The Network for the biology of Zinc: Zinc-net

Index Letter from Prof. Nicola Lowe Chair of COST Action TD1304……………..……… 3 Welcome Message…………………………………………………………………… 5 Time Schedule & Program Overview…………….…………………………..……… 6 Abstracts of Lectures…………………………………………………………………. 8 List of Practical Courses ……………………………………………………...……. 15 Program summary…………………………………………...……………………… 16

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Letter from Prof. Nicola Lowe Chair of COST Action TD1304 Dear Trainees, Trainers and Working Group 5 members, On behalf of the Zinc-Net (COST Action TD1304) Management Committee, I would like to welcome you to our meetings in Oviedo. We are holding two events in parallel here, a Training School, and a Working Group 5 workshop and are expecting around 50 delegates in total.

The workshop program has been expertly put together by Dr Hector Gonzalez and his team, at the Institute of Ophthalmology Fernández-Vega – Foundation of Ophthalmological Investigation and the University of Oviedo. He has invited expert speakers and trainers from his own institution (Alfredo Sanz-Medel, María Montes Bayón, Beatriz Fernández, Jorge Ruiz Encinar, Pablo Rodríguez González, Sergio Cueto, Marisa Fernández, Francisco Calderón, Amanda Suárez, Sara Rodríguez and María Cruz) as well as from the UK (Prof. Neville Osborne), Denmark (Tine E. Thingholm), Poland (Artur Krężel) and the USA (Lee Goldstein). We are very grateful to all of these experts who have agreed to share their knowledge with us. The central mission of our COST Action has been to defragment knowledge, create a panEuropean research platform and to train the next generation of zinc scientists. This workshop goes a long way towards achieving these core objectives.

Another of the key objectives for Zinc-Net was to create a Virtual Institute of Zinc Biology, VIZIBI. This is being realised through the creation and development of our VIZIBI web portal, under the guidance of our Working Group 5 (WG5) leader, Dr Imre Lengyel. The WG5 workshop that will also be taking place at the University of Oviedo has been designed to give time and space for network members to enhance the material that is currently available through this portal and to generate new material that we hope will be useful for our network members both now and in the future. Our aim is that VIZIBI remains a sustainable presence and resource for zinc scientists well beyond the life of our COST Action.

On that note, you will be aware that we are now entering the final 6 months of our 4-Year funding period from COST. It is hard to believe that our 4 years is nearly finished, but we have achieved a lot in this short time, both in terms of scientific 3

collaborations, publication outputs, but perhaps most importantly, new friendships. I hope that the new friendships forged in our time together in Oviedo will last many years and that we can continue to develop our research collaborations well into the future.

Finally, I must extend my personal thanks to Hector who has worked so hard to put these meetings together, and has been able to maintain a sense of humour throughout! Also to Imre Lengyel for co-ordinating the WG5 workshop and for all that he and Alberto Massarotti do to maintain our VIZBI web portal.

I very much hope that you enjoy our time together in Oviedo.

Kind regards, Nicola

Professor Nicola M.Lowe, B.Sc, PhD, R.Nutr, FAfN, FHEA, Chair of Management Committee, Zinc-Net COST Action TD1304

COST is supported by the EU Framework Program Horizon 2020

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Welcome Message

Oviedo (Spain), April 4, 2017

Dear Colleagues and Zinc-Net family, We are very proud and excited to host this Training School in Mass Spectrometry and the Working Group 5 Workshop, as part of the activities of the Zinc-Net: The network for the Biology of Zinc. We would like to share with you our laboratory expertise in mass spectrometry-based technologies to study total zinc, zinc distribution in biological tissues, zinc quantitative speciation and zinc-proteome.

We would like to thank the participation and implication of the speakers and trainers, and the support of the Institute of Ophthalmology Fernández-Vega and the University of Oviedo.

We hope that along this meeting you will have the opportunity to discuss present research highlights and applications, but also to get new ideas about how to handle problems and limitations that arise in this field. We also hope that during these days you will enjoy the hospitality of Asturian’s citizens as well as our gastronomy.

