Senior Researchers

Alina Astefanei
Research
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Alina Astefanei, PhD
University of Amsterdam
Van 't Hoff Institute for Molecular Sciences (HIMS)

Postal address
Postbus 94157
1090 GD Amsterdam

Visiting address
Science Park 904
1098 XH Amsterdam

email: A.Astefanei@uva.nl
researchgate.net/profile/Alina_Astefanei

Luitzen de Jong
Research

Mass spectrometric analysis of in vivo cross-linked protein complexes
After my retirement in November 2012, I continued as a guest worker my research activities arising from the work described in the thesis by Hansuk Buncherd entitled “Development of enrichment methods for cross-linked peptides to study the dynamic topology of large protein complexes by mass spectrometry”, defended December 2013. The aim of the project is to obtain a work flow enabling application of the technology developed in this thesis to an in vivo cross-linking approach of exponentially growing bacteria directly in the growth medium. This project in the field of analytical (bio)chemistry has a multidisciplinary nature and is carried out in close collaboration with microbiologists, organic chemists and mass spectrometrists.

For cross-linking we developed bis(succinimidy)-3-azidomethyl-glurtarate (BAMG). This cross-linker reacts with ε-aminogroups of juxtaposed lysine residues on the surface of proteins and protein complexes. Upon digestion following the cross-linking reaction, cross-linked peptides are generated along with an excess of unmodified peptides.

dejong-researchAim of the mass spectrometric analysis is to determine which lysine residues from which proteins became covalently linked during reaction with BAMG. This information can reveal the topology (i.e., spatial arrangement of composing proteins) of biological assemblies and is a prerequisite to understand the molecular mechanisms of complex processes, like transcription, translation and cell division. These are examples of biological processes accomplished by large protein complexes. The azido group in the spacer of BAMG enables isolation of cross-linked peptides and tremendously facilitates their identification by mass spectrometry and database searching.

The choice to use in vivo cross-linking directly in the growth medium is based on the notion that in vivo protein-protein interactions can be easily interrupted upon protein isolation or upon harvesting bacteria from the medium by centrifugation or filtering.

For growth, bacteria require, a.o., certain amines in the growth medium as a source of nitrogen. For in vivo cross-linking of exponentially growing bacteria, a medium is needed with a low concentration of compounds with amine groups, to prevent reaction with the cross-linker, but still enables fast growth. At present we are optimizing conditions for (i) growth, (ii) cross-linking directly in a bacterium culture, (iii) protein extraction and (iv), enrichment of certain cross-linked protein complexes and their digestion. The focus will be on protein complexes involved in cell division.

The work will shed new light on the molecular mechanisms underlying the cleavage process whereby one mother cell gives rise to two daughter cells. This knowledge can give clues towards the development of new antibiotics. The work flow to be developed can also be used to study several other processes and organizing principles in living cells.

A key reference is : Buncherd H, Roseboom W, De Koning LJ, De Koster CG, De Jong L (2014). A gas phase cleavage reaction of cross-linked peptides for protein complex topology studies by peptide fragment fingerprinting from large sequence database. J Proteomics 108, 65-77.

Contact details

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Luitzen de Jong, PhD
University of Amsterdam
Swammerdam Institute of Life Sciences (SILS)
Mass Spectrometry of Biomacromolecules

Visiting address
Sciencepark 904 (room C2.267)
1098 XH Amsterdam

email: l.dejong@uva.nl

Eva de Rijke
Research

Eva is an analytical chemist with over 10 years experience after her PhD as a scientist/project leader in both government and industrial R&D in the area of food quality and safety. During her research she has gained broad (hands-on) knowledge of a variety of analytical techniques such as LC-MS and GC-(IR)MS, but also spectroscopic techniques such as NMR, UV and fluorescence detection, to determine both the positive (e.g. nutritional and antioxidant values, taste, odour) and negative (contaminants, additives, off-flavours, veterinary drugs, adulteration/mislabeling) aspects of food. Currently she works as researcher in the Mass Spectrometry of Biomacromolecules group of the Swammerdam Institute of Life Sciences (SILS) on the project ‘Traceability of horticultural products based on natural isotopic composition’. This is part of the project Green Forensics within the Amsterdam Green Campus of which the main goal is to share knowledge of knowledge institutes with green companies in the region to drive innovation. Green Forensics is a consortium of Naktuinbouw, UvA and VU for forensic research in the green sector which offers services, education and R&D.

