spontaneous deamidation of asparagine and glutamine residues in proteins leads to degradation.
• Deamidation of asparaginyl residues produces isomeric N-aspartyl and isoaspartyl forms, implicated in neurodegenerative diseases.
• Accurate proteomic analysis of deamidation is hindered by rapid artifactual deamidation during sample preparation and co-elution of isomers in RPLC-MS/MS.

Purpose of the Study:

• To develop novel protocols for minimizing artificial deamidation during proteomic sample preparation.
• To establish an effective strategy for separating and quantifying all three deamidation products (original, N-aspartyl, isoaspartyl) from the same peptide.
• To enable accurate, proteome-wide analysis of deamidation.

Main Methods:

• Development of a modified sample preparation protocol to reduce artifactual deamidation.
• Implementation of an offline RP-ERLIC-MS/MS strategy combining reversed-phase chromatography fractionation with electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) and tandem mass spectrometry (MS/MS).

Main Results:

• The novel protocols effectively minimize artificial deamidation during sample processing.
• The RP-ERLIC-MS/MS strategy successfully separates the three isomeric deamidation products of peptides.
• This approach allows for the precise identification and quantification of deamidation on a proteome-wide scale.

Conclusions:

• The developed sample preparation and offline RP-ERLIC-MS/MS methods overcome the challenges of analyzing protein deamidation.
• These protocols provide a robust platform for accurate proteomic studies of deamidation, crucial for understanding its role in diseases.
• This advancement facilitates deeper insights into protein stability and age-related modifications.