PROJECT FINAL REPORT
Grant Agreement number: 303344
Project acronym: ENIGMAS
Project title: Explicitly Normalized Imaging Mass Spectrometry
Funding Scheme: FP7-PEOPLE-2011-IEF
Period covered:
from 1st October 2012
to 30th September 2014
Name of the scientific representative of the project's co-ordinator1, Title and Organisation:
Dr. Liam A. McDonnell
Leiden University Medical Center
Albinusdreef 2, Postzone L4-Q
Postbus 9600
2300 RC Leiden
Tel: +31(0)715268744
Fax:
E-mail:
Project websiteError! Bookmark not defined. address:
1
Usually the contact person of the coordinator as specified in Art. 8.1. of the Grant Agreement.
4.1
Final publishable summary report
- Executive Summary (1 page)
The Explicitly Normalized ImaGing MAss Spectrometry (ENIGMAS) project aimed at developing
methodologies for imaging mass spectrometry (MS) to enable its application to the study of preclinical animal models of neurological disease.
Main developments/achievements include:
- Reproducible on-tissue digestion protocols for protein analysis in imaging MS.
- Reproducible sample preparation protocols for metabolite, neuropeptide and intact protein analysis
by imaging MS.
- Comprehensive database of peptides and proteins identified by imaging MS in mouse brain tissue
sections.
- Bio informatics pipelines for automated and anatomical driven analysis of imaging MS datasets
from large animal cohorts.
- Application of imaging MS to pre-clinical models of neurological disease.
- Novel biomolecular insights regarding the changes produced in mouse brain by cortical spreading
depression, the electrophysiological equivalent of migraine aura in animals.
The work developed during the grant period was enthusiastically disseminated across the imaging
MS and neuroscience communities through the publication of peer reviewed manuscripts (2
published manuscripts, 1 submitted manuscript, 1 manuscript in preparation), participation in
scientific conferences (oral and poster communications), participation in workshops and advanced
training across Europe through the COST programme BM1104 – Imaging Mass Spectrometry: New
Tools For Healthcare Research. The technological improvements will be made publically available
through the imaging MS website (maldi-msi.org) for widespread application across different imaging
MS laboratories.
The innovative technologies/methodologies developed will have a profound impact on the imaging
MS and on the neurosciences field. Most important, they will allow:
- Facile protein identification through the developed imaging MS methods and protein databases;
- Automatic annotation of imaging MS datasets with the tissue histology/anatomy;
- Facile comparison of animal cohorts in pre-clinical models of neurological disease, whether by
extracting data from specific regions in the mouse brain or comparing distributions in different
animals;
- Alignment of imaging MS datasets to the Allen Brain Atlas, a genome-wide repository of data from
the mouse brain, allows the comparison of imaging MS data with genomic information, thereby
increasing the impact of imaging MS data and biological information retrieved;
- Widespread application of imaging MS to other neurological diseases of present day importance,
such as stroke, seizure, Alzheimer’s, Parkinson’s.
- Summary of the project: scientific context and objectives (3 pages)
The project entitled Revealing the molecular image of migraine through Explicitly Normalized
Imaging Mass Spectrometry – ENIGMAS aimed at developing and improving methodologies for
imaging MS analyses of biomolecules directly from tissue with a practical application to a very
common, severe, neurological disorder: migraine. This report presents a detailed breakdown of the
research activities and results obtained during the 24-month period of the FP7-PEOPLE-2011-IEF
Marie Curie grant, starting on the 1st October 2012 and terminating on 1st October 2014.
Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) can generate profiles
directly from tissue that contain hundreds of distinct biomolecular ions. Spatially-correlated analysis,
imaging MS, can simultaneously reveal how the intensity of each of these biomolecular ions varies
in the tissue samples. Imaging MS can be directly applied to tissue samples and is an untargeted
multiplex analysis, thus enabling the different regions of the tissue to be differentiated on the basis of
their endogenous biomolecular content. Furthermore, using essentially the same technique but
different preparation protocols imaging MS can analyse peptide, proteins or metabolites. One of the
advantages of imaging MS is that it can define the regions of a tissue based on their MS profiles and
thereby distinguish biomolecularly different regions even if they are not histologically distinct using
established tools. These capabilities offer enormous potential to investigate biomolecular changes
that occur prior to (or without) morphological change, or those for which a molecular specific stain is
unavailable. Also the study of neurological diseases for which the pathophysiology is not entirely
known, such as migraine, could benefit of systematic investigations of biomolecules that are different
between diseased and health tissue.
Migraine is a paroxysmal neurological disorder characterized by disabling attacks of headache and
associated neurological symptoms. The migraine aura, which occurs in one-third of migraine patients
and can lead to migraine headaches, is caused by cortical spreading depression (CSD), a selfpropagating wave of neuronal and glial cell depolarization travelling at about 3 to 5 mm/min in the
cerebral cortex. CSD causes a temporary dramatic failure of brain homeostasis, efflux of excitatory
amino acids and increased energy metabolism. Numerous neurotransmitters and metabolites are
associated with CSD including labile-phosphate compounds (ATP, ADP, AMP, cGMP,
phosphocreatine) and glycolytic metabolites (lactate, pyruvate, glucose, glycogen), many of which
are detectable by imaging MS. Experimental and clinical evidence strongly suggests that CSD, when
induced in one hemisphere of wild type mice, does not cross to the other hemisphere. The migraine
research group in Leiden University Medical Center (LUMC) has generated transgenic mice bearing
human pathogenic migraine mutations with increased susceptibility to CSD. Depending on the
severity of the mutation CSD waves can reach subcortical areas, which correlate with clinical
phenotypes.
As the full potential of imaging MS is presently not being used due to lack of an effective
normalization methodology nor is it (widely) applied to neurological disorders, I designed the
Explicitly Normalized ImaGing MAss Spectrometry (ENIGMAS) project which strives to combine
technical improvements of imaging MS with a practical application to a neurological disorder,
namely migraine. Importantly, the technologically development part of ENIGMAS has the potential
to be applied to other diseases areas and therefore has broad application potential.
Despite a few changes to the initial work plan, made to accommodate the current state of research in
the MSI and neurosciences fields, most major objectives were accomplished during the grant period.
All changes were made upon agreement between researcher and scientific coordinator. The main
objectives were as follows:
i) development of quantitative imaging MS platforms for protein analysis;
ii) determination of spatio-temporal changes following cortical spreading depression in
transgenic knock-in mouse models of migraine aura;
iii) development of data reduction tools for analysis of imaging MS datasets;
iv) MSI-directed micro-dissection for independent corroboration;
v) development of a universal method for quantitative imaging MS of proteins.
Objective 1: Quantitative imaging MS of proteins.
Objective 1 aimed at developing a reference standard based on SILAC mouse brain for the explicit
normalization and quantification of the tryptic peptides generated by on-tissue digestion. Currently,
on-tissue digestion is the method of choice for protein identification in imaging MS. However, ontissue digestion creates a large number of tryptic peptides, many of which are isobaric peptides. The
presence of multiple peaks at every mass precludes the correct identification of the tryptic peptides
generated, even with the lower dynamic range of an imaging MS experiment (due the very small
number of cells in each pixel). Therefore, careful characterization of the tryptic peptides detected
from the mouse brain tissue and their full resolution is of outmost importance for the success of the
imaging MS methodology for the analysis of proteins.
This work package was divided in 4 tasks:
1.1 – Identification of tryptic peptides produced by on-tissue digestion (month 2);
1.2 – Accurate mass, ultrahigh mass resolution imaging MS (month 3);
1.3 – Limits of detection, quantification, dynamic range using SILAC peptides (month 5);
1.4 – Reproducibility of on-tissue digestion (month 7).
Objective 2: Quantitative imaging MS of proteins in dynamic systems – determination of
spatio-temporal changes following migraine aura in transgenic migraine mice (month 13).
Objective 2 aimed at analysing the biomolecular changes induced by cortical spreading depression
(CSD) in wild-type and transgenic mouse brains with imaging MS. The transgenic mice use in this
study carried human pathogenic missense mutations (R192Q and S218L) previously identified in
patients with a rare subtype of migraine with hemiplegia during the aura phase of the migraine
attack. Briefly, mutation R192Q causes pure hemiplegic migraine whereas mutation S218L is
associated with cerebellar ataxia and an increased sensitivity to epileptic attacks and mild head
trauma triggered delayed edema formation, which can be fatal. In the mutant mice CSD propagates
not only faster, but can also spread to subcortical areas. The experiments planned included the
analysis of wild type, and the R192Q and S218L transgenic animals at 4 time points (2 and 15 min, 1
hour and 24 hours, reflecting immediate changes and less acute changes which are relevant as CSD
may change the network properties of the cortex) to determine the effects of the pathogenic missense
mutations on the spatio-chemical extent of the perturbations and their evolution.
Objective 3: Data reduction and imaging MS-based molecular histology of CSD datasets.
The purpose of this work package was to perform molecular histological annotation of the tissues,
indicating regions with perturbed tryptic peptide MS signatures and the identities of the perturbed
tryptic peptide ions, assess the temporal evolution and the statistical significance of the molecular
