Multiplexed Comparative Analysis of Intact Glycopeptides Using Electron-Transfer Dissociation and Synchronous...

Area of Science:

• Proteomics
• Glycomics
• Mass Spectrometry

Background:

• Tandem mass tags (TMT) enable multiplexed protein quantification.
• Challenges exist in simultaneous TMT quantification and identification of intact glycopeptides due to inefficient fragmentation.

Purpose of the Study:

• To develop an improved workflow for TMT-based intact glycopeptide quantification and identification.
• To overcome limitations of collision-induced dissociation (CID) in peptide backbone fragmentation.

Main Methods:

• Implemented an improved synchronous precursor selection/electron-transfer dissociation (SPS/ETD) workflow on an Orbitrap Tribrid mass spectrometer.
• Utilized CID for MS2 fragmentation and higher-energy collisional dissociation (HCD) for MS3 fragmentation.
• Integrated ETD for enhanced glycopeptide identification and TMT reporter ions from HCD MS3 for quantification.

Main Results:

• The SPS/ETD approach demonstrated improved accuracy, precision, and sensitivity for intact glycopeptide quantification compared to traditional methods.
• The workflow successfully maintained glycopeptide identification capabilities.
• Applied the workflow for site-specific quantitative comparison of glycoforms in therapeutic enzymes (Cerezyme and VPRIV).

Conclusions:

• The developed SPS/ETD workflow is suitable for TMT-based intact glycopeptide characterization of glycoproteins.
• This method enhances the accuracy and efficiency of glycopeptide analysis.
• The workflow facilitates detailed glycoform profiling of therapeutic proteins.