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Peptide mapping of 125I-labelled membrane protein of influenza viruses by reverse-phase high-performance liquid
Abstract:
The resolution potential of reverse-phase high-performance liquid chromatography (HPLC) for peptide analysis of hydrophobic viral membranes has been investigated, using as model the membrane (M) protein of influenza virus. Proteolytic digests of 125I-labelled M protein and CNBr fragments, extracted from radioiodinated whole virus, have been separated on a uBondapak C18 column with an isopropanol or acetonitrile solvent system. Peptide mapping of trypsin digests of M protein from A/PR/8/34 (H1N1) and A/chicken/Germany/N/49 (H10N7) viruses was identical, whereas Staphylococcus aureus V8 protease digests showed minor differences in at least two peptides. The results also show that HPLC is a powerful tool for the separation of proteolytic digests of viral proteins, since the peptide maps are highly reproducible and recovery was greater than 85%.
Insights
Reverse-phase high-performance liquid chromatography (HPLC) effectively analyzes viral membrane proteins. This method provides reproducible peptide maps for influenza M protein, demonstrating its power in viral protein separation.
Area of Science:
- Biochemistry
- Virology
- Analytical Chemistry
Background:
- Hydrophobic viral membrane proteins present challenges for analysis.
- The influenza virus membrane (M) protein serves as a model for studying these proteins.
Purpose of the Study:
- To investigate the resolution potential of reverse-phase high-performance liquid chromatography (HPLC) for analyzing viral membrane protein digests.
- To assess the reproducibility and recovery of HPLC for peptide mapping.
Main Methods:
- Separation of proteolytic digests and cyanogen bromide fragments of 125I-labelled influenza M protein using reverse-phase HPLC on a C18 column.
- Utilized isopropanol or acetonitrile solvent systems for elution.
- Peptide mapping was performed using trypsin and Staphylococcus aureus V8 protease.
Main Results:
- Identical peptide maps were observed for trypsin digests of M protein from two different influenza strains (H1N1 and H10N7).
- Minor differences in at least two peptides were detected in Staphylococcus aureus V8 protease digests.
- HPLC demonstrated high reproducibility and recovery rates exceeding 85% for peptide separations.
Conclusions:
- Reverse-phase HPLC is a powerful and reproducible technique for the separation and analysis of viral protein proteolytic digests.
- HPLC facilitates detailed peptide mapping, aiding in the characterization of viral proteins and potentially identifying strain-specific differences.