Tag: Andrea Gargano

Categories
Publications

HILIC-MS impurity profiling of therapeutic PS- oligonucleotides

DNA complex spiral structure , medical, science, genetic biotechnology, gene cell concept ,3D rendering .

Ion-Pairing Hydrophilic Interaction Chromatography for Impurity Profiling of Therapeutic Phosphorothioated Oligonucleotides

Oligonucleotides are short strands of synthetic DNA or RNA that are synthesized via a solid-phase synthesis, in which numerous of closely-related impurities are generated. Ion-pairing reversed-phase liquid chromatography (IP-RPLC), anion exchange chromatography (AEX), and hydrophilic interaction chromatography (HILIC) are often used to profile these impurities, which allows for good separation of impurities comprising different number of nucleotides as the full-length product (FLP). However, impurities comprising the same number of nucleotides as the FLP are often not separated. Therefore, ion-paring HILIC (IP-HILIC) was explored as an alternative separation mode to overcome these challenges.

Key points:

  • Changed selectivity: by adding ion-pairing reagents (IPRs) to the HILIC eluent, the relative contribution of the highly polar phosphate moieties on HILIC retention is reduced and, thereby, increasing the relative contribution of the nucleobase composition and conjugated groups.
  • Suppressed diastereomer separation: Phosphorothioation of the phosphate groups results in the formation of diastereomers, with 2n possible diastereomers (n = phosphorothioate groups). IPRs in the HILIC eluent reduced diastereomer separation, leading to sharper peaks.
  • Separation of same-length impurities: IP-HILIC shows increased separation of impurities comprising of the same number of nucleotides as the FLP, such as deaminated products that differ less than 1 Da from the FLP. This is noteworthy as no other MS-compatible, one-dimensional LC separation can achieve this.

Figure 1: IP-HILIC-MS total and extracted ion chromatograms of GalNAc-conjugated oligonucleotides (top) and non-conjugated oligonucleotides (bottom) and the mass spectra of peaks A-D (A & C: FLPs, B & D: deaminated products)

The developed IP-HILIC method shows great potential as a screening method for quality control. The work is published in Analytical Chemistry and can be found with the following link: https://pubs.acs.org/doi/full/10.1021/acs.analchem.5c01407

 

Categories
Publications

New Frontiers in  Intact Protein Characterization by LC-MS at CAST

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:

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

Categories
Conferences

CAST group participates in full force at HPLC 2025 with posters and lectures

CAST Group 2025

It is a tradition within our CAST group to attend one major scientific conference each year as a unified team. This provides an opportunity to present our work, strengthen internal cohesion, and engage with the broader research community. With liquid-phase separations as a common thread across our projects, all scientists from the CAST group, as well as those involved in related projects embedded within the Centre for Analytical Sciences Amsterdam, were therefore present at the HPLC 2025 symposium in Bruges, Belgium.

The full CAST group with fellow CASA members at the conference dinner party on Wednesday at HPLC2025 in Bruges, Belgium.

Four poster nominations for the Gargano team

The CAST group contributed to the symposium with a large number of posters. On Monday and Tuesday, our young scientists presented and discussed their work with the delegates. All four posters of the team of Andrea Gargano were nominated for the Best Poster Award.

Improving IgG Glycoform Analysis with Advanced HILIC-MS

Thomas Holmark presented his latest advances in improving the sensitivity and reliability of IgG glycoform analysis. Their work focuses on developing capillary-based HILIC-MS methods to better characterize glycosylation patterns in immunoglobulin G (IgG), which are important biomarkers for disease progression. By optimizing monolithic column synthesis and implementing self-packed trap columns, the team significantly reduced sample carryover and enhanced detection sensitivity—paving the way for more accurate, low-input analysis of IgGs secreted by in-vitro stimulated B-cells.

Masa Sawisawa, Thomas Holmark, Annika van der Zon and Ziran Zhai were nominated for a Best Poster Award.

Annika van der Zon receiving her best poster pitch prize.

