Iris Brémaud

Elham Karami

Sandrine Bardet

Nicolas Gilles

François Perego

Samad Zare

Joseph Gril

Changes in vibrational properties of coated wood through time from application of varnish, with recipes used in European or Iranian string instruments making

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Surface finish is usually one of the last steps in instrument making. Depending on types of instruments, as well as on cultures, wood finish can consist of different processes. In stringed instruments, in many cases, wood is coated, which can be done with different products, including a wide variety of varnishes. Although the primary goal is probably more related to protection and/or aesthetics, varnishes can also affect the vibrational properties. The acoustical effects of several violin-making varnishes has been analysed regarding their significances in regard to instrument behaviour [START_REF] Schelleng | Acoustical effects of violin varnish[END_REF]] and to the diversity of ingredients and combinations found in historical recipes [START_REF] Schleske | On the acoustical properties of violin varnish[END_REF]]. These studies noticed the evolution through time (years) after varnish application, however the ambient humidity conditions were not regulated, although they have a strong influence [START_REF] Brémaud | Effect of transitional moisture change on the vibrational properties of violin-making wood[END_REF], and/or the steps or measurement were not very detailed over time. Fine kinetics of changes in vibrational properties were analysed in the case of some more industrially used varnishes [START_REF] Minato | Dependence of vibrational properties of wood on varnishing during its drying process in Violin manufacturing[END_REF] or in the case of the oriental laquer Urushi [START_REF] Obataya | Effects of oriental lacquer (urushi) coating on the vibrational properties of wood used for the soundboards of musical instruments[END_REF][START_REF] Brémaud | Effects of aging and moisture on the dynamic viscoelastic properties of Oriental Lacquer (Urushi) film[END_REF]. However, different types of coatings or varnishes, due to their different chemical nature, should show different effects on the wood+coating properties [START_REF] Simonnet | Physical and chemical properties of varnishes and their vibrational consequences[END_REF], as well as different kinetics of changes during time after application. These changes through time are possibly related to the opin-ions about changing response of newly built instruments. The objective of the present work is to analyse the detailed kinetics of these changes, starting from the different steps of the varnishing procedure, and followed over long times in controlled climatic conditions. The study includes violinmaking as well as Iranian instruments making varnishing processes.

Materials and Methods

Studied material included wood+coating systems that are representative of European, and Iranian, traditions of instrument making. The wood species were White Mulberry (Morus alba) and Spruce (Picea abies). Material for both species was selected as high-grade for instrument making. 184 specimens (for Spruce) and 96 (for Mulberry) specimens were prepared, in the longitudinal (L) and radial (R) directions of wood. After measurements of the untreated physical and vibrational properties, they were distributed into matched groups of (6L+6R) specimens each through a careful statistical procedure. Each group is intended for a different modality of the varnishing process: 12+10 groups for Spruce+Violin making varnishes, 5+3 groups for Mulberry+Iranian long-necked lutes making varnishes. Vibrational properties were measured by non-contact forced vibrations of free-free bars, using the device and protocol described in [START_REF] Brémaud | Diversity of woods used or usable in musical instruments making[END_REF][START_REF] Brémaud | Characterisation and categorisation of the diversity in viscoelastic vibrational properties between 98 wood types[END_REF]. They included longitudinal and radial specific modulus of elasticity (E'/γ, proportional to resonance frequencies) and damping coefficient (tanδ, related to vibration decay and to the sharpness of resonances also called quality factor). Tested varnishing procedures and recipes were as follow: -Spruce was coated according to varnishing procedures used in violin making. 3 kinds of ground layers were tested. 5 recipes of siccative-oil based varnishes were applied, using different oils and resins and proportions. All the steps of application and curing (by UV-light) were done inside a climateregulated room. The changes in mass and in vibrational properties were monitored during all steps, and then followed during more than 12 months after application (still in progress).

-Mulberry was coated by alcohol-based sandarac varnish, following the same step-by-step monitoring through time, in regulated conditions, as above. Solvent alone, and 1, 3, 6 varnish layers were tested. Monitoring of changes in properties was conducted for about 8 months.

-An additional study on Mulberry was based on actual application by a skilled Tar and Setar maker of 3 different varnishes (2 alcohol-based: Sandarac and Shellac, and 1 artificial: polyester). Application was conducted in the workshop, then properties were measured in lab-conditions after 2.5 and 7 months.

Results and discussion

After applying on mulberry wood a single layer of Sandarac/ethanol varnish, the resonance frequency was barely affected in L direction but clearly in-Iris Brémaud, Elham Karami, Sandrine Bardet, Nicolas Gilles, François Perego, Samad Zare, Joseph Gril creased in R, and damping was increased by up to 50% in both R and L (Figure 1). Ethanol solvent alone had very limited effect. With 6 layers (similar to actual instrument making), immediate effects were an increase in frequency in R (+6%) and very strong increase in damping (+100 in L to +160% in R). Over time, frequency continued to increase while damping redecreased, starting to stabilize only after 2 months. Sandarac-ethanol varnish appears to have, when stabilized or "dry" -which took about 7monthsa stiffening effect with nearly no final increase in damping. Workshop application by a skilled maker gave similar results for Sandarac and for Shellac, while polyester proved to induce a more permanent increase in vibration damping.