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Eva de Rijke

Eva de Rijke, PhD
University of Amsterdam
Swammerdam Institute of Life Sciences (SILS)
Mass Spectrometry of Biomacromolecules

Visiting address
Sciencepark 904 (room C2.267)
1098 XH Amsterdam

telephone: +31 20 525 6969
email: E.deRijke@uva.nl

Andrea Gargano
Research

Smart Stationary Phases, Multidimensional Separations, Intact Proteins
Before moving to Amsterdam, I’ve been a fanciful mono-dimensional (separation) scientist for a number of years. In my PhD (Vienna, Austria) I worked at the synthesis and characterization of alternative stationary phase supports and chemistries (HILIC, mixed mode, ion exchange and chiral chromatography). My Post-Doctorate took me to Amsterdam at the HIMS institute and was my chance to learn and work in collaboration with several industrial partners (COAST project: HYPERformance LC) at the development and application of two-dimensional liquid chromatographic separations.

In 2015 I was awarded with a VENI grant by NWO for my research proposal on improving the separation technology for the characterization of Intact Protein Analysis (IPA). My research take place in the CASA laboratories (UvA and VU) and is performed in collaboration with international research laboratories (Pacific North West Laboratory, Richland WA, USA).

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gargano-andrea

Andrea Gargano, PhD
University of Amsterdam
Van 't Hoff Institute for Molecular Sciences (HIMS)

Postal address
Postbus 94157
1090 GD Amsterdam

Visiting address
Science Park 904 (room C2.248A)
1098 XH Amsterdam

telephone: +31 20 525 7040
email: a.gargano@uva.nl
researchgate.net/profile/Andrea_Gargano

Rob Haselberg
Research

I am an all-round analytical chemist with a strong background in capillary electrophoresis and liquid chromatography hyphenated with mass spectrometry. I use these technologies to characterize intact proteins. This ranges from proteins as small as insulin up to large protein complexes. I am interested in determining protein heterogeneity, getting insights in degradation processes, mapping intentional modifications and studying protein-protein interactions. Recently, my first personal research project was granted in which I will also focus on the bioanalysis of proteins. In all this research the goal is to keep the protein intact from sample to detector, as this reduces data complexity and minimizes risk of introducing artefacts along the way. As studying these properties of intact proteins is not straightforward, I develop and apply the required advanced hyphenated analytics. All of this is done in the context of biopharmaceutical, clinical, and doping analysis, often in collaboration with companies and other university groups. Besides my research, I also supervise the CE and CE-MS laboratory of the group.
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Rob Haselberg, PhD
Vrije Universiteit Amsterdam
Amsterdam Institute for Molecules, Medicines and Systems

Postal address
de Boelelaan 1085
1081 HV Amsterdam

Visiting address
de Boelelaan 1108
1081 HZ Amsterdam

telephone: +31 20 598 7536
email: e r.haselberg@vu.nl

Suhas Nawada
Research
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Suhas Nawada, MSc
University of Amsterdam
Van 't Hoff Institute for Molecular Sciences (HIMS)

Postal address
Postbus 94157
1090 GD Amsterdam

Visiting address
Science Park 904 (room C2.248a)
1098 XH Amsterdam

email: S.H.Nawada@uva.nl

Bert Wouters
Research

I am currently working as a post doc researcher at the Van 't Hoff Institute for Molecular Sciences (HIMS) at the University of Amsterdam under the supervision of Prof. Schoenmakers. Previously, I have completed a Master of Industrial Engineering (Chemistry), a Master of Biomolecular Sciences and a PhD of Chemical Engineering.

During my doctoral research at the Free University of Brussels, I have investigated the i) development of novel polymer monolithic stationary phases to enhance proteomics profiling, ii) a fundamental approach to study to the effects of using smaller particle technology and their chromatographic performance at higher operating pressures and temperatures, and iii) the development of novel microfluidic technology and chromatography concepts to realize spatial multi-dimensional chromatography separations.

My current research is situated in the context of the MAnIAC project (Making Analytically Incompatible Approaches Compatible), and includes the development of an immobilized enzyme reactor (IMER).

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Bert Wouters

Bert Wouters, Dr.
University of Amsterdam
Van 't Hoff Institute for Molecular Sciences (HIMS)

Visiting address
Science Park 904 (room C2.258A)
1098 XH Amsterdam

telephone: +31 20 525 6416
email: B.Wouters@uva.nl
researchgate.net/profile/Bert_Wouters