Third poster-pitch prize for monolithic HILIC-MS for Monoclonal Antibody Glycoform Profiling

Annika van der Zon introduced an acrylamide-based monolithic HILIC stationary phase, specifically designed to overcome the limitations of conventional silica-based materials. This new column enables high-resolution separation of intact mAb glycoforms, significantly outperforming traditional LC-MS methods such as RPLC. Using trastuzumab as a model, the team achieved chromatographic separation and mass spectrometric identification of up to 17 N-glycoforms—demonstrating enhanced sensitivity to minor variants. This development represents a major step forward in therapeutic antibody characterization, offering a powerful tool for quality control and drug development

NanoSEC-nMS Enhances Native Protein Complex Characterization

Ziran Zhai was also nominated for the poster-pitch prize and presented an innovative nanoflow size-exclusion chromatography-native mass spectrometry (nanoSEC-nMS) method at HPLC 2025, pushing the boundaries of intact protein and protein complex analysis. By operating at ultra-low flow rates (500 nL/min) and using capillary-format columns, the method drastically reduces sample requirements while preserving native protein structures and non-covalent interactions. This approach enables sensitive, high-resolution analysis of complex biomolecular assemblies, even from limited or biologically relevant samples like urine. With enhanced ionization stability and compatibility with high-salt buffers, nanoSEC-nMS represents a powerful advancement for native top-down proteomics and structural biology workflows.

Pharmaceutical Analysis Poster Award for Masa Serizawa

Masa Serizawa showcased a major advance in the analysis of biodegradable polymers, focusing on poly(lactic-co-glycolic acid) (PLGA)—a cornerstone material in drug delivery systems. The team developed optimized SEC-MS and normal-phase LC methods to achieve precise characterization of PLGA microstructure, including molecular weight distribution (MWD), chemical composition distribution (CCD), and functionality-type distribution (FTD). By minimizing fragmentation during electrospray ionization with cesium iodide, they enabled reliable identification of PLGA isomers and block structures. Furthermore, their new NPLC approach extends analysis to high-molecular-weight PLGAs (up to 185 kDa), offering detailed insight into both end-group functionality and lactic/glycolic acid ratios. These innovations pave the way for the design of more effective, tailored PLGA-based drug delivery platforms. He was awarded the Best Pharmaceutical Analysis Poster Award for his great work.

Masa Serizawa receiving the Best Pharmaceutical Analysis Poster Award.

Other poster contributions from PhD candidates included those of Sanne Boot, who showcased her efforts to simulate LC×LC-MS data for optimizing oligonucleotide separations, enabling the exploration of separation conditions within complex chemical spaces. PhD candidate Gerben van Henten showed his work on the evaluation of chromatographic response functions. His poster explained how some optimization methods struggle with chromatographic response functions that fundamentally were believed to be suitable.

Several CAST graduate students were also present at HPLC2025. Lonneke van Dalen presented her poster on developing a new peak integration method using neural networks in collaboration with Unilever.

Merel Konings demonstrated the optimization of light degradation reactors to conduct degradation studies in liquid-phase separations, and Rebecca Gibkes presented her work on plate-height modeling and peak parking as part of a collaboration between the University of Amsterdam, the Vrije Universiteit Brussel and Gustavus Adolphus College.

Rick van den Hurk as Csaba Horvath Finalist

Rick van den Hurk presenting his latest work on the effect of a lack of radial mixing in systems where flows are combined.

Rick van den Hurk was nominated as Csaba-Horváth finalist and addressed a key obstacle in expanding two-dimensional liquid chromatography (2D-LC): the solvent mismatch between coupled separation dimensions. His work focused on active modulation strategies, such as SPAM, ACD, and ASM, that dilute the first-dimension effluent with a weaker solvent to improve retention and peak shape in the second dimension. Using experimental setups and computational fluid dynamics simulations, the team investigated how radial mixing at T-junction interfaces impacts analyte retention. Van den Hurk explained how these findings provide valuable insights into optimizing flow conditions and mixer design, paving the way for broader and more robust application of 2D-LC in industrial and research settings. This work is conducted in collaboration with the group of Dwight Stoll at Gustavus Adolphus College.

Bob Pirok was invited to present a lecture on the state of machine learning in chromatography, and explained during his presentation the importance of equiping machine-learning workflows with suitable data processing strategies. A key part of the lecture focused on the work by CAST researcher Nino Milano and his efforts to simulate realistic 2D chromatographic data for the evaluation of peak detection algorithms.