The effects of application on spruce wood of violin-making varnishes is illustrated, in Figure 2, by the example of a linseed-oil + Sandarac resin varnish.

The vibrational properties changed little just after application, when varnish is still quite liquid. After UV-accelerated curing, damping is very strongly increased (+40% to +80% in radial direction) for a single layer of varnish applied. On Spruce, the differential of effects between along the grain and cross-grain is much more pronounced than on Mulberry. The protocol we designed allows to separate the effects of varnish itself from effects of moisture changes (due to UV or to slight de-regulations of climatic room). One thick layer of varnish evolves much more slowly than a thin one. Changes in damping are only partially related to added mass of varnish: 2 thin layers result in more added mass than 1 thick layer, but has similar effects on vibration damping. The kinetics of changes in properties after coat- ing with siccative-oil based varnish is obviously much slower than the previous example on alcohol-based varnish. With this example of "fatty" varnish, the properties continue to evolve, without yet clear signs of stabilization, after one year from initial varnishing. The monitoring of changes in properties through time is intended to be continued for several years. To complete the example described here, the presentation will compare the same protocol and obtained results with 5 different kinds of "siccative oil + resins" violin-making varnishes.

Figure 1 :

 1 Figure 1: Changes trough time (7months) of vibration damping of mulberry wood varnished with Sandarac dissolved in ethanol. 1 data point = mean of 6 specimens, Time axis in Log-scale (0=1day, 1=10days, 2=100days).


Stéphane Roche

Laurent Tiers

Monique Provansal

Martial Seveno d Marie-Thérèse Piva

Patrick Jouin

Sylvain Lehmann
email: sylvain.lehmann@igh.cnrs.fr

Depletion of one, six, twelve or twenty major blood proteins before proteomic analysis: The more the better?

Keywords:

Depletion of major blood proteins is one of the most promising approaches to access low abundant biomarkers using proteomics. Immunocapture columns often used for this purpose exist in different formats depending on the number of major proteins removed. In this article, we compared the relative interest of depleting either one (albumin), six (albumin, IgG, IgA, transferrin, α1-antitrypsin, and haptoglobin), twelve (the previous six and apo A-I and -II, orosomucoid, α2-macroglobulin, fibrinogen, IgM) or twenty blood proteins (the previous twelve and IgD, ceruloplasmin, apo B, complement C1q, C3, C4, plasminogen, and prealbumin). Such study raises interesting issues related to the reproducibility, practicability, specificity of the immunocapture, and to the impact of removing not only the selected molecules, but also associated peptides and proteins. Depleted sera were here analysed using different proteomic approaches, including two dimensional electrophoresis and SELDI-TOF. Altogether, our results clearly confirmed the interest of depleting major blood proteins for the proteomic detection of low abundant components. However, we observed that increasing the number of depleted proteins from twelve to twenty had a limited beneficial impact and might increase drawbacks in removing associated peptides and proteins. This conclusion is however related to the technologies that we have used, and we believe that it is necessary to adapt the immunocapture to the analytical method employed, and to the ratio between wanted and unwanted proteins removed.

Introduction

Proteomic analysis of serum and plasma is of major interest for the identification and the detection of new clinical bio-markers. Many studies are focusing on these fluids to detect the presence of peptides and proteins which changes in concentration or post-translational modification are linked to a particular disease state. However, the proteomic analysis of these blood fractions is analytically challenging due to the high dynamic concentration range (over 10 orders of magnitude) of constituent protein/peptide species [START_REF] Anderson | The human plasma proteome: history, character, and diagnostic prospects[END_REF]. Difficulties are even increased by the fact that the most abundant proteins that constitute 95% of the bulk mass of proteins represent less than 0.1% of the total number of proteins. Serum and plasma have a very high concentration of proteins, typically in the range of 50-80 g/L and the abundant proteins like albumin produce in most proteomic approaches large signals which mask or interfere with the detection of low amount protein components.

