Determining Copper and Zinc Content in Superoxide Dismutase Using Electron Capture Dissociation Under Native Spray...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Proteomics

Background:

• Localizing metal ions in proteins is analytically challenging, especially during mass spectrometry fragmentation.
• Standard fragmentation methods like collision-induced dissociation (CID) can detach or scramble metal ions.
• Preserving metal binding sites is crucial for understanding metalloprotein function and disease mechanisms.

Purpose of the Study:

• To develop and apply a novel electron capture dissociation (ECD) method for precise localization of metal binding sites in metalloproteins.
• To investigate copper and zinc binding in Cu, Zn superoxide dismutase (SOD1), an enzyme implicated in amyotrophic lateral sclerosis (ALS).
• To differentiate between copper and zinc loss in SOD1 using mass spectrometry.

Main Methods:

• Utilized a newly available electron capture dissociation (ECD) tool retrofitted on quadrupole time-of-flight (QTOF) mass spectrometers.
• Applied ECD fragmentation to intact Cu, Zn superoxide dismutase (SOD1) to analyze metal binding sites.
• Compared ECD fragmentation with traditional methods to assess metal ion preservation.

Main Results:

• Electron capture dissociation (ECD) successfully localized copper and zinc binding sites in SOD1 with high precision.
• Distinct ECD fragments allowed differentiation between copper and zinc loss from SOD1.
• The ECD method demonstrated superior preservation of metal binding compared to collision-induced dissociation (CID).

Conclusions:

• Electron capture dissociation (ECD) is a powerful technique for localizing metal binding sites in metalloproteins.
• This method enables the study of metal ion dynamics in enzymes like SOD1, relevant to neurodegenerative diseases such as ALS.
• The described approach is broadly applicable to various metalloproteins for detailed structural and functional analysis.