Peptide quantification using 8-plex isobaric tags and electron transfer dissociation tandem mass spectrometry

Area of Science:

• Proteomics
• Analytical Chemistry
• Mass Spectrometry

Background:

• Isobaric tags for absolute and relative quantitation (iTRAQ) are widely used for peptide quantification.
• Traditional beam-type collision activated dissociation (CAD) of 8-plex iTRAQ tags allows for eight-channel relative quantification.
• Electron transfer dissociation (ETD) has shown potential for increased quantification channels with 4-plex iTRAQ reagents.

Purpose of the Study:

• To investigate the fragmentation patterns of 8-plex iTRAQ labeled peptides using ETD.
• To determine the number of quantification channels achievable with ETD of 8-plex iTRAQ labeled peptides.
• To compare ETD-based quantification with traditional beam-type CAD methods.

Main Methods:

• Labeling of peptides with 8-plex iTRAQ tags.
• Fragmentation of labeled peptides using electron transfer dissociation (ETD).
• Analysis of reporter ion intensities and fragmentation patterns using mass spectrometry (MS/MS).
• Subsequent resonant excitation (CAD) of a specific m/z peak for enhanced quantification.

Main Results:

• ETD of 8-plex iTRAQ labeled peptides produces unique reporter ions enabling five-channel quantification.
• A subsequent CAD step on a 322 m/z fragment allows for eight-channel quantification.
• ETD-based quantification shows good correlation with traditional beam-type CAD methods (R² = 0.9383).

Conclusions:

• ETD offers a viable alternative fragmentation method for 8-plex iTRAQ labeled peptides.
• A two-step ETD followed by CAD approach enables full eight-channel quantification.
• This method provides a reliable alternative for relative peptide quantification in complex proteomic samples.