To minimize these problems, proteomic techniques are constantly improving to access to a wider range of low concentration candidates [START_REF] Rai | Analysis of Human Proteome Organization Plasma Proteome Project (HUPO PPP) reference specimens using surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry: multi-institution correlation of spectra and identification of biomarkers[END_REF][START_REF] States | Challenges in deriving high-confidence protein identifications from data gathered by a HUPO plasma proteome collaborative study[END_REF]. Many methods rely on a multidimensional separation scheme combining for example multidimensional chromatography and electrophoresis or mass spectrometry (MS) methods [START_REF] Pieper | The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins[END_REF][START_REF] Yang | A study of glycoproteins in human serum and plasma reference standards (HUPO) using multilectin affinity chromatography coupled with RPLC-MS/MS[END_REF]. This is the case of the Surface-Enhanced Laser Desorption/Ionization Time-of-flight (SELDI-TOF) method [START_REF] Clarke | SELDI-TOF-MS proteomics of breast cancer[END_REF][START_REF] Tang | Current developments in SELDI affinity technology[END_REF] that relies on MS to detect proteins and peptides initially retained by binding to various chromatographic surfaces (anionic, cationic, IMAC, hydrophobic).

When dealing with complex biological fluid like blood, most available proteomic technologies will however benefit from the preanalytical use of various pre-fractionation techniques [START_REF] Issaq | Methods for fractionation, separation and profiling of proteins and peptides[END_REF]. They are represented for example by liquid chromatography [START_REF] Guerrier | A simplified monobuffer multidimensional chromatography for high-throughput proteome fractionation[END_REF], binding to solid-phase libraries [START_REF] Guerrier | Reducing protein concentration range of biological samples using solid-phase ligand libraries[END_REF][START_REF] Thulasiraman | Reduction of the concentration difference of proteins in biological liquids using a library of combinatorial ligands[END_REF], enrichment of low molecular weight proteins [START_REF] Orvisky | Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma[END_REF] or the prior removal of major proteins. The latter is often achieved using immobilized dye [START_REF] Leatherbarrow | Studies on the mechanism of binding of serum albumins to immobilized cibacron blue F3G A[END_REF][START_REF] Birch | Enrichment of Escherichia coli proteins by column chromatography on reactive dye columns[END_REF] or immunoaffinity [START_REF] Maccarrone | Mining the human cerebrospinal fluid proteome by immunodepletion and shotgun mass spectrometry[END_REF][START_REF] Bjorhall | Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples[END_REF][START_REF] Roche | Interest of major serum protein removal for Surface-Enhanced Laser Desorption/Ionization-Time Of Flight (SELDI-TOF) proteomic blood profiling[END_REF]. Ideally, a depletion technique should be selective and remove 100% of the targeted protein (or proteins) without retaining non-targeted proteins. A number of commercial products have been designed for the depletion of several proteins from human or mammals serum and plasma. Most of the products are based upon specific antibodies to the high-abundance proteins present in these fluids. In theory, to access to low abundant biomarkers, the more high-abundance proteins that can be removed or depleted in a single experiment, the better. This general assumption is based at the peptide level diversity by LC-MS/ MS analysis of the tryptic digest of plasma proteins after 6 vs. 12 protein immunoaffinity subtraction [START_REF] Liu | Evaluation of multiprotein immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry[END_REF], and in the case of cerebral spinal fluid using plasma-designed 6 vs. 20 different antibody sites on a single column [START_REF] Thouvenot | Enhanced detection of CNS cell secretome in plasma protein-depleted cerebrospinal fluid[END_REF]. Nevertheless, issues like reproducibility, practicability, specificity of the immunocapture, impact of removing not only the selected molecules, but also peptides and proteins associated with them, need to be carefully taken into account. In this manuscript we described for the first time in parallel, the comparison of different commercial columns capturing either one (albumin), six (albumin, IgG, IgA, transferrin, α1-antitrypsin, haptoglobin), twelve (the previous six and apo A-I and-II, orosomucoid, α2-macroglobulin, fibrinogen, IgM) or twenty blood proteins (the previous twelve and IgD, ceruloplasmin, apo B, complement C1q, C3, C4, plasminogen, and prealbumin). Depleted sera were analysed using different proteomic approaches including two dimensional electrophoresis and SELDI-TOF. Altogether, our results clearly confirmed the utility of depleting major blood proteins for the proteomic detection of low abundant components. However, we observed that increasing the number of depleted proteins from twelve to twenty had a limited beneficial impact and might increase drawbacks in removing associated peptides and proteins.

2.

Materials and methods ). Fractionation of serum proteins was performed as described by the manufacturers using buffers provided with the columns (Table 1). Briefly, serum was mixed in dilution buffer, filtered at 0.22 µm, added to immunoaffinity microbead spin column and incubated at room temperature. The unbound proteins were then collected by centrifugation. After washes with the dilution buffer, bound proteins were eluted in one or two steps with the stripping buffer. After depletion, the unbound proteins were concentrated and submitted to a buffer exchange with 50 mM Tris pH 8.8 at 4 °C by a 45 min centrifugation on PES ultrafiltration columns with a 3 kDa cut-off (Vivaspin 500, Vivascience ref VS0192) for SELDI-TOF and 10 kDa cut-off (Vivaspin 500, Vivascience) for electrophoresis. Protein quantitation was performed using BCA kit (Sigma Aldrich). Both the standard curve and the samples before and after immunodepletion were diluted in 50 mM Tris pH 8.8 prior the analysis.

