Tag: Andrea Gargano

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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

 

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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

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Funding

COAST TAFU-XL grant for Andrea Gargano

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.

CAST researcher Dr. Andrea Gargano has been awarded a 50,000 euro TAFU-XL grant from the Community of Innovation for Comprehensive Analytical Science and Technology (COAST). Together with Pieter van Delft of the company Corbion, he will further develop the novel concept of polymer sequencing by enzymatic hydrolysis.

When developing biodegradable co-polymers for applications such as drug delivery systems, but also when designing new plastic materials that are more easily recyclable, it is crucial to relate their properties (e.g. tensile characteristics and degradability) to their composition and molecular structure. However, current state-of-the-art methods for polymer analysis provide only limited information about the chemical composition and molecular weight distribution of the polymers. 

Enzymes

In a novel approach, researchers Dr Andrea Gargano (Analytical Chemistry) and Prof. Francesco Mutti (Biocatalysis) at the Van ‘t Hoff Institute for Molecular Sciences (HIMS) have called enzymes to the rescue. Together with industrial partners (DSM, Covestro and InnoSyn), they have developed novel cutinase enzymes that selectively depolymerize polyester (co)polymers. Since the resulting oligomers are less complex and more soluble, they open a path for more detailed analysis of the original polymer strands. The researchers have dubbed their method POLY-SEQU-ENCHY which is an acronym for polymer sequencing by enzymatic hydrolysis.

In the Talent Fund (TAFU) XL project of COAST, the HIMS researchers will now team up with Corbion to elucidate the structure of the polyester-based poly lactic-co-glycolic acid copolymers by means of POLY-SEQU-ENCY. This will then be related to the use of these polymers for applications such as drug-delivery systems. The grant of 50,000 euros is intended to establish the ‘proof-of principle’ that will support subsequent public-private-partnership (PPP) grant applications.

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Recognition

Gargano and Pirok featured in ‘Top 40 Under 40’ of analytical scientists worldwide

The Analytical Scientist Power List

CAST researchers Andrea Gargano and Bob Pirok were featured for the second time in a row in the Top 40 Under 40 by The Analytical Scientists. The magazine has published profiles of all listed scientists, including their analyses of the current state of affairs, their predictions for the future, and their personal mission statements. 

The magazine presents the list as a celebration of ‘analytical science’s rising stars, who will, hopefully, provide the answers to the 21st century’s biggest questions’. It was compiled upon nomination by the readers of the magazine and shortlisting by an independent panel of expert judges.

"Modern separation technology, especially multi-dimensional, has the capabilities to crack many analytical problems in public and private labs. It is unacceptable that you rarely see this stuff applied in routine environments."

Bob Pirok

I think analytical science needs to grow even more into a more multidisciplinary and broader community rather than following a spiral of hyper-specialization.

Andrea Gargano

Further Reading