Best wishes,

The Local Organizing Committee Dr. Beatriz Fernández García Dr. Héctor González Iglesias

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Time Schedule & Program Overview 4th APRIL – INSTITUTE FERNÁNDEZ-VEGA

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OPHTHALOMOLOGY

7:30 – 8:30

Registration of participants at Silken Monumental Naranco Hotel

9:00 – 9:20

Training School Opening – Prof. Jesús Merayo Lloves. Institute of Ophthalmology Fernández-Vega & Foundation of Ophthalmological Investigation

Lectures 9:20 – 10:10

Plenary Lecture - MS-based Metallomics and Heteroatom-Tagged Targeted Proteomics. Prof. Alfredo Sanz-Medel

10:10 – 10:45

Key Note – High Resolution Laser-Ablation Metallomic Imaging Mass Spectrometry (MIMS) Tissue Mapping: A powerful Bioanalytical Technique for investigating zinc and other biometals in human and animal biospecimens. Prof Lee E. Goldstein

10:45 – 11:20 Key Note - ICP-MS based strategies to monitor cellular uptake and management of metal species. Prof. María Montes Bayón 11:20 – 12:00 Coffee Break 12:00 – 12:35 Key Note - Quantitative Phosphoproteomics. Prof. Tine E. Thingholm.

12.35 – 13:10 Key Note - Zinc and the retina. Prof. Neville N. Osborne 13:10 – 13:45 Key Note - Mass spectrometry in understanding of zinc protein folding and stability. Prof Artur Krezel 14:00 – 15:30 Lunch at Silken Monumental Naranco Hotel 15:30 – 17:30 Laboratory Tour of Mass Spectrometry Technologies – Faculty of Chemistry – University of Oviedo 18:00

Brainstorming Session & Dinner at Silken Monumental Naranco Hotel

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5th APRIL - FACULTY OF CHEMISTRY – UNIVERSITY OF OVIEDO 9:00 – 9:30

Allocation of trainees into groups – Faculty of Chemistry

9:30 – 11:30

Practical Course

11:30 – 12:00 Coffee Break 12:00 – 13:00 Practical Course 13:00 – 14:00 Lunch 14:00 – 15:30 Practical Course 15:30 – 16:00 Coffee Break 16:00 – 17:30 Practical Course 18:00 – 20:00 City Tour 20:00

Dinner at Silken Monumental Naranco Hotel

6th APRIL 9:30 – 11:00

Practical Course – Faculty of Chemistry

11:00 – 11:30 Coffee Break 12:00 – 13:00 Practical Course 13:00 – 14:00 Lunch 14:00 – 15:30 Practical Course 15:30 – 16:00 Coffee Break 16:00 – 17:30 Practical Course 18:00

Training School Closing

20:00

Dinner at Tierra Astur Águila (Colloto)

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Abstracts of Lectures

4th April Institute of Ophthalmology Fernández-Vega Foundation of Ophthalmological Investigation

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MS-based Metallomics and Heteroatom-tagged Targeted Proteomics

Alfredo Sanz Medel Department of Physical and Analytical Chemistry. Faculty of Chemistry. University of Oviedo. asm@uniovi.es

Contributors: A. Sanz-Medel, J.M. Costa, J. Ruiz, F. Calderón and M. García.

The classical boundaries and tools of Atomic Spectrometry are blurring nowadays.

Atomic spectroscopists one day studying photon/atoms interactions, are today investigating also the molecules constituting their samples (i.e. chemical speciation).

On the other hand, more and more bioscientists are learning to make use of analytical strategies of atomic spectrometry and chemical speciation (e.g. in proteomics). The outstanding analytical potential of mass spectrometry (MS) is becoming the chemical ladder to scalate from isotopes, atoms, elements and molecules up to nanoparticles and nanostructures of matter. An atmospheric plasma ion source (e.g. ICP) with a mass analyser and an appropriate separation could form the basis for analysing directly metalloproteins. In any case that elemental speciation using ICP-MS, should be completed with the corresponding molecular (MALDI- or ESI-MS) information for “integrated” chemical speciation. These combinations are opening new avenues and insights in bioscience. Traditionally, the biologically relevant non-metals S and P have been elusive to sensitive ICP-MS determinations. However, the new ICP(QQQ)MS instruments enable S and P analysis at very low limits of detection (orders of magnitude better than ICP-MS used before). This sensitive S and P determinations turns out to be of critical importance to allow direct absolute quantification of proteins or of protein phosphorylations (by S and P simultaneous determinations) for targeted proteomics1. Also, highly amplified ICP-MS detections can be achieved by tagging the protein of interest with metal nanoparticles (NPs). The high number of ICP-MS detectable atoms, per NP tag, enables a much higher sensitivity of the corresponding proteins by metal NPs tagged immunoassays. What is more, such amplifications can be enhanced by orders of magnitude if we select a special NP (e.g. ZnS(Mn) QDs) which, by a catalytic reduction, deposits on its surface relatively huge amounts of native gold form a Au(III) proper solution.