SDS-PAGE electrophoresis

NuPAGE Electrophoresis system (Invitrogen) was used. Briefly, following the manufacturer procedure, 60 µg of protein in 30 µl of LDS buffer 1X were loaded on 4-12% gradient gel. The running buffer used was MOPS and the migration was performed under 100 V current during 1 h. Proteins were stained by colloidal Coomassie following manufacturer procedure (GelCode, Fermentas) and the gels were scanned using an Epson 1680 scanner.

2D electrophoresis

Samples were recovered in 200 µl of solubilising buffer (8 M urea, 1 M thiourea, 4.8% CHAPS, 50 mM DTT). For the first dimension, 50 µg of proteins were diluted in 125 µl of rehydratation buffer (9.8 M urea 4% CHAPS 50 mM DTT and 0.5% IPG buffer 4-7). IPG strips (Amersham ref 17-6001-10), 7 cm, covering a pH range of 3-10 were rehydrated with this solution during 18 h under low viscosity paraffin oil. For focalisation, the following voltage/time profile was used on a IPGPhor II: 300 V for 2 h, a gradient until 1000 V for 1 h, a gradient until 5000 V for 1 h 30 and 5000 V during 3 h 30. A total of 23250 Vh was obtained. For the second dimension, strips were equilibrated for 30 min in 6 M urea, 30% glycerol, 2% SDS, 50 mM Tris pH 8.8, 1% DTT and then incubated for an additional 30 min in the same solution except that DTT was replaced by 5% iodoacetamide. After equilibration, proteins were separated in the second dimension in 4-12% NuPAGE gels (Invitrogen). Gels were stained with a silver nitrate procedure [START_REF] Shevchenko | Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels[END_REF][START_REF] Roche | Comparative proteomic analysis of human mesenchymal and embryonic stem cells: towards the definition of a mesenchymal stem cell proteomic signature[END_REF] and scanned at 300 dots/in. using the Labscan 3 software (GE Healthcare) after a procedure of calibration using the kaleidoscope LaserSoft Imaging (Kodak Ref: R020123).

SELDI-TOF analysis

Each sample was 1.5 times with a solution of 8 M Urea, 1% CHAPS and shaken 15 min at room temperature. Denaturated samples equivalent to 40 µg of protein were then mixed with 200 µl of array specific binding buffer.

The following binding buffers were used for washing and protein binding on the arrays: weak cationic exchange (CM10) with 100 mM Ammonium Acetate pH 4, 0.1% Triton and strong anion exchange array (Q10) with 100 mM Tris, 0.1% Triton pH 9.0. ProteinChips arrays were pre-equilibrated with 150 µl of binding buffer in a 96 wells bioprocessor and incubated 5 min with gentle agitation. After removing binding buffers from the wells, denaturated samples were added and incubated for 1 h on a plate shaker at room temperature. The wells were washed two times with the binding buffer and one time with the binding buffer minus triton during 5 min, followed by a final brief rinse with water. ProteinChip arrays were removed from the bioprocessor and air-dried. Finally, 0.8 µl of saturated sinapinic acid solution was applied twice to each spot and the chips were allowed to air-dry again. Mass spectrometric analysis was performed by SELDI-TOF mass spectrometry in a PBS-IIc Protein-Chip reader (Bio-Rad). All data collection settings were similar (calibration, focusing mass, laser intensity and detector sensitivity). Each spectrum was the result of an average of at least 100 laser shots and was externally calibrated with the All-in-1 Protein Standard II (Bio-Rad). Spectra analysis was carried out using the ProteinChip software version 3.2 (Bio-Rad). The background was subtracted using the default software settings. Peaks with a ratio signal/noise above 3 were identified by the ProteinChip Software. After normalization on Total Ion Current (TIC) and quantification, the data were exported to Hierarchical Clustering Explorer Software (HCE v3 [START_REF] Seo | Interactively optimizing signal-to-noise ratios in expression profiling: project-specific algorithm selection and detection p-value weighting in Affymetrix microarrays[END_REF]). Clusters were obtained using a Manhattan Distance test.

Protein identification by MALDI-TOF

Protein spots were excised from 1D gels and were washed in 15 µl of 100 mM NH 4 HC0 3 during 10 min. After addition and incubation with 15 µl of acetonitrile (ACN) during 10 min, supernatants were removed and the procedure repeated. After speedvac treatment, spots were re-hydrated in 10 µl of trypsin solution (15 ng/µl, Promega) and digested overnight at 25 °C in 10 µl of 100 mM NH 4 HC0 3 5 mM CaCl 2 buffer. The tryptic peptides were extracted in a two-step procedure: the first step was composed of addition of 10 µl of 100 mM NH 4 HC0 3 and 10 µl of ACN for 10 min. This step was repeated twice and collected supernatants were pooled. The second step was represented by an incubation of 10 min with 10 µl of 5% formic acid and an addition of 10 µl of ACN for 10 min. This step was repeated twice and the two supernatants were also pooled. After complete drying, the pellet was resuspended in 10% formic acid and was desalted by using Millipore ZipTip C18 columns (Milipore, Bedford, MA).