perturbations. This included the development of automated feature detection and extraction routines
of imaging MS datasets for data analysis.
This work package was divided in 2 tasks:
3.1 – Data reduction and imaging MS-based molecular histology of CSD datasets (month
14);
3.2 – Probabilistic Latent Semantic Analysis of reduced datasets (month 16).
Objective 4: Imaging MS-directed microdissection for independent corroboration.
Objective 4 aimed at performing imaging MS-based molecular histology analysis to define tissue
regions with distinct MS profiles. These regions would be extracted from the brain tissue by laser
capture microdissection and then analysed by common proteomics techniques for independent
corroboration of the CSD induced changes.
This work package was comprised of 3 main tasks:
4.1 – Alignment of adjacent tissue sections (month 18);
4.2 – Microdissection of areas highlighted by imaging MS analysis (month 20);
4.3 – Quantitative proteomics analysis of microdissected samples (month 24).
Objective 5: Development of universal method for quantitative protein imaging MS.
The SILAC mouse methodology is broadly applicable and can be used for any imaging MS
investigation using mouse models (e.g. neurodegeneration, obesity, cancer). However, a significant
fraction of imaging MS research is presently focused on human tissue; consequently a universal
method for explicit protein quantitation would significantly increase the scope and impact of the
project. Therefore, the development of reference sample based on 18O-enzymatic labelling of tryptic
peptides from on-tissue digestion was proposed.
This work package was divided in 2 tasks:
5.1 – Trypsin catalysed 18O-labelling of tryptic peptides (month 20);
5.2 – Limits of detection, quantification, dynamic range using 18O-labelled peptides (month
24).
- Main scientific and technological results/foregrounds (6 pages)
Objective 1: Quantitative imaging MS of proteins.
This work package was divided into 4 tasks as previously described. Three out of the 4 tasks initially
described were successfully accomplished within the 24 month period of the grant. Work related
with task 1.3 (Limits of detection, quantification, dynamic range using SILAC peptides) is still
ongoing. One extra task was added to objective 1, as explained below. Changes to the original work
plan were made to address the developments in technology and methodologies that occurred in the
mass spectrometry imaging and migraine research fields during the time between submission and
start date of the grant. All modifications to the planned experiments were made upon agreement
between researcher and scientific coordinator.
- Task 1.a: Assessment of effects of heat induced tissue stabilization (not in the original work
package).
The quality of the tissue sample is fundamental for the success of an imaging MS experiment. It is
well documented that post-mortem degradation of tissue can lead to profound changes in the
composition of the mass spectra, the intensities of the peaks and distributions of the biomolecular
ions. The episodic nature of CSD and rapid response of metabolites and peptides to post-mortem
degradation means that many of the changes may be diluted or overwhelmed by changes following
animal sacrifice, due to the normal evolution of CSD as well as post-mortem degradation. Therefore,
for studying such episodic disturbances it may be essential to ‘freeze’ the biochemical effects of CSD
as soon as possible by deactivating the tissue’s constituent enzymes. A high-power thermal contact
device (Denator Stabilizor T1) that can denature proteins of a whole mouse brain in 1-2 minutes has
been recently developed for tissue stabilization. In order to test its applicability to imaging MS
studies and assess the effects on tissue structure and histology a set of experiments were planned in
collaboration with Prof. Per Andrén, leader in the analysis of neuropeptides and small metabolites in
models of neurological disease by imaging MS, from the University of Uppsala. Briefly, 16 wild
type mice were sacrificed by cervical dislocation and their brains excised at different post-mortem
time points: 0, 3, 10 and 30 min (4 animals per time point). The brains were excised, bisected and
one hemisphere was heat-stabilized with the Denator Stabilizor T1, while the other was flash frozen
in dry ice. The brains were stored at -80 ˚C until MSI analysis of proteins, metabolites and peptides.
Preliminary results show the effect of post-mortem degradation at the metabolite level with
increasing post-mortem time. Changes at protein and peptide level were negligible when compared
with the flash freezing in dry ice method. However, the effect of the tissue stabilization procedure
resulted in massive changes in the morphology of the tissue, compromising any subsequent
histological analysis. In the context of the analysis of animal cohorts of CSD (objective 2 and 3), the
morphological deformations induced by the heat stabilization procedure mean that the correlation of
imaging MS datasets from different animals and correlation of imaging MS with histology are
compromised. Therefore, flash freezing in dry ice was selected as the tissue stabilization method to
be used in all subsequent experiments.
- Task 1.1: Identification of tryptic peptides produced by on-tissue digestion (month 2)
There was no universal procedure for on-tissue digestion or peptide identification by imaging MS at
the start of the grant. Many methodologies had been described but most were based on a common
workflow, as follows: i) washing tissue sections in aqueous/organic solvents; ii) enzyme deposition
onto the tissue section followed by incubation; iii) matrix deposition onto tissue sections; iv) imaging
MS analysis by MALDI-MS. Therefore the first part of the research activities was focused on the
development and establishment of a methodology for reproducible on-tissue digestion and imaging
MS analysis.
One of the problems encountered during the development of on-tissue digestion methodologies is
related with the MALDI bias towards arginine terminated peptides. Tryptic peptides have a Cterminal lysine (Lys) or arginine (Arg) amino acid, which facilitates the formation of singly charged
ions by MALDI and analysis by MS in the positive ion mode. The natural occurrence of Arg and Lys
in the eukaryotic proteome is 5.4 % and 6.4 %, respectively, which corresponds to 46 % Argterminated and 54 % Lys-terminated tryptic peptides (considering 100 % theoretical trypsin
efficiency). However, it has been reported that MALDI-MS analysis is biased towards Arg-tryptic
peptides. These results were further confirmed in the laboratory after analysis of on-tissue digest
extracts by LC-MALDI-MS/MS and LC-ESI-MS/MS: 70 % of all tryptic peptides sampled by
MALDI are C-terminated Arg-peptides in contrast to the 42 % C-terminated Arg-peptides sampled
by ESI. Although this fact per se is not an issue regarding the analysis of proteolytic peptides by
MSI, it becomes critical when analysing SILAC tissue (to be used in tasks 1.3, 1.4, 2.1 for
quantitative experiments). SILAC mouse brain tissue is commercially available but only in the 13CLys labelled form, a factor which was not expected in the original work plan. Thus, if on-tissue
digestion would be performed with trypsin as originally described in the proposal, most labelled
peptides would not be detected due to the severe under sampling of C-terminal 13C-Lys-terminated
tryptic peptides by MALDI. To address this issue, improve the identification of proteolytic peptides
by MS/MS and establish the best methodology for MSI analysis after on-tissue digestion a number of
different experiments initially not planned in the proposed work plan were performed:
- Optimization of tissue washing conditions with different buffer/solvent systems to remove
contaminants and interferences with MS analysis;
- Optimization of on-tissue digestion with different enzymes, with distinct cleavage
specificity, namely: i) trypsin; ii) a cocktail of trypsin/Lys-C; iii) Lys-C; iv) Lys-N; v) Arg-C.
- Optimization of enzyme and matrix deposition protocols with the SunCollect (SunChrom,
Germany) spraying device.
- Extraction of on-tissue digested peptides and analysis by LC-MS/MS on a Q-Exactive
Orbitrap (Thermo)
The addition of an extra task (1.a) and the need to overcome unpredicted problems, as explained
above, contributed for the delay and deviations between the planned and actual work performed. In
addition, this contributed to the delay in accomplishing tasks 1.2 and 1.3 (see below).: Limits of
detection, quantification, dynamic range using SILAC peptides. Nevertheless, the original planned
deliverable – Database of peptide identifications, was successfully accomplished.
Most significant results are highlighted below:
- Development of robust buffer-free protocol for on-tissue digestion.
- Comprehensive identification of peptides from on-tissue digestion – total combined
number of identified peptides with different enzymes was 5339, which represents an increase of
180% identified peptides when compared to the classical on-tissue digestion approach with trypsin
alone.
- Comprehensive identification of proteins – total combined number of proteins identified
with multiple enzyme approach was 1194, ca. 52% more than the classical on-tissue digestion
approach with trypsin alone.
- KEGG pathway analysis revealed that most of the identified proteins are involved in
neurodegenerative disorders (e.g. Alzheimer’s disease, Parkinson’s disease and Huntington’s
disease) as well as in long-term depression, showing that the developed approach is applicable to the
study of other neurological disorders by imaging MS.
The developed procedure is the most comprehensive collection of peptide identifications after ontissue digestion reported to date. It is now used as the standard on-tissue digestion method in our
laboratory at the Leiden University Medical Center, and has been communicated to the imaging MS
community in several conferences (50th Anniversary of the Dutch Society for Mass Spectrometry –
Rolduc, Netherlands, 2014; 62nd American Society for Mass Spectrometry Conference on Mass
Spectrometry & Allied Topics, Baltimore, USA, 2014; OurCon II Meeting – Mass Spectrometry
Imaging, Antalya, Turkey, 2014); several workshops sponsored by the COST program BM1104:
Imaging Mass Spectrometry: New Tools For Healthcare Research (Stevenage, UK, 2012; Lille,
France, 2014); and through publication in peer reviewed journals (one submitted manuscript).
- Task 1.2: Accurate mass, ultrahigh mass resolution imaging MS (month 3)