Subsequent elemental detection of this Au by ICP-MS allows an ultra-sensitive indirect determination of the NP-tagged protein (analyte). We will illustrate our developed immunoplatform and its extraordinary performance for real-life determinations of extremely low levels in human serum of “early alarm biomarkers” of cancer (e.g. prostate specific antigen, PSA) and its enormous potential as a possible “early alarm biomarker” of breast cancer in women2.

1. F. Calderón-Celis, S. Diez-Fernández, J.M Costa-Fernández, J.Ruiz Encinar, Juan J. Calvete and A. Sanz-Medel. Analytical Chemistry, 88, 9699-9706, (2016).

2. M. García-Cortés, J. Ruiz Encinar, J.M. Costa, A. Sanz-Medel. Biosensors and Bioelectronics, 85, 128-134, (2016).

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High Resolution Laser-Ablation Metallomic Imaging Mass Spectrometry (MIMS) Tissue Mapping: A Powerful Bioanalytical Technique for Investigating Zinc and Other Biometals in Human and Animal Biospecimens

Lee E. Goldstein Lee E. Goldstein, MD, PhD, Associate Professor of Psychiatry, Neurology, Pathology & Laboratory Medicine, Ophthalmology, Biomedical Engineering, Electrical & Computer Engineering, Boston University School of Medicine, College of Engineering, NIH Alzheimer’s Disease Center; Director, Boston University Center for Biometallomics, Boston, MA USA 02118. lgold@bu.edu

The Boston University Center for Biometallomics (CBM) is a unique bioanalytical core resource dedicated to promoting lead-edge biomedical research requiring advanced ultra-trace elemental and isotopic analytical capabilities and highresolution, laser ablation-assisted metallomic imaging mass spectrometry (MIMS) tissue mapping. The CBM, the first and only dedicated core resource of its kind in the nation. MIMS capabilities at the CBM provide previously unachievable tissue mapping with definitive, interference-free elemental and isotopic identification (mass resolution, >0.001 amu) and ultra-trace analytical sensitivity (ppt-ppq) at single-cell spatial resolution (~5 µm). MIMS tissue mapping enables accurate elemental-isotopic MIMS mapping in a wide variety of biospecimens (tissue sections, whole organs, fixed specimens, etc.) across widely varying elemental-isotopic tissue concentrations (>1012 LDR, <0.2 cps dark noise). Multiple elements and isotopes can be accurately mapped simultaneously. Core technologies at the CBM include: (i) double-focusing magnetic sector field ICP-mass spectrometer (Element XR, Thermo Scientific), (ii) quadrupole ICP-mass spectrometer (iCAP-Q, Thermo Scientific), (iii) ICP-optical emission spectrometer (7600 ICP-OES, Thermo Scientific), (iv) custom-designed laser-induced breakdown spectrometer (Applied Spectra), (v) femtosecond IR and nanosecond UV laser ablation platforms (FIRLA and NULA, respectively), (vi) interlocked ultra-trace analytical clean rooms dedicated to state-of-the-art metallomic analyses of human and animal biospecimens. The CBM also conducts a wide variety of allied bioanalytical, biochemical, and mass spectrometry assays through collaborative affiliation with the Boston University Biomedical Mass Spectrometry Resource, NMR-MRI High-Field Imaging Core, NIH-NIA Boston University Alzheimer’s Disease Center & Brain Banks, Harvard University Electron Microscopy Facility, and other affiliated core facilities. The CBM supports an array of state-ofthe-art bioanalytical and metallomic analytical capabilities,including multi-elemental and isotopic profiling, high-resolution MIMS tissue mapping, custom tissue processing and histopathology. In this presentation, the MIMS technique will be introduced and explained in details with examples of applications relevant to zinc biology in brain and eye research.

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ICP-MS based strategies to monitor cellular uptake and management of metal species

María Montes Bayón Department of Physical and Analytical Chemistry.

Faculty of Chemistry.

University of Oviedo montesmaria@uniovi.es

Contributors: M. Montes-Bayón, F. J. García Alonso, M. Corte, R. ÁlvarezFernández, A. Sanz-Medel, J. Bettmer, E. Blanco.

Iron is an essential element for cell growth and division. Recent experiments have linked a deregulation of iron’s metabolism with breast cancer progression, aggressiveness and recurrence. In fact, it is conceived that chronic failure in the redox balance due to the presence of high intracellular concentration of this metal has the potential to modulate specific signaling networks associated with cancer malignancy.