Mass of peptides was determined in the positive-ion reflector mode of an UltraFlex I MALDI-TOF-TOF mass spectrometer (Bruker Daltonics). Peptide mass fingerprint identification of proteins was performed by searching against the HUMAN entries of databases (SwissProt and TrEMBL) and by Fig. 1 -Comparison of the initial serum and the unbound fractions collected after depletion of 1, 6, 12 and 20 major blood proteins using commercial columns (Table 1). Panel A: The protein concentration (g/L) of the samples was measured with the BCA kit (Sigma) after buffer exchange. Results were obtained from 4 independent depletion experiments. The curve with the diamonds represents the theoretical amount of proteins remaining after depletion of the corresponding molecules. Statistical analysis were performed using the unpaired t test and were considered significant for a p value of <0.05. Panel B: Coomassie stained SDS-PAGE gel of the samples ran in duplicate. A fixed amount of protein (60 µg) was loaded per lane for all the samples. This amount corresponded to 1 µl of the initial serum and 3.2 µl, 5 µl, 7.3 µl and 7.5 µl of serum equivalent for the column removing 1, 6, 12 and 20 proteins, respectively. The numbered arrows on the left indicated the bands that were selected for protein identification (Sup Table 1). Stars and crosses indicated bands that were specifically discussed in the text.

Fig. 2 -Silver stained 2D electrophoresis gels of the initial serum and the unbound fractions collected after depletion of 1, 6, 12 and 20 major blood proteins using commercial columns (Table 1). A fixed amount of protein (50 µg) was analysed for all the samples. This amount corresponded to 0.8 µl of the initial serum and 2.7 µl, 4.2 µl, 6.1 µl and 6.3 µl of serum equivalent for the column removing 1, 6, 12 and 20 proteins, respectively. 2D electrophoresis was performed form pH3 to pH10 in the horizontal dimension and from 150 kDa to 15 kDa in the vertical dimension. To illustrate the disappearance of series of proteins upon depletion, some spots were labeled based on our own proteomic identification (not shown) confirmed by data of the literature.

using the Mascot V 2.2 algorithm with one missing trypsin cleavage site and a mass tolerance of 50 ppm.

Results and discussion

3.1.

Quantification of unbound proteins

The outcome of the capture was first controlled and quantified by evaluating the percentage of proteins recov-ered from each column (Fig. 1A). After immunocapture of 1, 6, 12 or 20 proteins (Table 1), unbound proteins represented respectively: 30.97% ±0.35, 19.93% ± 3.48, 13.65% ± 2.62 and 13.28% ± 1.26 of the initial protein content. Reproducibility and recovery were in the range of what have been obtained before with the different columns [START_REF] Bjorhall | Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples[END_REF][START_REF] Roche | Interest of major serum protein removal for Surface-Enhanced Laser Desorption/Ionization-Time Of Flight (SELDI-TOF) proteomic blood profiling[END_REF][START_REF] Fu | A robust, streamlined, and reproducible method for proteomic analysis of serum by delipidation, albumin and IgG depletion, and two-dimensional gel electrophoresis[END_REF][START_REF] Steel | Efficient and specific removal of albumin from human serum samples[END_REF][START_REF] Huang | Immunoaffinity separation of plasma proteins by IgY microbeads: meeting the needs of proteomic sample preparation and analysis[END_REF]. The decrease in protein content followed the expected theoretical removal of proteins based on the normal serum protein composition (diamond curve, Fig. 1A). Statistical analysis confirmed significant differences in recovery between the columns, for all but the 12 and 20 columns. When compared to the 12 column, the 20 column removed 8 additional proteins (IgD, ceruloplasmin, apolipoprotein B, complement C1q, C3, C4, plasminogen, and prealbumin) (Table 1). It is important to note that all these proteins represent only a minor fraction of the total serum protein content. This explains why the difference between the amounts of unbound protein was not significantly different between the two columns [START_REF] Roche | Clinical proteomics of the cerebrospinal fluid: towards the discovery of new biomarkers[END_REF]. Nevertheless, the higher diminution of the protein diversity brought out by the column removing 20 proteins could be interesting for further proteomic analyses.

