With reversed phase liquid chromatography (RPLC)-MS worklows, the study of complex samples of proteins above 30 kDa is challenging and often not possible. The most frequently used approaches rely on the enzymatic hydrolyzation of biopolymers and the analysis of their fragments (e.g. bottom-up proteomics), inferring the characteristics of the (intact) proteins and losing information on their distribution in post-translational modifications (proteoforms). Tens of thousands of post-translational-modifications have been reported in the human proteome. However, there is little knowledge about the role of proteoform heterogeneity
In our research, we develop methods that can handle and give mass information on large proteoforms and protein assembly (up to over 200 kDa).
We are working on the study of proteins and proteoforms at the intact level (intact protein MS and/ or top-down MS) to help understand processes of disease development or diagnosis. In particular we are working at the application of low-flow methods, such as HILIC, native SEC and IEC-MS, to study glycoform and allotype distribution of intact proteoforms such as serum IgG . Our aim is to apply such methods to the study of the development of immune and autoimmune diseases. We also plan to expand the area to other proteoform targets that are currently unexplored.
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:
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:
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:
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:
CAST scientist Luca Tutiš together with Govert Somsen and Andrea Gargano as well as AstraZeneca, Novartis and GSK have just published a manuscript describing a novel LC-MS method for impurity profiling of therapeutic phosphorothioated oligonucleotides.
CAST scientists Annika van der Zon and Ziran Zhai have just published two manuscriptsshowcasing significant advances in the low flow analysis of intact antibodies and
Scientists from the CAST team and related projects within the Center of Analytical Science Amsterdam actively participated with lectures, posters and social contributions to the HPLC2025 conference in Brugge, Belgium.