Thus, this work has been focused on the comparative evaluation of part of the Fe metallome in two breast cancer cell lines of different malignancy: MCF-7 and MDAMB-231. Evaluation of the total cytosolic iron as well as the ultrafiltrable iron content has been conducted using inductively coupled plasma mass spectrometry (ICP-MS) as Fe selective detector. It will be illustrated that a significantly higher total Fe concentration can be found in the less malignant phenotype. Additionally, Fefractionation experiments, conducted by coupling size exclusion chromatography (SEC) to ICP-MS and the ferritin concentration as well as the Fe:ferritin ratios in both cell phenotypes are significantly different.

Despite these experiments have been conducted in bulk cell cultures, it has been long known that biological populations are heterogeneous and that it is more interesting to obtain information about individual cells rather than settling for average information.

Therefore, some new experimental set-up to permit metal content determination in individual cells will be also presented in the second part of the talk using specific sample introduction strategies with ICP-MS detection.1 1. Wang, H.; Wang, B.; Wang, M.; Zheng, L.; Chen, H.; Chai, Z.; Zhao, Y.; Feng, W. Analyst 2015, 140, 523-531.

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Quantitative Phosphoproteomics

Tine Engberg Thingholm Associate Professor Department of Biochemistry and Molecular Biology University of Southern Denmark tthing@bmb.sdu.dk

Protein phosphorylation is one of the key post-translational modifications in nature; it plays a central role in many cellular processes such as transcriptional and translational regulation, cellular signaling and communication, proliferation, differentiation, and cell survival. It has been suggested that 30% of all proteins expressed in a eukaryotic cell are phosphorylated at some point, emphasizing the ubiquitous role of protein phosphorylation. The importance of protein phosphorylation on the function and regulation of cellular processes underlines the need for a highly selective and efficient method for enriching phosphorylated proteins and identifying the phosphorylation sites in these proteins to understand the biological role of this modification.

Phosphoproteomics is a tool that has become increasingly powerful over the last decade as new enrichment strategies emerged; suddenly making it possible to perform selective enrichments of phosphorylated peptides from highly complex samples even from very small amounts of starting materials. The introduction of robust methods for fishing out phosphorylated peptides and combining them with mass spectrometry made it possible to perform large-scale screening of protein phosphorylation in the cell. When also combining these methods with quantitative strategies like isobaric or isotopic labeling or label-free quantitation, one is now able to make a comparison of protein phosphorylation profiles in different samples, at different disease stages, in different cell types etc.

Furthermore, protein phosphorylation has been indicated as a key player in zinc signaling. Several zinc transporter proteins have been identified as linked to different protein phosphorylation signaling cascades. The use of quantitative phosphoproteomics strategies is still very new to the zinc research field, however, it offers an powerful tool for studying the link between protein phosphorylation and zinc signaling.

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Zinc and the retina

Neville N. Osborne, PhD., DSc. Fundación de Investigación Oftalmológica, Oviedo, Spain and Nuffield Department of Clinical Neurosciences, Oxford University, UK. neville.osborne@eye.ox.ac.uk Zinc is the most abundant trace metal in the retina where its distribution is nonuniform. Zinc is also essential for appropriate delivery of vitamin A from the liver to the retina to produce the protective pigment melanin of the eye. Bound-zinc is particularly located in the inner nuclear layer, (e.g. forming part of the structure of zinc finger transcription factors), while loosely-bound zinc is prominent in the retinal pigment epithelium and photoreceptor layers. Loosely-bound zinc ions in the photoreceptors might play a role in the phototransduction cascade and rhodopsin regeneration. Loosely-bound zinc is also found in presynaptic vesicles of photoreceptor cells in the outer plexiform and inner plexiform layers and can be synaptically released to affect both ionotropic and metabotropic receptors and also ion channels to modulate neurotransmission. The correct amount of loosely-bound zinc ions is maintained by regulating the function of zinc transporters, sensors and trafficking/storage proteins (i.e. metallothionein). Retinal homeostasis of zinc may be dysregulated because of systemic zinc depletion, aging and diseases such as agerelated macular degeneration. It remains however a goal to determine clearly the role of zinc in defined ophthalmological diseases. While basic sciences suggest that manipulation of retinal zinc metabolism in defined situations might improve visual function they also show that too much zinc is toxic to the retina. Significantly, the landmark age-related disease study (AREDS) showed a slowing down of the deterioration of visual acuity in patients with advanced age-related macular degeneration when taking 40-80mg zinc/day along with certain antioxidants. The recommended daily intake for zinc is around 10mg, achieved with a healthy diet.