1D and 2D electrophoresis protein profiles

The efficiency and the reproducibility of the depletion were also evaluated by the duplicate analysis of the unbound protein Fig. 3 -SELDI-TOF analysis. Panel A: SELDI-TOF spectra (between 2500 and 15,000 m/z) of the initial serum and the unbound fractions collected after depletion of 1, 6, 12 and 20 major blood proteins using commercial columns (Table 1). CM10 and Q10 proteinchips corresponding to anionic and cationic chromatographic surfaces were used. A fixed amount of protein (40 µg) was analysed for all the samples. This amount corresponded to 0.7 µl of the initial serum and 2.1 µl, 3.3 µl, 4.9 µl and 5.0 µl of serum equivalent for the column removing 1, 6, 12 and 20 proteins, respectively. Stars and diamonds indicated peaks which decreased or emerged along with the depletion. Panel B: Bar graph of the number of peaks with signal/noise ratio above 3 detected in the CM10 condition of the panel A. The data were compiled from 4 independent experiments. Statistical analysis were performed using the unpaired t test and were considered significant for a p value of <0.05.

fractions on 1D NuPAGE gels (Fig. 1B). No obvious differences between duplicates were observed. Interestingly, the removal of a major protein, like albumin, clearly allowed the detection of new proteins as revealed by PMT and MALDI-TOF analysis (Fig. 1B, see Sup Table 1). In fact, after spot picking in the section of the gel where the albumin was located before its depletion (stars), α-1-antichymotripsin, immunoglobulin C gamma 1 and 2 could be identified. When 6, 12 or 20 proteins were removed, additional proteins such as antithrombin-III, vitamin D-binding protein, α-1-acid glycoprotein 1 and 2 could be detected (Sup Table 1). At the α2-macroglobulin position (cross), an additional protein (factor H complement protein) could be detected by PMT only after depletion of this protein by the two relevant columns (depleting 12 or 20 proteins). Overall, these PMT results indicated that the removal of, at least 6 major proteins, allowed the detection of many low abundant components.

The interest of protein depletion was also illustrated by the 2DE analysis that clearly revealed, as demonstrated in many previous works, an increase in the number of spots detected after depletion, and a disappearance of known proteins (Fig. 2). The ability to detect more 2D spots seemed related to the capacity to load more equivalent serum on the gels, and to the "unmasking" of spots following the removal of major proteins.

SELDI-TOF profiles

To further address the proteomic complexity of the samples, unbound serum fractions were analysed using SELDI-TOF CM10 and Q10 ProteinChips (Bio-Rad). As illustrated Table 2 and Fig. 3 for m/z ratio ranging from 2500 to 15,000, significantly more peaks with signal/noise ratio above 3 could be detected in the depleted fractions (Fig. 3). This is illustrated on low magnification spectra where some peaks decreased (Fig. 3A, diamonds) while others emerged along with the depletion (Fig. 3A, stars). Importantly, in our hand, the number of peaks detected after removal of 20 proteins was lower than that obtained with 6 or 12 proteins (p = 0.012 and p = 0.00013).

This result might be related to the different commercial origins of the columns, or to the removal not only of the 20 selected proteins but also of peptides and proteins associated with them. Parallel analysis of the retained fractions (not shown) was however not informative to confirm this possibility. As expected, the use of depletion columns that involved additional manipulation of the samples increased by at least 5% the variability of the data (Table 2). This was observed for all the columns and this needs to be taken into account in proteomic workflows including immunocapture. The global reproducibility of the SELDI-TOF analysis was however satisfactory with Pearson factors calculated for the same samples above 0.9 (Sup Table 2). Finally, hierarchical cluster analysis (HCE v3 [START_REF] Seo | Interactively optimizing signal-to-noise ratios in expression profiling: project-specific algorithm selection and detection p-value weighting in Affymetrix microarrays[END_REF]) of the SELDI-TOF data revealed the presence of two main clusters, one being composed by the 1 and 6 depleted protein profiles, and the second one by the 12 and 20 depleted protein profiles (Fig. 4). This distribution was confirmed by Pearson correlation factors (Sup Table 2).

Conclusion

Altogether, our results clearly confirmed the interest of depleting major blood proteins for the proteomic detection of low abundant components. However, we observed that increasing the number of depleted proteins from twelve to twenty had a limited beneficial impact and might increase drawbacks in removing associated peptides and proteins. This risk is linked to the fact that, in nondenaturing conditions, the immunocaptured proteins remained associated with an all range of peptides and proteins. In any case, in this study, as well as in a previous one using LC-MS/MS in CSF [START_REF] Thouvenot | Enhanced detection of CNS cell secretome in plasma protein-depleted cerebrospinal fluid[END_REF], the removal of 20 major proteins was more interesting than just removing 6 of them. The threshold between the gain of removing many proteins, and the drawbacks of removing associated proteins depend most likely on the design of the experiment, the analytical methods, and the choice between wanted and unwanted proteins removed. 3A). Clustering of the data generated with the Q10 chips were similar (not shown).