Ultrahigh mass resolution FTICR-MS is necessary to resolve the isobaric proteolytic peptides and
reference standards (from the SILAC mouse). As the isobaric ions can differ in mass by just several
mDa it is essential to ensure high mass accuracy throughout the imaging MS experiment. High
resolution imaging MS measurements of on-tissue digestion experiments were performed on a 9.4 T
Fourier transform ion cyclotron resonance (FTICR).
Measurements were performed after the optimization of the on-tissue digestion procedure (see task
1.1), which explains the delay in the schedule. Nevertheless, the original planned deliverable –
Accurate mass, ultrahigh mass resolution imaging MS, was successfully accomplished.
The results obtained show a successful assignment of 23.5% of the total number of proteins
identified by LC-MS/MS (see task 1.1), corresponding to 280 proteins when the results of the
different enzymes used for on-tissue digestion were combined. These results show the high quality of
data produced and the robustness of the on-tissue digestion procedure and FTICR-MS analysis
performed.
The developed procedure represents the most comprehensive database of peptide identification by
high resolution imaging MS performed to date. It is now used as the standard imaging MS analysis
protocol for on-tissue digestion in our laboratory at the Leiden University Medical Center, and has
been communicated to the imaging MS community in several conferences (50th Anniversary of the
Dutch Society for Mass Spectrometry – Rolduc, Netherlands, 2014; 62nd American Society for Mass
Spectrometry Conference on Mass Spectrometry & Allied Topics, Baltimore, USA, 2014; OurCon II
Meeting – Mass Spectrometry Imaging, Antalya, Turkey, 2014); several workshops sponsored by the
COST program BM1104: Imaging Mass Spectrometry: New Tools For Healthcare Research
(Stevenage, UK, 2012; Lille, France, 2014); and through publication in peer reviewed journals (one
submitted manuscript – the same as task 1.1).
- Task 1.3: Limits of detection, quantification, dynamic range using SILAC peptides (month 5)
One of the main objectives of the proposal was the development of a methodology for quantitative
imaging MS of hundreds of proteins in tissues using the SILAC mouse brain as reference standard.
Although commercially available SILAC mouse brain tissue is expensive – a 13C-labeled mouse
brain (450 mg) costs ca. 4000 € (880 € per 100 mg). In addition, SILAC mouse brain is only
available with 13C labelled Lys, which was not predicted in the original work plan. Therefore, an
optimized protocol for on-tissue digestion with enzymes with cleavage specificity for Lys had to be
developed, since the on-tissue digestion with trypsin would produce non-labelled Arg peptides,
compromising the protein quantification (see task 1.1 and 1.2). This explains the delay in the
accomplishment of this objective.
In a parallel study, a strategy for quantification based on peptide dimethylation is currently being
developed. Briefly, a standard solution of proteolytic peptides was generated by on-tissue digestion
of WT mouse brain sections, according to the developed standard protocol (task 1.1), and the
proteolytic peptides extracted. The extracted peptides were then dimethylated and homogeneously
sprayed over the on-tissue digested mouse brain sections (sample tissue). Dimethylation of peptide
extracts introduces 2 methyl groups and each N-terminus and 2 methyl groups at each Lys side chain,
therefore increasing the peptides’ mass by 28 Da (Arg-terminated tryptic peptides with no missed
cleavage) or by 56 Da (Lys-terminated tryptic peptides with no missed cleavage). The mass
difference allows for standard proteolytic peptides to be distinguished from their homologue peptides
from the on-tissue digested sample, thus allowing normalization of the m/z features on a peptide-topeptide basis. Imaging MS analysis was performed with a 9.4T FTICR mass spectrometer equipped
with a MALDI source. Once optimized, the normalization protocol based on dimethylated peptide
analogs will be applied to SILAC mouse brain tissue. The efficiency of both approaches will be
compared, not only in terms of normalization efficiency, but also time and cost efficiency.
- Task 1.4: Reproducibility of on-tissue digestion (month 7).
The reproducibility of on-tissue digestion was originally planned to be performed with SILAC tryptic
peptide reference standards. However, due to the costs associated with SILAC mouse brain, this task
was addressed concurrently with task 1.1 and 1.2 as explained above. Reproducibility of the ontissue digestion approach was assessed by examining the results obtained from peptide and protein
identification.
The developed procedure represents the most comprehensive database of peptide and protein
identification by high resolution imaging MS performed to date. It is now used as the standard
imaging MS analysis protocol for on-tissue digestion in our laboratory at the Leiden University
Medical Center, and has been communicated to the imaging MS community in several conferences
(50th Anniversary of the Dutch Society for Mass Spectrometry – Rolduc, Netherlands, 2014; 62nd
American Society for Mass Spectrometry Conference on Mass Spectrometry & Allied Topics,
Baltimore, USA, 2014; OurCon II Meeting – Mass Spectrometry Imaging, Antalya, Turkey, 2014);
several workshops sponsored by the COST program BM1104: Imaging Mass Spectrometry: New
Tools For Healthcare Research (Stevenage, UK, 2012; Lille, France, 2014); and through publication
in peer reviewed journals (one submitted manuscript – the same as task 1.1).
Objective 2: Quantitative imaging MS of proteins in dynamic systems – determination of
spatio-temporal changes following migraine aura in transgenic migraine mice (month 13)
The original planned experiments focused only on imaging MS protein analysis of 2 transgenic
mouse models of migraine, R192Q and S218L mice, at different time points after CSD (2 and 15
min, 1 hour and 24 hours). However, the initial planned experiments were changed due to
unpredicted factors associated with i) experimental method development; ii) development of methods
for high-resolution mass spectrometry (FTICR) analysis of peptide samples; iii) upgrade of the
FTICR measurement cell, which prevented the use of the instrument for several months; and iv) new
data obtained from experiments performed by the imaging MS group and the migraine group in the
period covering grant submission, approval and actual start date (approximately 1 year and 2
months). Therefore, to accommodate the current state of the art and to avoid further delays a new
experimental plan was put into practice after agreement between researcher and scientific
coordinator.
Instead of 2 mouse models of migraine, a single transgenic mouse model carrying the knock-in
human pathogenic mutation R192Q, which in patients is associated with pure hemiplegic migraine,
was used. To understand the biomolecular changes associated with CSD and with the migraine
genotype, a series of experiments were carried in which different groups of wild-type and R192Q
mice were analysed. Seven CSD events (separated by 5 min) were evoked in the right hemisphere of
the cortex of wild-type and R192Q mice with 1 M KCl (5 animals per group). The animals were
sacrificed after the last CSD event and the brains extracted and immediately flash frozen in dry ice to
prevent post-mortem degradation. Equivalent sham experiments utilizing aqueous NaCl (1 M) in
place of aqueous KCl were performed in parallel (6 animals per group), and naïve mouse controls
were also analysed (5 animals per group) so as to clearly differentiate CSD related biomolecular
changes. Experiments performed after grant submission and before the grant start date revealed
biomolecular changes at metabolite and neuropeptide level, but the absence of protein changes in
wild-type mouse after CSD. Therefore, instead of focusing only on protein analysis, as originally
planned, imaging MS experiments focused on the analysis of 3 different molecular classes, proteins,
peptides and metabolites. To efficiently handle the imaging datasets and to allow comparison
between the different animals, all imaging datasets and correspondent histological images were
registered to the ABA (see objective 3). A large number of metabolites and peptides showed
substantial distribution changes in the brain associated with CSD. Among those, different mass
spectral features showed significant (t-test, p < 0.05) changes in the isocortex, 146 Da and 377 Da
(metabolite mass range), 11344 Da (protein mass range); and in the thalamus, 1820 Da and 1834 Da
(peptide mass range), of the CSD affected brain hemisphere of R192Q mice. Based on high mass
resolution experiments and database search, m/z features 146 Da and 377 Da were putatively
identified as glutamate and fructose 1,6-bisphosphate, respectively. Based on previous imaging MS
work and on in-situ hybridization experiments (Allen Brain Atlas) and on BLAST search (in UniProt
database), m/z feature 11344 Da was putatively identified as an acetylated form of Histone H4. In
particular, the identification of glutamate changes correlate with the current migraine paradigm in
which glutamate plays an essential role in the CSD event. One manuscript is currently in preparation.
Objective 3: Data reduction and imaging MS-based molecular histology of CSD datasets.
This work package was performed concurrently with the tasks in objective 1and 2, for better
integration of experimental work and data analysis. Due to the extended biological variability
encountered while developing work packages 1 and 2 and the need to develop efficient and
automated platforms for imaging MS data analysis, the researcher and coordinator agreed to add an
extra task to the original planned experiments: the development of a pipeline for automatic
registration of multiple imaging MS datasets with their respective histological images and a single
reference system, the Allen Brain Atlas.
One of the main difficulties in imaging MS is the related with the handling of large datasets
generated. Alignment of imaging MS datasets with tissue histology is a fundamental step in the data
analysis pipeline since it allows the interpretation of the spatially-correlated MS data in the
biological context of the tissue. To address this topic, a tool was developed that allows the
registration of imaging MS datasets from multiple mouse brain tissues to the same coordinate space,
the Allen Brain reference Atlas (ABA). The main elements of the registration pipeline are: i) preprocessing of the histological sample image; ii) selection of the closest mouse brain tissue section
