This is an Application Brief and does not contain a detailed Experimental section.
The global COVID-19 pandemic has resulted in extensive efforts to develop vaccines for the novel coronavirus. Identifying vaccine targets relies on robust analytical methods to understand SARS-CoV-2 structural biology. This study focuses on reversed-phase liquid chromatographic analysis of the intact SARS-CoV-2 spike protein, which has emerged as a potential target for vaccine development due to its role in viral pathogenesis.1,2 This work demonstrates that using difluoroacetic acid (DFA) as a mobile phase modifier in place of formic acid (FA) results in increased chromatographic resolution during intact protein analysis. Furthermore, the results suggest that pairing this approach with N- and O-glycosidase treatments may enable more detailed intact protein MS investigations.
Using DFA instead of FA as the mobile phase modifier achieves:
The SARS-CoV-2 spike protein, which facilitates host cell infection, has become a subject of detailed study due to its potential as a COVID-19 vaccine target. Proper characterization of this novel coronavirus protein relies on robust identity and purity tests. While extensive characterization work is underway to study the SARS-CoV-2 spike protein’s glycans and glycopeptides, intact protein analysis using reversed-phase liquid chromatography (RPLC), either with or without the combined use of endoglycosidases, may offer unique analytical insights.3,4
Figure 1. The SARS-CoV-2 spike protein (gray) with glycans modeled on its surface. Lorenzo Casalino, Zied Gaieb, and Rommie Amaro, UC San Diego.
To aid this effort, Waters shares the following method:
The following experimental conditions were used for RPLC-FLR-MS intact protein analysis of the SARS-CoV-2 spike protein.
|
LC system: |
ACQUITY UPLC I-Class |
|
Detection: |
FLR (280 nm emission, 320 nm excitation) |
|
Vials: |
QuanRecovery vials |
|
Column(s): |
BioResolve RP mAb Polyphenyl, 2.7 μm, 450 Å, 2.1 x 50 mm |
|
Column temp.: |
80 °C |
|
Sample temp.: |
8 °C |
|
Injection volume: |
1 µL |
|
Flow rate: |
0.2 mL/min |
|
Mobile phase A: |
0.1% IonHance DFA or FA in water |
|
Mobile phase B: |
0.1% IonHance DFA or FA in acetonitrile |
|
Gradient: |
15–55% Mobile phase B in 20 minutes |
|
MS system: |
Vion IMS QToF Mass Spectrometer |
|
Ionization mode: |
ESI+ |
|
Acquisition range: |
1500–4000 m/z |
|
Capillary voltage: |
2.25 kV |
|
Collision energy: |
6 V |
|
Cone voltage: |
140 V |
Figure 2. Comparison of FA and DFA Intact Protein Fluorescence Signal.
Figure 3. Comparison of FA and DFA Intact Protein Total Ion Chromatograms.
Employing DFA as a mobile phase modifier resulted in a comparatively higher resolution chromatogram. Compared to using FA as a mobile phase modifier, gradient peak capacity increased by over three-fold while the less abundant proteoforms were better resolved. Pairing this chromatographic approach with N- and O-glycosidase treatments may enable more detailed MS investigations at the intact protein level of analysis.
Because the SARS-CoV-2 spike protein is implicated in viral pathogenesis, it has become a target for vaccine development. Efficient therapeutic development relies on a solid structural and functional understanding of the SARS-CoV-2 spike protein target. Intact protein analysis using RPLC can be used to refine our understanding of the SARS-CoV-2 spike protein and thus help to identify and develop promising new COVID-19 therapies. This work demonstrates that the use of DFA instead of FA as mobile phase modifier enhances method resolving power while maintaining MS-compatibility.
720006907, Revised December 2020