The Manhattan distance was used as the correlation factor. Two main clusters were observed, one being composed by the 1 and 6 depleted protein profiles, and the second one by the 12 and 20 depleted protein profiles.

Fig. 4 -

 4 Fig.4-Hierarchical clustering of the SELDI-TOF peaks detected in the CM10 condition for the initial serum and the unbound fractions collected after depletion (see Fig.3A). Clustering of the data generated with the Q10 chips were similar (not shown). The Manhattan distance was used as the correlation factor. Two main clusters were observed, one being composed by the 1 and 6 depleted protein profiles, and the second one by the 12 and 20 depleted protein profiles.

Table 1 -

 1 Description of the different commercial kits and their characteristics as indicated by the manufacturers.

	Column	1	6	12	20
	Capacity (serum)	20 µl	14 µl	10 µl	8 µl
	Unbound elution buffer 10 mM Tris-HCl, pH 7.4,	Undisclosed buffer composition 10 mM Tris-HCl,	Phosphate buffered saline
		150 mM NaCl		pH 7.4, 150 mM NaCl
	Elution volume	500 µl	200 µl	500 µl	100 µl
	Bound elution buffer	0.1 M Glycine, pH 2.5	Undisclosed buffer composition 0.1 M Glycine, pH 2.5	0.1 M Glycine-HCl, pH 2.5,
					and Tween 20
	Elution volume	500 µl	1000 µl	500 µl	300 µl
	Commercial name	ProteomLab IgY-HAS	Hu-6HC (Agilent)	ProteomLab IgY-12	ProteoPrep20 (SIGMA)
		(Beckman Coulter)		(Beckman Coulter)
	Proteins removed	Albumin	Albumin, IgG, IgA, transferrin,	Albumin, IgG, IgA, IgM,	Albumin, IgG, IgA, IgM, IgD,
			α1-antitrypsin, and haptoglobin	transferrin, fibrinogen,	transferrin, fibrinogen,
				α 2-macroglobulin,	α 2-macroglobulin,
				α1-antitrypsin,	α1-antitrypsin, haptoglobin,
				haptoglobin,	orosomucoid, ceruloplasmin,
				apolipoprotein A1, A2,	apolipoprotein A1, A2, B,
				and orosomucoid	complement C1q, C3, C4,
					plasminogen, and prealbumin

Table 2 -

 2 Statistical analysis of SELDI-TOF peaks with signal/noise ratio above 3 detected on CM10 biochips.

	Number of	Number	Minimal	Maximal	Mean
	proteins	of peaks	SD	SD	SD
	depleted	detected
	0 (serum)	18.8 ± 1.3	5.22%	33.59%	14.53%
	1	27 ± 1.4	3.63%	54.19%	22.26%
	6	29.5 ± 1.7	2.15%	51.33%	19.15%
	12	30.8 ± 0.5	4.67%	66.54%	28.36%
	20	26.5 ± 1	5.70%	39.43%	23.04%
	Results were obtained from 4 independent depletion experiments
	of the same samples. (SD: standard deviation).

Please cite this article as: Roche S, et al, Depletion of one, six, twelve or twenty major blood proteins before proteomic analysis: The more the better? J Prot (2009), doi:10.1016/j.jprot.2009.03.008

J O U R N A L O F P R O T E O M I C S X X ( 2 0 0 9 ) X X X -X X X

Acknowledgements

This study was supported by grants from the EC FP6 program cNEUPRO, the "Institut National contre le Cancer" (INCa), the CHRU of Montpellier and the CNRS.

Competing interests

For analytical evaluation, the protein depletion columns used in this study were provided by the different companies at a reduced price.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jprot.2009.03.008.


Michael Hutchinson
email: michael.hutchinson@nsl.eu.com

Juliette Marais
email: juliette.marais@ifsttar.fr

Émilie Masson

Jaizki Mendizabal
email: jmendizabal@ceit.es

Meyer Zu Hörste

Precise and reliable localization as a core of railway automation (Rail 4.0)

High Speed Railway services have shown that Railways are a competitive and, at the same time, an environmentally friendly transport system. The next level of improvement will be a higher degree of automation, with partial or complete automatic train operation up to fully automatic unattended driverless operation to reduce energy consumption and noise, as well as improving punctuality and comfort. Based on extensive experience in separated railway systems such as Metros, VAL (Véhicule automatique léger) and subways, today's discussion focuses on the fully automatic train operation on regular railway lines. This introduces some

questions that are more complex than in today's systems: the performance, length and weight of the trains spreads in a wider range; there are other and more complex operations; components such as level crossings in different equipment variants are added; requirements are higher and the technological equipment point is more heterogeneous.

