Defining intact protein primary structures from saliva: a step toward the human proteome project
F Halgand1, V Zabrouskov, S Bassilian
1NPI-Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California 90024, United States. frederic.halgand@univ-rennes1.fr
Analytical Chemistry
|April 19, 2012
Summary
Top-down mass spectrometry identified novel salivary protein structures, including isoforms and modified proteins. This study proposes an iterative workflow for accurate protein identification and structural assignment using advanced MS techniques.
Area of Science:
- Proteomics
- Biochemistry
- Analytical Chemistry
Background:
- Salivary proteins exhibit structural diversity.
- Top-down mass spectrometry (MS) is crucial for characterizing protein structures.
Purpose of the Study:
- To investigate structural diversity within abundant salivary protein families.
- To identify and characterize novel salivary proteins and their modifications.
- To develop an optimized workflow for protein structure elucidation.
Main Methods:
- Top-down mass spectrometry utilizing hybrid quadrupole time-of-flight (Q-TOF) and Fourier-transform ion cyclotron resonance (FTICR) instruments.
- High-resolution, high mass accuracy MS with electron-capture dissociation (ECD) for detailed structural analysis.
- Iterative manual refinement of MS/MS data due to complex post-translational modifications.
Main Results:
- Identification of two protein II-2 isoforms differing by less than 1 Da.
- Determination of a sequence for protein IB8a including a mutation and C-terminal covalent modification.
- Assignment of a sequence for a previously unreported 10433 Da protein.
- Characterization of Peptide P-J and discovery of its truncated form.
- Demonstrated the utility of FTICR-MS and ECD for unambiguous primary structure determination.
Conclusions:
- Advanced MS techniques, particularly FTICR-MS with ECD, are essential for detailed protein structural analysis.
- The presence of diverse post-translational modifications necessitates manual data refinement.
- An iterative MS/MS data processing workflow is proposed for complete protein identity and structure assignment.
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