Andrea Gargano presented a novel HILIC-MS approach using custom-designed acrylamide-based monolithic columns to improve the analysis of intact biomacromolecules. These columns offer enhanced selectivity and reduced secondary interactions, enabling the separation of closely related variants that are challenging for traditional RPLC-MS. Gargano demonstrated applications which included the resolution of oligonucleotide impurities, glycated protein isomers, and monoclonal antibody glycoforms, advancing analytical capabilities in therapeutic development and quality control. 

Luca Tutis’s contribution on the development of an ion-pair HILIC method for oligonucleotide impurity analysis was selected for a talk. This method significantly alters HILIC selectivity for ONs by emphasizing nucleobases and conjugated groups (GalNAc) over highly polar phosphate groups. This enabled the resolution of deamination, non-conjugated, and PS-PO converted impurities. Importantly, this IP-HILIC approach is fully compatible with MS, crucial for accurate impurity identification.

Honorary CAST member and emeritus professor Peter Schoenmakers lectured on the sense and nonsense of artificial intelligence in chromatography and gave a historical overview in his lecture along with applications that he placed into context using fundamental principles known to chromatographers.

Special events

Advancing Education in Separation Science

Andrea Gargano, Simone Dimartino and Martina Catani organized the Education session workshop, introducing in the HPLC program a new space to discuss innovation in education. Many delegates joined discussing innovations in separation science education (including teaching materials, software based tools, project based learning and a session discussing perspectives in skills and knowledge that the analytical scientist of the future should be educated on) including Bob Pirok.

Second prize in HPLC Tube

Dissemination of scientific work is incredible important to inform society about the latest progress. In this context, the symposium annually hosts the HPLC Tube where researchers can make brief videos to explain their work. CAST researchers Andrea Gargano, Annika van der Zon, Ziran Zhai and Thomas Holmark won the third prize in this category on their video entitled: “Stay Intact: AcryHILIC’ Glycoform Impact.”

Scientists discussed the state of current education practice of chromatography and how to improve this further.

Bob dedicating the first official copy to CASA leader and co-teacher Prof. Govert Somsen.

Launch of educational textbook Analytical Separation Science

Bob Pirok and Peter Schoenmakers launched their textbook Analytical Separation Science. The book is designed to support students, educators, and practitioners by offering a comprehensive and structured overview of modern separation techniques. With clear explanations, didactic modules, and academic exercises, it serves as a valuable resource for classroom teaching and self-study alike. The book is accompanied by an interactive website (https://ass-ets.org), featuring figures, lectures, and supporting materials to bridge the gap between theory and practice.

Short course in AI in Chromatatography

Bob Pirok and Tijmen Bos delivered a well-attended short course on Artificial Intelligence in Chromatography, introducing participants to the growing role of AI in analytical method development and data processing. The course covered foundational concepts in machine learning, modern optimization techniques, and practical applications across the chromatographic workflow, from peak detection to intelligent gradient design. Through interactive examples and real-world case studies, the session provided researchers and practitioners with the tools to understand and begin applying AI in their own laboratory environments.

Scientists discussed the state of current education practice of chromatography and how to improve this further.

Categories
Publications

SEC-MS and Enzymes for polyester polymers analysis

Abstract representation of a molecular connection crystallized in the hexagonal system.. 3d illustration.

CAST scientist Masashi Serizawa recently published a manuscript in which he investigated a novel size-exclusion chromatography hyphenated with mass spectrometry and ultraviolet (SEC-MS/UV) method to characterize poly lactide-co-glycolide (PLGA), and also co-authored work on the use of enzymes for polyester polymer degradation.

 

·         SEC-MS characterization of PLGA:

Size-exclusion chromatography (SEC) hyphenated with MS is valuable for microstructure analysis. While SEC-UV/RI determines molecular weight distribution (MWD), SEC-MS is used for chemical composition distribution (CCD) and functionality-type distribution (FTD). However, previous applications of SEC-MS have failed to address the risk of polymer fragmentation during the analysis process. It is crucial to establish whether SEC-MS methods can be applied to biodegradable polymers and to recognize if fragmentation processes occurred during the SEC separation or during the ESI-MS process.

 

This study addresses fragmentation in PLGA analysis by optimizing SEC-MS conditions. We demonstrate that cesium iodide (CsI) minimizes fragmentation during electrospray ionization (ESI-MS), simplifying spectra and enabling differentiation of PLGA isomers. This facilitates accurate determination of CCD and FTD, even revealing “blockiness” when coupled with selective degradation.

