CAST scientists Annika van der Zon and Ziran Zhai have just published two manuscripts
showcasing significant advances in the low flow analysis of intact antibodies and protein complexes, offering improved sensitivity and performance. The CAST team will utilize these novel nano SEC-MS and HILIC-MS methods in future bioanalysis projects.
Analyzing Minute Amounts of Protein Complexes with Nanoflow Size Exclusion Chromatography–Native Mass Spectrometry
Characterizing intact proteoforms and protein complexes often faces challenges in maintaining native structures and high sample requirements. CAST scientist Ziran Zhai developed a novel nanoflow size exclusion chromatography–native mass spectrometry (nanoSEC-nMS) method to overcome these limitations.
Key Advancements:
- Optimized Capillary SEC Columns & Reduced Peak Broadening: The method includes techniques for preparing high-performance capillary SEC columns and optimizing injection to reduce peak widths.
- Direct Coupling under Challenging Conditions: It enables direct coupling of nanoflow SEC with native MS even in salt-rich environments.
- Milder Desolvation for Native Structures: Nanoflow allows for milder ESI desolvation, preserving the native structures of proteins and complexes.
- High Sensitivity and Throughput: The method requires limited sample (approx. 100 nL per injection) and significantly enhances native MS throughput, enabling online desalting and oligomer separations within 25 minutes.
Figure 1: Analysis of urine samples and Ovitrelle with the nanoSEC-nMS: (a) EIC of the urine hCG samples; (b) MS spectrum of hCG proteins; (c) deconvoluted results of hCG proteins; (d) EIC of the Ovitrelle sample; (e) MS spectrum of Ovitrelle; (f) deconvoluted results of Ovitrelle.
This nanoSEC-nMS method enables the analysis of proteins and complexes across a broad molecular weight range (10 to 250 kDa) in their native states, preserving noncovalently bound metal ions. This study was published in Analytical Chemistry and can be accessed freely at the link below:
https://pubs.acs.org/doi/10.1021/acs.analchem.5c01019
Precise Glycoform Profiling of Intact Antibodies with HILIC-MS
Traditional methods struggle with comprehensive intact antibody glycoform profiling. To address this,CAST scientist Annika van der Zon et al. at developed a novel hydrophilic interaction chromatography (HILIC) method based on lab made acrylamide-based monolithic columns directly coupled to mass spectrometry.
Key Innovations:
- Optimized Monolithic Stationary Phase: The porogen composition was optimized, enhancing separation efficiency
- Enhanced Glycoform Resolution: The method achieved baseline separations for single and double Fc glycosylation, and partial separations for glycoforms differing by a single glycan unit.
- Sensitive Detection of Minor Glycoforms: It enabled sensitive measurement of low-abundance glycoforms in the nanogram injection range.
Figure 2. Analysis of intact trastuzumAb at the intact level. Base Peak Chromatogram of the analysis and Extracted Ion Currents of selected glycoforms are shown.
This HILIC-MS method significantly enhances glycoform selectivity for intact antibodies, providing a more comprehensive characterization essential for bioanalytical applications. This work was published in the Journal of Analytical Chemistry and can be accessed freely at the link below:
