Absolute Quantitation of Phosphopeptides and Glycopeptides Using Coulometric Mass Spectrometry

Area of Science:

• Biochemistry
• Analytical Chemistry
• Proteomics

Background:

• Protein post-translational modifications (PTMs), including phosphorylation and glycosylation, are crucial for biological functions.
• Quantifying PTMs is challenging due to the absence of specific peptide or protein standards.

Purpose of the Study:

• To develop a novel, standard-free method for the absolute quantification of phosphopeptides and glycopeptides.
• To overcome the limitations of current PTM quantification techniques.

Main Methods:

• Enzymatic dephosphorylation of phosphotyrosine peptides followed by coulometric mass spectrometry (CMS).
• N-terminal derivatization of electrochemically inactive phosphopeptides and glycopeptides with a catechol tag (DPDP) for CMS quantification.
• Application of the method to quantify glycopeptides from a NIST monoclonal antibody (mAb).

Main Results:

• Accurate quantification of phosphopeptides with errors as low as 0.3%.
• Successful quantification of derivatized phosphopeptides and glycopeptides with errors below 6%.
• Quantification of five N297 glycopeptides from a monoclonal antibody using CMS without standards.

Conclusions:

• The developed CMS-based approach enables standard-free absolute quantification of phosphopeptides and glycopeptides.
• This method significantly enhances the potential for accurate quantitative proteomics and biological research involving PTMs.