Figure 1: schematic illustration of in-source fragmentation in SEC-MS, depending on ionization agents

The study is supported by the COAST/TKI-Chemistry POLY-SEQU-ENCHY project between the UvA and Corbion and is funded by Mitsubishi Chemical Corporation. This work was recently published in the Journal of the American Society for Mass Spectrometry and can be accessed freely at the link below:

https://doi.org/10.1021/jasms.4c00447

 

·         Insights in the selectivity of enzymes for polyester co-polymer degradation

Another example of the SEC-MS/UV polymer application conducted by our group is the structural analysis of aromatic/aliphatic polyesters. To understand the polymer chain structure of aromatic/aliphatic polyesters, Eman et al. successfully developed a two novel thermostable cutinase that primarily degrade aliphatic ester bonds. These enzymes maintain activity at elevated temperatures of up to 90 °C thanks to enzyme engineering.

For both enzymes, higher hydrolysis rates were observed for aliphatic compared to aromatic homo-polyesters. SEC-MS analysis revealed that the hydrolysis of aliphatic/aromatic co-polyesters occurred at the aliphatic monomers, significantly reducing the molecular weight and changing the end-group composition. These results underline the importance of co-polymer composition in the biodegradation of co-polymer systems and demonstrate the applicability of enzymes for the analytical characterization of synthetic polymers by selectively reducing their molecular weight.

Figure 2: Results of SEC-MS/UV analysis of a copolymer containing aromatic/aliphatic polyesters,
comparing between before and after enzymatic degradation (The degradations were
performed at 71°C, using thermostable cutinase)

This research was funded by Topconsortium voor Kennis en Innovatie (TKI) Chemie, deployment project PPS-programma toeslag 2019 (CHEMIE.PGT.2020.020). This work was recently published in the Chemistry – A European Journal and can be accessed at the link below:

https://doi.org/10.1002/chem.202403879

Categories
Publications

Separation of mAb charge variants by CZE-MS method under near-native pH conditions

CAST scientist Annika van der Zon recently introduced a near-native separation method for characterizing charge variants of intact monoclonal antibodies (mAbs) using capillary zone electrophoresis (CZE) coupled with mass spectrometry (MS). In this study we used a nanoflow sheath liquid interface, known as nanoCEasy applied thanks to the collaboration via the Uniiversity of Aalen.

The CZE-MS method, employs a neutral static capillary coating made of hydroxypropyl methylcellulose, combined with 50 mM acetic acid at pH 5.0, to create MS-compatible conditions for separating mAb charge variants. Currently, the pharmaceutical industry uses the EACA method of He et al. (2011) method to routinely profile charge variants, but this method relies on a non-volatile background electrolyte (BGE), making it incompatible with MS and hindering the identification of separated charge variants.

The MS-compatible CZE method we introduce allows to obtain similar charge variant profiling as the EACA method but allows for MS analysis. The CZE-MS coupling, enabled by nanoCEasy’s low-flow sheath liquid interface, successfully identified and quantified basic and acidic variants, incomplete pyroglutamate variants, and glycoforms of the mAbs tested. This CZE-MS method provides a powerful tool for assessing mAb heterogeneity and achieving charge variant separation.

 

Figure: Schematic representation of the CZE-UV/MS separation of charge variants of mAbs.

 

This study is published in the journal Analytical Chimica Acta, see here:
Thanks to all the co-authors for their contribution to this study.
Categories
Publications

NPLC method to characterize end groups of poly lactic acid co-glycolic acid copolymers

Vertical image laboratory test tubes for research with white plastic polymer or medical supplies lie on a black background. Conceptual image.

The CAST scientist Masashi Serizawa recently published a manuscript in which he investigated a novel method of using gradient elution normal-phase liquid chromatography with basic and acidic additives to separate PLGAs in the different end groups and in the different chemical compositions at the same time.

PLGA is an important material in drug delivery systems. It is used in nanoparticle-containing drugs to prevent a sudden increase in drug concentration in the body when the drug is ingested. The LA/GA ratio and differences in the terminal structure of PLGA have a significant effect on the degradation rate of PLGA in the body.

To distinguish these distinctions, we created a unique ternary gradient liquid chromatography method utilizing base and acid additives. Initially, we used a gradient of hexane, a poor solvent, and ethyl acetate, a good solvent, with a mobile phase containing a base additive to separate non-ester-terminated PLGAs (ester-terminated PLGA and cyclic PLGA) based on their chemical composition. Subsequently, by switching the mobile phase to THF containing an acid additive, we were able to elute acid-terminated PLGA.

