Are Internal Fragments Observable in Electron Based Top-Down Mass Spectrometry?

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Protein tandem mass spectrometry (MS/MS) typically yields terminal fragments, limiting interior sequence coverage.
• Native top-down mass spectrometry (MS) particularly struggles with internal fragmentation for comprehensive protein analysis.
• Accurate annotation of posttranslational modifications and sequence variants requires improved sequence coverage via internal fragments.

Purpose of the Study:

• To investigate internal fragment formation in electron capture dissociation (ECD) and electron transfer dissociation (ETD).
• To evaluate the reliability of internal fragment assignments under various mass spectrometry conditions.
• To explore the influence of activation strategies on internal fragment generation and identify atypical fragmentation patterns.

Main Methods:

• Experiments conducted on quadrupole time-of-flight, Orbitrap, and FT-ICR mass spectrometers across four laboratories.
• Electron capture dissociation (ECD) performed under ultrahigh vacuum and ETD-comparable pressure conditions.
• Analysis using two complementary software packages, with ETD-higher energy collision dissociation MS3 for validation.

Main Results:

• Under typical ECD and ETD conditions, internal fragments cannot be confidently assigned.
• Atypical ECD conditions produced internal fragments and terminal b-type fragments, indicating a different ion-electron activation process.
• Atypical conditions and ETD-higher energy collision dissociation (EThcD) revealed unusual fragment ion types like x ions, likely from radical z-type ion vibrational activation.

Conclusions:

• Standard ECD and ETD are insufficient for reliable internal fragment assignment in protein MS/MS.
• Modified or atypical ECD conditions can generate internal fragments and novel ion types, suggesting alternative fragmentation pathways.
• Further research into these atypical conditions may enhance protein sequence coverage and variant analysis.