from the ABA; iii) affine registration to capture global deformations (e.g. scale, translation, and
rotation); and iv) non-linear (elastic) image registration to capture local deformations (e.g. local
compression or tears) and account for inter-individual variability. A quantitative comparison of the
registration process demonstrated that the mean error after the registration was approximately 30 µm,
smaller than the pixel size of most MALDI imaging MS experiments. This pipeline enables facile
and rapid inter animal comparisons by first testing if each animal’s tissue section was sampled at a
similar location and enabling the extraction of the biomolecular signatures from specific brain
regions.
Despite not being present in the original proposal, the development of this tool has a number of
advantages not only for the development of the ENIGMAS project but for the imaging MS field in
general. The main benefits are:
- Automatic annotation of imaging MS datasets with the tissue histology/anatomy;
- Automatic determination of tissue sampling location;
- Facile comparison of animal cohorts, whether by extracting data from specific regions in the
mouse brain or comparing distributions in different animals;
- The alignment pipeline is not biased towards the quality of the imaging MS data and is
applicable to imaging MS datasets obtained using any ionization method and mass spectrometer.
- Alignment of imaging MS datasets to the ABA, a genome-wide repository of data from the
mouse brain, allows the comparison of imaging MS data with genomic information, thereby
increasing the impact of imaging MS data and biological information retrieved.
- The use of public atlases/databases to allow the rapid dissemination of this tool and the
widespread use of imaging MS within the analytical and neurosciences communities.
Two manuscripts have been published.
Objective 4: Imaging MS-directed microdissection for independent corroboration.
This work package was divided into 3 tasks as previously described. Task 4.1 was performed in
parallel with work package 2 and 3, as explained above. Tasks 4.2 and 4.3, comprising the
microdissection of areas highlighted by imaging MS and quantitative proteomics were not
accomplished during duration of the grant and are currently being performed. Expected results are
the definitive identification of the molecular features associated with CSD induced changes observed
in work package 2.
Objective 5: Development of universal method for quantitative protein imaging MS.
Due to difficulties in controlling the extent of 18O labelling of on-tissue digestion extracts, a
universal method for quantitative protein imaging MS relying on dimethylation of on-tissue digest
extracts is currently being developed. Peptide 18O labelling with trypsin suffers from back-exchange,
which means that the labelling reaction is reversible and therefore 100 % labelling efficiency (double
18
O labelling) cannot be obtained, thus generating a number of contaminants (single 18O labelled
peptides) that compromise the quantitative experiment. Therefore, upon agreement between
researcher and coordinator, an alternative quantification method relying on dimethylation is currently
being developed. See task 1.3 for details.
- Potential impact and main dissemination activities (2 pages)
The work developed during the grant period will have an impact at different levels:
- Scientific impact
i) The methods developed for on-tissue protein digestion and the database of peptides and proteins
identified will be made public through the publication of peer reviewed manuscripts (one manuscript
submitted). In addition, the database of m/z features identified will be made public and accessible to
the analytical sciences community through the http://www.maldi-msi.org/ website.
ii) The pipeline developed for registration of imaging MS datasets with histological images and the
ABA enables facile and rapid inter animal comparisons, quantification of regional biomolecular
content, and in the future correlation of the imaging MS data to the gene-expression image data
provided by the ABA. Furthermore, this pipeline makes possible the comprehensive analysis of
animal cohorts in pre-clinical studies of other neurological disease ssuch as stroke, Parkinson’s,
Alzheimer’s and seizure. (two peer reviewed publications)
iii) The analysis of an animal model of migraine revealed changes in metabolites, peptides and
proteins associated with CSD. Currently, complementary analysis aiming to identify and validate the
biomolecular changes observed by imaging MS are being performed and are expected to bring new
insights into the underlying biochemical mechanisms of CSD. (one manuscript in preparation)
- Socioeconomic impact
i) Migraine has been estimated to be the most costly neurological disorder in the European
Community (> €27 billion p.a.). The methodologies developed and the results obtained from
transgenic models of migraine by imaging MS are being validated by complementary techniques,
such as LC-MS/MS and immunohistochemistry and will help elucidate the molecular mechanism of
migraine susceptibility and thus help identify biomarkers and potential therapeutic targets for
alleviating the consequences of migraine.
ii) The capabilities developed during the grant period have been and are being disseminated through
the imaging MS community. As such the results obtainedt will impact on healthcare and provide
extra impetus to the already significant role biotechnology plays within Europe.
The work developed and the results obtained were disseminated via different activities and
platforms, as described below:
- Peer reviewed manuscripts
1) Walid M. Abdelmoula*, Ricardo J. Carreira*, Reinald Shyti, Benjamin Balluff, René J. M. van
Zeijl, Else Tolner, Boudewijn F.P. Lelieveldt, Arn M.J.M. van den Maagdenberg, Liam A.
McDonnell, Jouke Dijkstra, Automatic Registration of Mass Spectrometry Imaging Datasets to the
Allen Brain Atlas, Analytical Chemistry, 86 (8), 2014, 3947-3954. DOI: 10.1021/ac500148a
*equal contributions
2) Walid M. Abdelmoula, Karolina Škrášková, Benjamin Balluff, Ricardo J. Carreira, Else A.
Tolner, Boudewijn F.P. Lelieveldt, Laurens van der Maaten, Hans Morreau, Arn M.J.M. van den
Maagdenberg, Ron M.A. Heeren, Liam A. McDonnell, Jouke Dijkstra, Automatic Generic
Registration of Mass Spectrometry Imaging Data to Histology using Nonlinear Stochastic
Embedding, Analytical Chemistry, 86 (18), 2014, 9204-9211. DOI: 10.1021/ac502170f
3) Bram Heijs, Ricardo J. Carreira, Else A. Tolner, Arnoud H. de Ru, Arn M.J.M. van den
Maagdenberg, Peter A. van Veelen, Liam A. McDonnell, A comprehensive analysis of the mouse brain
proteome sampled in mass spectrometry imaging, Analytical Chemistry (submitted)
4) Ricardo J. Carreira*, Reinald Shyti*, Walid M. Abdelmoula, Benjamin Balluff, Sandra van
Heiningen, Ludo Broos, René J. M. van Zeil, Jouke Dijkstra, Michel Ferrari, Else Tolner, Liam
McDonnell, Arn M.J.M. van den Maagdenberg, Mass spectrometry imaging investigation of cortical
spreading depression in transgenic models of migraine. (in preparation)
*equal contributions
- Scientific communications (poster and oral)
1) Registration of Mass Spectrometry Imaging datasets to the Allen Brain Atlas. 62nd ASMS
Conference on Mass Spectrometry and Allied Topics, Baltimore (MD), USA (June 2014).
2) On-tissue digestion MALDI MSI analysis of FFPE mouse brain. On-Tissue Digestion Initiative:
Round Robin, Lille, France (June 2014)
3) Mass Spectrometry Imaging and the Allen Brain Atlas. Dutch Society for Mass Spectrometry
meeting, NVMS 2014, Rolduc, Netherlands (April 2014).
4) The effect of heat-induced tissue stabilization on the MS- and histo-architecture of mouse brain. 61st
ASMS Conference on Mass Spectrometry and Allied Topics, Minneapolis (MN), USA (June 2013).
5) The molecular image of migraine revealed through Imaging Mass Spectrometry. Biomedical
Research Centre, Sheffield Hallam University, Sheffield, United Kingdom (May 2013).
6) The effects of heat-induced tissue stabilization. Emerging Technologies and Multimodal Imaging
Workshop, Vienna, Austria (February 2013).
Additionally, the researcher further disseminated the results obtained and work performed during the
period of the grant through lectures in numerous advances courses:
1) Applications of mass spectrometry imaging. Frontiers of Science Course for biomedical students,
Leiden, Netherlands (September 2014).
2) Mass spectrometry imaging in biomarker discovery. Biomolecular Mass Spectrometry Course,
Utrecht, Netherlands (September 2014).
3) Mass spectrometry imaging: integration with the Allen Brain Atlas, 1st EU Course on Imaging Mass
Spectrometry Data Analysis, Sheffield, UK (September 2014)
4) MALDI-MS Imaging of Metabolites. Advanced Course Metabolomics for Microbial Systems Biology
2013, TU Delft, Delft, Netherlands (November 2013).
The researcher was also actively involved in training courses and workshops sponsored by the COST
action BM1104 – Imaging Mass Spectrometry: New Tools For Healthcare Research.
1) On-Tissue Digestion Initiative: Round Robin, Lille, France (Workshop, 2014)
2) Mass spectrometry imaging quantitation workshop. University of Geneva, Geneva, Switzerland.
(Workshop, 2013)
3) Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) for MS-Imaging
analysis of elemental tags in tissue samples. Dr. Norbert Jakubowski’s laboratory; Federal Institute for
Materials Research and Testing (BAM); Berlin, Germany. (Advanced training, 2013)
4) On-tissue enzymatic digestion for protein identification in mouse brain tissue sections by MALDI-Ion
Mobility Mass Spectrometry Imaging. Prof. Malcolm Clench’s laboratory; Sheffield Hallam University;
Sheffield, United Kingdom. (Advanced training, 2013)
5) Emerging Technologies and Multimodal Imaging Workshop, Vienna, Austria (Workshop, 2013)
6) Stabilization of tissues: assessment of effects of heat-induced tissue stabilization. Prof. Per Andrén’s
laboratory; University of Uppsala; Uppsala, Sweden. (Advanced training, 2012)
7) 1st EU-COST Seed Course on Imaging Mass Spectrometry. FOM-AMOLF; Amsterdam, The
Netherlands. (Advanced training, 2012)
4.2
Use and dissemination of foreground
A plan for use and dissemination of foreground (including socio-economic impact and target groups
for the results of the research) shall be established at the end of the project. It should, where
appropriate, be an update of the initial plan in Annex I for use and dissemination of foreground and
be consistent with the report on societal implications on the use and dissemination of foreground
(section 4.3 – H).
The plan should consist of:

Section A
This section should describe the dissemination measures, including any scientific publications
relating to foreground. Its content will be made available in the public domain thus
demonstrating the added-value and positive impact of the project on the European Union.

Section B
This section should specify the exploitable foreground and provide the plans for exploitation. All
these data can be public or confidential; the report must clearly mark non-publishable
(confidential) parts that will be treated as such by the Commission. Information under Section B
that is not marked as confidential will be made available in the public domain thus
demonstrating the added-value and positive impact of the project on the European Union.
Section A (public)
This section includes two templates

Template A1: List of all scientific (peer reviewed) publications relating to the foreground of the project.

Template A2: List of all dissemination activities (publications, conferences, workshops, web sites/applications, press releases, flyers,
articles published in the popular press, videos, media briefings, presentations, exhibitions, thesis, interviews, films, TV clips, posters).
These tables are cumulative, which means that they should always show all publications and activities from the beginning until after the end of
the project. Updates are possible at any time.
TEMPLATE A1: LIST OF SCIENTIFIC (PEER REVIEWED) PUBLICATIONS, STARTING WITH THE MOST IMPORTANT ONES
Title of
the
Main
periodical
author
or the
series
Number, date or
frequency
Place of
publication
Year of
publication
Relevant
pages
Is/Will open
access3
provided to
this
publication?
NO.
Title
1
Automatic Registration of Mass
Spectrometry Imaging Datasets
to the Allen Brain Atlas
Analytical
Chemistry
No 86 (8), 2014
American
Chemical
Society
2014
2
A comprehensive analysis of
the mouse brain proteome
sampled in mass spectrometry
imaging
Mass spectrometry imaging
investigation of cortical
spreading depression in
transgenic models of migraine
Analytical
Chemistry
Submitted for
publication
American
Chemical
Society
Expected
late 2014
yes
JASMS
In Preparation
Springer
Expected
beginning
2015
yes
3
2
Publisher
Permanent
identifiers2
(if available)
3947-3954
DOI:
10.1021/ac500148a
yes
A permanent identifier should be a persistent link to the published version full text if open access or abstract if article is pay per view) or to the final manuscript accepted for publication (link to
article in repository).
3 Open Access is defined as free of charge access for anyone via Internet. Please answer "yes" if the open access to the publication is already established and also if the embargo period for open
access is not yet over but you intend to establish open access afterwards.
4
Registration of Mass
Spectrometry Imaging Data to
Histology using Nonlinear
Stochastic Embedding
Analytical
Chemistry
No 86 (18), 2014
American
Chemical
Society
2014
9204-9211
DOI:
10.1021/ac502170f
yes
TEMPLATE A2: LIST OF DISSEMINATION ACTIVITIES
NO.
Type of activities4
Main
leader
Title
Date/Period
Place
1
Symposium/ workshop
Frontiers of
Science Course
for biomedical
students
September 2014
Leiden
(Netherlands)
2
Symposium/ workshop
Biomolecular
Mass
Spectrometry
Course
September 2014
Utrecht
(Netherlands)
3
Symposium/ workshop
1st EU Course on
Imaging Mass
Spectrometry
Data Analysis
September 2014
Sheffield
(UK)
4
Conference
62nd ASMS
Conference on
Mass
June 2014
Baltimore
(USA)
Type of
audience5
Scientific
Community
(higher
education,
Research)
Scientific
Community
(higher
education,
Research)
Scientific
Community
(higher
education,
Research),
Industry
Scientific
Community
(higher
Countries
addressed
Size of
audience
< 50
International
course
< 50
International
course
< 50
International
course
> 3000
International
conference
4
A drop down list allows choosing the dissemination activity: publications, conferences, workshops, web, press releases, flyers, articles published in the popular press, videos, media
briefings, presentations, exhibitions, thesis, interviews, films, TV clips, posters, Other.
5 A drop down list allows choosing the type of public: Scientific Community (higher education, Research), Industry, Civil Society, Policy makers, Medias, Other ('multiple choices' is
possible).
Spectrometry and
Allied Topics
5
Symposium/ workshop
On-Tissue
Digestion
Initiative: Round
Robin
June 2014
Lille
(France)
6
Conference
April 2014
Rolduc
(Netherlands)
7
Symposium/ workshop
Dutch Society for
Mass
Spectrometry
meeting, NVMS
2014, Rolduc,
Netherlands
Mass
spectrometry
imaging
quantitation
workshop
October 2013
Geneva
(Switzerland)
8
Symposium/ workshop
Advanced Course
Metabolomics for
Microbial
Systems Biology
November 2013
Delft
(Netherlands)
9
Conference
61st ASMS
Conference on
Mass
Spectrometry and
Allied Topics
June 2013
Minneapolis
(USA)
10
Symposium/ workshop
Emerging
Technologies and
Multimodal
Imaging
Workshop
January 2013
Vienna
(Austria)
11
Symposium/ workshop
1st EU-COST
Seed Course on
Imaging Mass
October 2012
Amsterdam
(Netherlands)
education,
Research),
Industry
Scientific
Community
(higher
education,
Research),
Industry
Scientific
Community
(higher
education,
Research),
Industry
Scientific
Community
(higher
education,
Research),
Industry
Scientific
Community
(higher
education,
Research)
Scientific
Community
(higher
education,
Research),
Industry
Scientific
Community
(higher
education,
Research),
Industry
Scientific
Community
(higher
< 50
International
conference
>100
International
conference
< 50
International
course
< 50
International
course
> 3000
International
conference
< 50
International
conference
< 50
International
course
Spectrometry
education,
Research)
Section B (Confidential6 or public: confidential information to be marked clearly)
Part B1
The applications for patents, trademarks, registered designs, etc. shall be listed according to the template B1 provided hereafter.
The list should, specify at least one unique identifier e.g. European Patent application reference. For patent applications, only if applicable,
contributions to standards should be specified. This table is cumulative, which means that it should always show all applications from the
beginning until after the end of the project.
TEMPLATE B1: LIST OF APPLICATIONS FOR PATENTS, TRADEMARKS, REGISTERED DESIGNS, ETC.
Confidential
Click on
YES/NO
Type of IP
Rights7:
Foreseen
embargo date
dd/mm/yyyy
Application
reference(s)
(e.g. EP123456)
Subject or title of application
6
Note to be confused with the "EU CONFIDENTIAL" classification for some security research projects.
7
A drop down list allows choosing the type of IP rights: Patents, Trademarks, Registered designs, Utility models, Others.
Applicant (s) (as on the application)
Part B2
Please complete the table hereafter:
Type of
Exploitable
Foreground8
Description
of
exploitable
foreground
Confidential
Click on
YES/NO
AUTOMATIC
YES
ALIGNMENT
OF
HISTOLOGIC
AL IMAGES
TO MSI
DATA
Foreseen
embargo
date
dd/mm/yyyy
Exploitable
product(s) or
measure(s)
Sector(s) of
application9
Timetable,
commercial or
any other use
IMAGE ANALYSIS
BIOMEDICAL
2016
Patents or
other IPR
exploitation
(licences)
NO
Owner & Other
Beneficiary(s)
involved
LUMC
SOFTWARE
In addition to the table, please provide a text to explain the exploitable foreground, in particular:





Its purpose
How the foreground might be exploited, when and by whom
IPR exploitable measures taken or intended
Further research necessary, if any
Potential/expected impact (quantify where possible)
19
A drop down list allows choosing the type of foreground: General advancement of knowledge, Commercial exploitation of R&D results, Exploitation of R&D results via standards,
exploitation of results through EU policies, exploitation of results through (social) innovation.
9 A drop down list allows choosing the type sector (NACE nomenclature) : http://ec.europa.eu/competition/mergers/cases/index/nace_all.html
4.3
Report on societal implications
Replies to the following questions will assist the Commission to obtain statistics and
indicators on societal and socio-economic issues addressed by projects. The questions are
arranged in a number of key themes. As well as producing certain statistics, the replies will
also help identify those projects that have shown a real engagement with wider societal issues,
and thereby identify interesting approaches to these issues and best practices. The replies for
individual projects will not be made public.
A
General Information (completed automatically when Grant Agreement number is
entered.
Grant Agreement Number:
Title of Project:
Name and Title of Coordinator:
B
303344
Explicitly Normalized Imaging Mass Spectrometry
Dr. Liam A. McDonnell
Ethics
1. Did your project undergo an Ethics Review (and/or Screening)?
*
If Yes: have you described the progress of compliance with the relevant Ethics
Review/Screening Requirements in the frame of the periodic/final project reports?
No
Special Reminder: the progress of compliance with the Ethics Review/Screening Requirements should be
described in the Period/Final Project Reports under the Section 3.2.2 'Work Progress and Achievements'
2.
Please indicate whether your project involved any of the following issues (tick
box) :
RESEARCH ON HUMANS
*
Did the project involve children?
* Did the project involve patients?
* Did the project involve persons not able to give consent?
* Did the project involve adult healthy volunteers?
* Did the project involve Human genetic material?
 Did the project involve Human biological samples?
 Did the project involve Human data collection?
RESEARCH ON HUMAN EMBRYO/FOETUS
*
Did the project involve Human Embryos?
*
Did the project involve Human Foetal Tissue / Cells?
*
Did the project involve Human Embryonic Stem Cells (hESCs)?
*
Did the project on human Embryonic Stem Cells involve cells in culture?
*
Did the project on human Embryonic Stem Cells involve the derivation of cells from Embryos?
PRIVACY
* Did the project involve processing of genetic information or personal data (eg. health, sexual
lifestyle, ethnicity, political opinion, religious or philosophical conviction)?
* Did the project involve tracking the location or observation of people?
RESEARCH ON ANIMALS
* Did the project involve research on animals?
* Were those animals transgenic small laboratory animals?
* Were those animals transgenic farm animals?
* Were those animals cloned farm animals?
x
x
* Were those animals non-human primates?
RESEARCH INVOLVING DEVELOPING COUNTRIES
* Did the project involve the use of local resources (genetic, animal, plant etc)?
* Was the project of benefit to local community (capacity building, access to healthcare, education
etc)?
DUAL USE
 Research having direct military use
* Research having the potential for terrorist abuse
No
No
C
Workforce Statistics
3.
Workforce statistics for the project: Please indicate in the table below the number of
people who worked on the project (on a headcount basis).
Type of Position
Scientific Coordinator
Work package leaders
Experienced researchers (i.e. PhD holders)
PhD Students
Other
4.
Number of Women
1
1
How many additional researchers (in companies and universities) were
recruited specifically for this project?
Of which, indicate the number of men:
Number of Men
3
2
0
D Gender Aspects
5.