This method offers the advantage of quick analysis compared to traditional NMR methods, making it potentially valuable for future industrial research. Furthermore, it can be applied to high molecular weight PLGA of 180 kDa, making it useful for the development of high molecular weight PLGA, which is challenging to analyze using mass spectrometry techniques such as MALDI-TOF-MS.

 

Figure: (left) schematic illustration of the working principle of the NPLC separation. (right): key results obtained in the study
Figure: (left) schematic illustration of the working principle of the NPLC separation. (right): key results obtained in the study

 

The study is supported by the COAST/ TKI-Chemistry POLY-SEQU-ENCHY project between the UvA and Corbion (Gorinchem, The Netherlands) and is funded by Mitsubishi Chemical Corporation.

The link to the publication is reported below.

https://doi.org/10.1016/j.chroma.2024.465137

Categories
Publications

Parallel Gradients 2DLC-HRMS of complex protein digest

2D-LC System
2D-LC System

Investigating the proteins in biological samples can help us understand and identify diseases and improve the effectiveness of medication. To study proteins in these samples, they are typically digested into peptides and subsequently analyzed by liquid chromatography (LC) hyphenated with high-resolution mass spectrometry (HRMS).

Comprehensive two-dimensional LC (LC×LC) offers increased separation power over traditional LC methods. However, most common gradient designs require re-equilibration of every second-dimension run, resulting in high flow rate operations to limit the empty separation space. This also limits MS sensitivity as flow splitting is required to handle such flow rates.

In this work, we developed an LC×LC method using a so-called parallel-gradient design, which omits the need for column re-equilibration and enables the use of the entire separation space. Moreover, this allows for lower flow rates and maintains the sensitivity for low-abundant analytes. The parallel-gradient design achieved higher surface coverages and sensitivity at lower effective peak capacities. Most importantly, both methods were applied to analyze a Human IMR90 lung fibroblast cell line digest to assess its applicability to real complex samples. The parallel-gradient method was able to identify significantly more proteins than the current state-of-the-art methods while using the same analysis time and at a lower solvent consumption. The applicability of the parallel-gradient design could be improved even further by shortening the modulation times, as it was not limited by column re-equilibration.

The study is a collaborative work done thanks for the contribution of many colleagues and students. The link to the publication is reported below.

https://doi.org/10.1021/acs.analchem.4c02172

 

 

 

Categories
Publications

Nanoflow IEC-HRMS to study complex proteoform mixtures

Clinical Materials Molecules
Clinical Materials Molecules

The CAST scientist Ziran Zhai published a manuscript investigating a novel method of using nanoflow strong cation exchange – native mass spectrometry to characterize non-denaturing complex proteoforms mixtures from the intact level. Zhai focuses on three critical aspects: i) extending the MW that can be observed by top-down proteomics, ii) increasing the MS sensitivity to create conditions of detecting low-abundant proteins, and iii) apply mild desolvation conditions to maintain the native structures of proteins and complexes. 

Proteoforms, which are protein products arising from homologous genes due to sequence variations, alternative splicing, and post-translational modifications, play a crucial role in a wide range of critical functions. However, the standard approach to characterize proteins, known as bottom-up proteomics, faces limitations. This approach cannot directly identify proteoforms as the presence of proteins is inferred from peptides. While top-down proteomics and intact protein mass spectrometry offer solutions to these limitations, the most common top-down methods employ denaturing LC-MS approaches, which unfold proteins and lead to the loss of non-covalent protein complexes.

In this work, we directly coupled nanoflow (250 or 500 nL min-1) strong cation exchange chromatography (SCX) to nano-electrospray-ionization (nESI) under native MS (nMS) conditions. Proteins were separated on packed capillary SCX columns and eluted according to their pI values by a salt-mediated pH gradient method. The low flow promoted desolvation/ionization efficiency allowing for sensitive detection of low-abundant proteins and complexes. We successfully applied our method to analyze an E. coli cell lysate and observed hundreds of proteins with masses up to 150 kDa. We believe that the proposed nanoSCX-nMS is a promising approach for characterizing proteoforms and provides a universal strategy to overcome detection limitations in native top-down proteomics.

 

Screenshot

The study is part of the FFF (From Form to Function) project of Zhai, Astefanei,
Corthals, and Gargano and was funded by the Chinese Scholarship Council (CSC) and was recently published in Analytica Chimica Acta and can be accessed freely at the link below.

https://pubs.acs.org/doi/10.1021/acs.analchem.4c01760.

 

Categories
Publications

Analysis of Heavily Glycated Proteins by HILIC and SEC-HRMS

Trypsin digestive enzyme molecule (human). Enzyme that contributes to the digestion of proteins in the digestive system. 3D rendering based on protein data bank entry 1trn. Ball-and-stick model, black background.

The CAST scientist Ziran Zhai recently published a manuscript in which he investigated the usefulness of two novel CAST methods, namely low-flow HILIC [1] and SEC-HRMS [2], to characterize extensively glycated proteins from the intact level. Zhai focuses on four critical aspects: i) using denaturing HILIC-MS to separate glycoconjugates (including, in some cases, the separation of isomers), ii) using native SEC-MS to study the aggregates formed during glycation, iii) identifying the advanced glycation end-products (AGEs), and iv) monitor the dynamic changes of AGEs.

Advanced glycation end products (AGEs) are a family of compounds of diverse chemical nature that are the products of nonenzymatic reactions between reducing sugars (here glucose) and, in the case of our study, proteins. Sugars can attach at different positions in a protein following a Maillard reaction, distributing over several amino acids and in many different chemical species.  Previous studies focused on digesting glycated proteins to identify the AGEs and glycoconjugates from the peptide level. However, these strategies make it difficult to monitor the co-occurrence of multiple glycation events and, therefore, cannot monitor the evolution of the glycation process.

In this study, three model proteins (RNase-A, hemoglobin, and NISTmab) were exposed to conditions that favored extensive glycation and the formation of AGEs. As shown, with HILIC-MS, the glycated forms of the proteins could be resolved based on the number of reducing monosaccharides, and the SEC-MS method under non-denaturing conditions provided insights into glycated aggregates (Figure 1). More than 25 different types of species were observed in both methods, among which 19 of these species have not been previously reported. By tracing the progress of glycation, the dynamic changes of the specific AGEs could be monitored over time.

Figure 1. BPC of non-glycated (A, C) and glycated (20 days, B and D) RNase-A acquired by HILIC-MS and SEC-MS. Deconvolution results of glycated RNase-A (20 days, E and F) obtained by HILIC-MS and SEC-MS methods.

The study is part of the FFF (From Form to Function) project of Zhai, Astefanei,
Corthals, and Gargano and was funded by the Chinese Scholarship Council (CSC) and was recently published in Analytica Chimica Acta and can be accessed freely at the link below.

https://www.sciencedirect.com/science/article/pii/S0003267024003441

References

 [1] https://pubs.acs.org/doi/full/10.1021/acs.analchem.1c03473

[2] https://www.sciencedirect.com/science/article/pii/S0003267023005457

 

Categories
Grants

Open Competition M2 grant for immunoglobulin analysis for Andrea Gargano

Grant for immunoglobulin analysis by Andrea Gargano
Grant for immunoglobulin analysis by Andrea Gargano

Dr. Andrea Gargano of the research group Analytical Chemistry together with Dr. Elena Dominguez Vega of the Center for Proteomics and Metabolomics at Leiden University Medical Center have been awarded an NWO Open Competition M2-grant. Together they receive about € 740,000 for their four-year HYPE-IMMUNe research project involving two PhD students.

The HYPE-IMMUNe project focuses on developing methods to characterize the structures and function of serum immunoglobulins, which are among the most important molecules of the immune system and exist in myriads of different variants. The projects will focus on the development of separation and affinity workflows coupled with mass spectrometry to disclose the structure-function relationship of the immunoglobulins responsible for the response to a disease or an autoimmune response. This technology will open new frontiers in advancing novel biotherapeutics and early disease diagnostics.

The M-grants issued by the Domain Board Science of the Dutch Research Council NWO are intended for innovative, high-quality, fundamental research and/or studies involving matters of scientific urgency. They offer researchers the possibility to elaborate creative and risky ideas and to realise scientific innovations that can form the basis for the research themes of the future. In the current round of the Open Competition Domain Science-M programme, eighteen grant applications were approved out of 72 applications.

Further Reading

NWO Press Release