MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation, T. Sénard, A.F.G. Gargano, D. Falck, S.W. de Taeye, T. Rispens, G. Vidarsson, M. Wuhrer, G.W. Somsen, E. Domínguez-Vega. Front Immunol 11 (2020) 1–13. https://doi.org/10.3389/fimmu.2020.02049.

In this paper, we present a middle-up strategy for the analysis of the intact fragment crystallizable (Fc) region of human plasma IgGs, with the aim of acquiring integrated information of the N-glycosylation and other PTMs of subclasses and allotypes. Human plasma IgG was isolated using Fc-specific beads followed by an on-bead C H 2 domain digestion with the enzyme IdeS. The obtained mixture of Fc subunits was analyzed by capillary electrophoresis (CE) and hydrophilic interaction liquid chromatography (HILIC) hyphenated with MS. CE-MS provided separation of different IgG-subclasses and allotypes, while HILIC-MS allowed resolution of the different glycoforms and their oxidized variants. The orthogonality of these techniques was key to reliably assign Fc allotypes. Five individual donors were analyzed using this approach. Heterozygosis was observed in all the analyzed donors resulting in a total of 12 allotypes identified. The assignments were further confirmed using recombinant monoclonal IgG allotypes as standards.

Characterization of Complex Proteoform Mixtures by Online Nanoflow Ion-Exchange Chromatography – Native Mass Spectrometry, Ziran Zhai Author,Despoina Mavridou Matteo Damian,Francesco G. Mutti ,Peter J. Schoenmakers ,Andrea F.G. Gargano. Anal. Chem. 2024, publication date May 21, 2024. DOI: 10.1021/acs.analchem.4c01760 

In this paper, we describe for the first time the use of nanoflow ion-exchange chromatography directly coupled with native MS to resolve mixtures of intact proteins. Reference proteins and protein complexes with molecular weight between 10 and 150 kDa and a model cell lysate were separated using a salt-mediated pH gradient method using volatile additives. The method allowed for low detection limits (0.22 pmol of monoclonal antibodies) while proteins presented non-denatured MS (low number of charges and limited charge state distributions) and the oligomeric state of the complexes analyzed was mostly kept. Excellent chromatographic separations including the resolution of different proteoforms of large proteins (> 140 kDa) and a peak capacity of 82 in a 30-minute gradient, were obtained. The proposed setup and workflows show great potential for analyzing diverse proteoforms in native top-down proteomics, opening unprecedented opportunities for clinical studies and other sample-limited applications.

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.

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 

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.

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:

https://doi.org/10.1021/acs.analchem.5c02033