x
Did you carry out specific Gender Equality Actions under the project?
6.
Yes
No
Which of the following actions did you carry out and how effective were they?
Not at all
effective





Design and implement an equal opportunity policy
Set targets to achieve a gender balance in the workforce
Organise conferences and workshops on gender
Actions to improve work-life balance




Very
effective




Other:
Was there a gender dimension associated with the research content – i.e. wherever people were
7.
the focus of the research as, for example, consumers, users, patients or in trials, was the issue of gender
considered and addressed?
 Yes- please specify
x
No
E
Synergies with Science Education
8.
Did your project involve working with students and/or school pupils (e.g. open days,
participation in science festivals and events, prizes/competitions or joint projects)?
Yes- please specify
Lectures in advanced courses
x

9.
No
Did the project generate any science education material (e.g. kits, websites, explanatory
booklets, DVDs)?
 Yes- please specify
x
No
F
Interdisciplinarity
10.
Which disciplines (see list below) are involved in your project?
 Main discipline10: 1.3

Associated discipline10: 1.1
 Associated discipline10: 1.5
G
Engaging with Civil society and policy makers
11a
Did your project engage with societal actors beyond the research
community? (if 'No', go to Question 14)

x
Yes
No
11b If yes, did you engage with citizens (citizens' panels / juries) or organised civil society
(NGOs, patients' groups etc.)?
No
x
 Yes- in determining what research should be performed
 Yes - in implementing the research
 Yes, in communicating /disseminating / using the results of the project
10
Insert number from list below (Frascati Manual).

