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Protein identification using sequential ion/ion reactions and tandem mass spectrometry
Joshua J Coon1, Beatrix Ueberheide, John E P Syka
1Department of Chemistry, and Engineering Physics Program, University of Virginia, Charlottesville, VA 22901, USA.
Summary
Rapid protein sequencing from both ends is now possible in seconds using a new mass spectrometry method. This technique aids in identifying protein modifications and gene family members.
Area of Science:
- Proteomics
- Mass Spectrometry
- Biochemistry
Background:
- Accurate protein sequencing is crucial for understanding biological functions.
- Existing methods for protein sequencing can be time-consuming and require large sample amounts.
- Characterizing posttranslational modifications and splice variants is essential for comprehensive proteome analysis.
Purpose of the Study:
- To develop a rapid method for sequencing intact proteins from both N and C termini.
- To apply this method for characterizing histone posttranslational modifications.
- To identify novel members of the H2A gene family.
Main Methods:
- Electrospray ionization of proteins into gas-phase multiply charged ions.
- Electron transfer with fluoranthene radical anions to induce backbone dissociation.
- Ion/ion reaction with benzoic acid for deprotonation of fragment ions.
- Analysis of singly and doubly charged ions to determine N- and C-terminal sequences (15-40 amino acids).
Main Results:
- Simultaneous sequencing of intact proteins from N and C termini in 1-2 seconds.
- Characterization of posttranslational modifications on histone H3.1.
- Identification of an additional member of the H2A gene family.
- Successful database searching using sequence and mass data for protein identification and variant analysis.
Conclusions:
- The developed method offers rapid and efficient protein sequencing from both termini.
- This technique is valuable for characterizing complex proteomic samples, including PTMs and splice variants.
- The approach facilitates quick identification of proteins and discovery of new gene family members.