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Mass spectrometry in understanding of zinc protein folding and stability

Artur Krężel Department of Chemical Biology, Faculty of Biotechnology University of Wrocław, Poland artur.krezel@uwr.edu.pl Zinc (formally Zn2+) is the most widespread transition metal in biology.1 In cells, it is present mostly in bound state with cellular proteins. Bioinformatic analysis suggests that at least 3000 protein in human protein participates in Zn2+ binding.2 Zinc in proteins plays catalytic, structural or regulatory function. In each class of proteins Zn2+ is bound in a different way which is expressed by different stability and reactivity of the binding site. Biophysical property of zinc binding sites are also linked with cellular Zn2+ status. In our study, we focus on the understanding molecular basis that drive stability and reactivity of zinc sites in proteins. Mass spectrometry is one of the most informative techniques that can be used for that purpose. By the differential modification of cysteine residues in partially metalated human zinc metallothionein, we aimed to structurally characterize Zn4-7-MT species that are believed to be the most important in cellular zinc buffering.3 Differently modified metallothionein was analyzed directly or after its trypsinization by MS/MS and zinc folding pathway was proposed. Our results allow to better understand dual nanomolar and picomolar zinc affinities of metallothionein. In another studies we applied hydrogen-deuterium exchange (HDX) reaction for mapping hydrogen bond network present in zinc sites of Rad50 zinc hook domain and classical zinc fingers in order to understand sequencedependent and structural factors that affect thermodynamic stability of those domains.1,4 We found that metal-coupled folding of zinc domains is most important driving force in elevation stability of structural zinc sites in proteins.

Financial support by National Science Centre (2014/13/B/NZ1/00935) is gratefully acknowledged.

1. Kochanczyk, T., Drozd, A., Krężel A. Metallomics 2015, 7, 244.

2. Andreini C. et al. J. Proteome Res. 2006, 5, 196.

3. Krężel A., Maret, W. J. Am. Chem. Soc. 2007, 129, 10911.

4. Kochańczyk T. et al. Sci. Rep. 2016, 6, 36346.

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Practical Courses 5th – 6th April Faculty of Chemistry University of Oviedo

PC1. Quantitative bio-imaging of zinc by Laser Ablation - ICP-MS. Beatriz Fernández-García and María Cruz-Alonso.

PC2. Total analysis and quantitative speciation of zinc by HPLC-ICP-MS. Héctor González-Iglesias, Sara M. Rodríguez and María Luisa Fernández-Sánchez.

PC3. ICP-QQQ: Clinical applications. Francisco Calderón Celis and Jorge Ruiz Encinar.

PC4. Absolute Quantification of Proteins by LC-ESI-MSMS. Amanda Suarez Fernández and Pablo Rodríguez-González.

PC5. Protein and peptide characterization by ESI-QTOF. Sergio Cueto Díaz.

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PROGRAM SUMMARY: DATE/ Schedule

03/April/17

MORNING 12:00-20:00 HOTEL REGISTRATION (Silken Monumental Naranco - SMN) AFTERNOON

EVENING

04/April/17 7:30-8:30 BREAKFAST at SMN & Meeting REGRISTRATION at SMN 9:00-14:00 TRAINING SCHOOL at IOFV 14:30-16:00 LUNCH at SMN 16:00-18:00 LABTOUR at FCH

20:00-22:00 DINNER at SMN

18:00-22:00 BRAINSTORMIN SESSION + DINNER at SMN

SMN: Silken Monumental Naranco Hotel: IOFV: Institute of Ophthalmology Fernández-Vega FCH: Faculty of Chemistry

05/April/17

06/April/17

07/April/17

8:00-9:00 BREAKFAST at SMN

7:00-9:00 BREAKFAST at SMN

9:30-13:00 TRAINING SCHOOL at FCH

9:30-13:00 TRAINING SCHOOL at FCH

ADIOS

13:00-14:00 LUNCH of TRAINING SCHOOL at FCH 14:00-18:00 LUNCH of TRAINING SCHOOL at FCH

13:00-14:00 LUNCH of TRAINING SCHOOL at FCH 14:00-18:00 LUNCH of TRAINING SCHOOL at FCH

20:00 DINNER at SMN

20:00 DINNER

8:00-9:00 BREAKFAST at SMN