Yes
11c In doing so, did your project involve actors whose role is mainly to
x
No
organise the dialogue with citizens and organised civil society (e.g.
professional mediator; communication company, science museums)?
12. Did you engage with government / public bodies or policy makers (including international
organisations)
x



No
Yes- in framing the research agenda
Yes - in implementing the research agenda
Yes, in communicating /disseminating / using the results of the project
13a Will the project generate outputs (expertise or scientific advice) which could be used by
policy makers?
 Yes – as a primary objective (please indicate areas below- multiple answers possible)
Yes – as a secondary objective (please indicate areas below - multiple answer possible)
x
 No
13b If Yes, in which fields?
Agriculture
Audiovisual and Media
Budget
Competition
Consumers
Culture
Customs
Development Economic and
Monetary Affairs
Education, Training, Youth
Employment and Social Affairs
Energy
Enlargement
Enterprise
Environment
External Relations
External Trade
Fisheries and Maritime Affairs
Food Safety
Foreign and Security Policy
Fraud
Humanitarian aid
Human rights
Information Society
Institutional affairs
Internal Market
Justice, freedom and security
Public Health
Regional Policy
Research and Innovation
Space
Taxation
Transport
X
13c If Yes, at which level?
 Local / regional levels
National level
x
 European level
International level
x
H
Use and dissemination
14.
How many Articles were published/accepted for publication in
peer-reviewed journals?
To how many of these is open access11 provided?
2
2
How many of these are published in open access journals?
How many of these are published in open repositories?
2
To how many of these is open access not provided?
0
Please check all applicable reasons for not providing open access:
 publisher's licensing agreement would not permit publishing in a repository
 no suitable repository available
 no suitable open access journal available
 no funds available to publish in an open access journal
 lack of time and resources
 lack of information on open access
 other12: ……………
How many new patent applications (‘priority filings’) have been made?
15.
0
("Technologically unique": multiple applications for the same invention in different
jurisdictions should be counted as just one application of grant).
16.
17.
Indicate how many of the following Intellectual
Property Rights were applied for (give number in
each box).
Trademark
0
Registered design
0
Other
0
How many spin-off companies were created / are planned as a direct
result of the project?
0
Indicate the approximate number of additional jobs in these companies:
18. Please indicate whether your project has a potential impact on employment, in comparison
with the situation before your project:

In small & medium-sized enterprises
 Increase in employment, or

Safeguard
employment,
or
In large companies

x
None of the above / not relevant to the project
 Decrease in employment,
 Difficult to estimate / not possible to quantify
Indicate figure:
19. For your project partnership please estimate the employment effect
resulting directly from your participation in Full Time Equivalent (FTE =
one person working fulltime for a year) jobs:
11
Open Access is defined as free of charge access for anyone via Internet.
12
For instance: classification for security project.
x
Difficult to estimate / not possible to quantify
I
Media and Communication to the general public
20.
As part of the project, were any of the beneficiaries professionals in communication or
media relations?
No
 Yes
x
21.
As part of the project, have any beneficiaries received professional media / communication
training / advice to improve communication with the general public?
No
 Yes
x
22
Which of the following have been used to communicate information about your project to
the general public, or have resulted from your project?

Coverage in specialist press
 Press Release

Coverage in general (non-specialist) press
 Media briefing

TV
coverage
/
report
Coverage in national press


Coverage in international press
 Radio coverage / report

Brochures /posters / flyers
Website for the general public / internet
x
DVD
/Film
/Multimedia
x
Event targeting general public (festival, conference,

exhibition, science café)
23
In which languages are the information products for the general public produced?


Language of the coordinator
Other language(s)
x
English
Question F-10: Classification of Scientific Disciplines according to the Frascati Manual 2002 (Proposed
Standard Practice for Surveys on Research and Experimental Development, OECD 2002):
FIELDS OF SCIENCE AND TECHNOLOGY
1.
1.1
1.2
1.3
1.4
1.5
2
2.1
2.2
2.3.
NATURAL SCIENCES
Mathematics and computer sciences [mathematics and other allied fields: computer sciences and other
allied subjects (software development only; hardware development should be classified in the
engineering fields)]
Physical sciences (astronomy and space sciences, physics and other allied subjects)
Chemical sciences (chemistry, other allied subjects)
Earth and related environmental sciences (geology, geophysics, mineralogy, physical geography and
other geosciences, meteorology and other atmospheric sciences including climatic research,
oceanography, vulcanology, palaeoecology, other allied sciences)
Biological sciences (biology, botany, bacteriology, microbiology, zoology, entomology, genetics,
biochemistry, biophysics, other allied sciences, excluding clinical and veterinary sciences)
ENGINEERING AND TECHNOLOGY
Civil engineering (architecture engineering, building science and engineering, construction engineering,
municipal and structural engineering and other allied subjects)
Electrical engineering, electronics [electrical engineering, electronics, communication engineering and
systems, computer engineering (hardware only) and other allied subjects]
Other engineering sciences (such as chemical, aeronautical and space, mechanical, metallurgical and
materials engineering, and their specialised subdivisions; forest products; applied sciences such as
geodesy, industrial chemistry, etc.; the science and technology of food production; specialised
technologies of interdisciplinary fields, e.g. systems analysis, metallurgy, mining, textile technology
and other applied subjects)
3.
3.1
3.2
3.3
4.
4.1
4.2
MEDICAL SCIENCES
Basic medicine (anatomy, cytology, physiology, genetics, pharmacy, pharmacology, toxicology,
immunology and immunohaematology, clinical chemistry, clinical microbiology, pathology)
Clinical medicine (anaesthesiology, paediatrics, obstetrics and gynaecology, internal medicine, surgery,
dentistry, neurology, psychiatry, radiology, therapeutics, otorhinolaryngology, ophthalmology)
Health sciences (public health services, social medicine, hygiene, nursing, epidemiology)
AGRICULTURAL SCIENCES
Agriculture, forestry, fisheries and allied sciences (agronomy, animal husbandry, fisheries, forestry,
horticulture, other allied subjects)
Veterinary medicine
5.
5.1
5.2
5.3
5.4
SOCIAL SCIENCES
Psychology
Economics
Educational sciences (education and training and other allied subjects)
Other social sciences [anthropology (social and cultural) and ethnology, demography, geography
(human, economic and social), town and country planning, management, law, linguistics, political
sciences, sociology, organisation and methods, miscellaneous social sciences and interdisciplinary ,
methodological and historical S1T activities relating to subjects in this group. Physical anthropology,
physical geography and psychophysiology should normally be classified with the natural sciences].
6.
6.1
HUMANITIES
History (history, prehistory and history, together with auxiliary historical disciplines such as
archaeology, numismatics, palaeography, genealogy, etc.)
Languages and literature (ancient and modern)
Other humanities [philosophy (including the history of science and technology) arts, history of art, art
criticism, painting, sculpture, musicology, dramatic art excluding artistic "research" of any kind,
religion, theology, other fields and subjects pertaining to the humanities, methodological, historical and
other S1T activities relating to the subjects in this group]
6.2
6.3
2.
FINAL REPORT ON THE DISTRIBUTION OF THE
EUROPEAN UNION FINANCIAL CONTRIBUTION
This report shall be submitted to the Commission within 30 days after receipt of the final
payment of the European Union financial contribution.
Report on the distribution of the European Union financial contribution
between beneficiaries
Name of beneficiary
1.
2.
n
Total
Final amount of EU
beneficiary in Euros
contribution
per