Many evolutionary or innovative approaches for railway automation rely on precise and reliable localization. Especially for higher levels of automation it is essential. Starting from existing technologies such as the European Train Control System (ETCS), moving on to advanced driver assistance systems for local automatic operation up to full automatic train operation (ATO), all of these solutions depend on knowledge of the position of the train on the network. Improved concepts for railway operation e.g. using moving block, on-board integrity supervision or virtual train sets will result in even higher requirements for accuracy and reliability of the localization.

The contribution shows an approach based on Global Navigation Satellite Systems (GNSS) as a priority source of information used in combination with sensor data fusion. Elements such as trackside augmentation as well as the use of digital maps will be discussed. Different approaches are considered, e.g. those currently under development by projects in the innovation program in the joint undertaking Shift2Rail.

Keywords: localisation, odometry, GALILEO, train control, digital route map

Introduction

The precise as well as reliable detection of the position and their safe communication play a key role for the train operation control system. In recent years, the tendency to shift more and more positioning equipment functionality for safety-critical applications from the track side to the vehicle on-board-side could be observed. Train-borne positioning systems enable a costeffective operation technology for railway lines with a low traffic density and a more flexible operation than track-side positioning systems can offer for lines with a high traffic density. For on-board positioning systems, such as satellite-based train positioning systems, the mapmatching process is of particular importance, since then the data provided by different positioning sensors, such as GALILEO receivers, Doppler radars, odometers and inertial sensors are related to a digital map of the respective railway network. This implies that the digital map is a key element in a positioning system for safety-critical applications.

State of the art

Train location is of main use in signalling applications. In Europe, the train position is traditionally processed with the help of equipment on tracks. The typical equipment is a track circuit, i.e. a simple electrical device used to detect the presence of a train on rail tracks. This equipment is thus not devoted to locating the train specifically but to locating it indirectly on a track portion. The location can also be determined with the help of detectors placed along the track, on which the train protection relies. These sensors can be transponders (Balises), which communicate with the train on-board equipment when the train runs over them. In order to harmonize European solutions, Europe has developed the European Train Control System (ETCS) for the signalling, control and train protection. In levels 1 and 2 of ETCS, a Balise or a group of Balises is installed on tracks to give a passing train a position reference. The Balise initializes the odometer, and the train position is computed by the odometer as a distance run since the last relevant Balise group. The system composed of a Balise and odometer computes the position of the train interfaced with the EVC (European Vital Computer) train-borne subsystem (cf. figure 1). In level 3 of ETCS, the train location shall be sent by the train itself to the ground [UNISIG SUB026]. No line side signals will be required for delivering movement authorities. All information will be exchanged between the ETCS on-board system and the Radio Block Center (RBC) trackside system through mobile networks. Two main pieces of information are communicated by the train to the RBC: its location and the confirmation that the train did not lose any wagon (train integrity). Fig. 1 shows the generic structure of the onboard train control system. The last level of ETCS shall also improve line capacity by making it possible to manage circulations with moving blocks. ERTMS Level 3 will allow reducing the number of trackside equipment on the one hand, and the implementation of moving block in order to reduce the train spacing and then enhance capacity on the other hand.

Mainly driven by economic reasons and thus, acceleration of ERTMS penetration into the networks, the use of GNSS-based systems and, in particular, their introduction in signalling systems is seriously investigated today as a positioning opportunity included in ETCS and tested all around the world. In this context, GNSS is investigated to be the basis for a new embedded train locator.

The use of GNSS for low cost signalling solution and, in particular, in the highest level of ETCS (level 3) and the ERTMS Regional operation context, is an issue that has been investigated since the beginning of the 2000's [START_REF] Raymond | Innovations Bring Satellite Control within Reach[END_REF]. The concept of "virtual Balises" emerged to allow this new technology to penetrate the railway operational modes. The virtual Balises are dematerialized points, recorded in an embedded geographic database as illustrated in figure 2. These points are basically coordinates of the real (removed) physical Balises. The goal of the virtual Balise concept is to detect the position of the train when passing over the identified points by comparing the GNSS-based location of the train with the database. The train delivers then the same telegram that would have been sent with the use of the physical Balise. As such, the process could be as transparent as possible for the global system. The virtual Balise has been investigated in lots of projects since the beginning of the studies such as in RUNE [START_REF] Albanese | The RUNE project: navigation performance of GNSS-based railway user navigation equipment[END_REF] or recently in 3InSat [START_REF] Rispoli | Recent Progress in Application of GNSS and Advanced Communications for Railway Signaling[END_REF] or ERSAT [START_REF] Facchinetti | Trends in GNSS Italian application scenarios in transportation[END_REF].

Figure 2: Virtual Balise use instead of physical trackside Balise

With several projects, the European Commission, through the successive Framework Programs, funded research in order to explore and promote the use of global navigation satellite systems for low cost signalling solutions and, in particular, in the highest level of ETCS (level 3) and the ERTMS Regional. First projects were APOLO [Filip, 2001], GADEROS [Urech 2002] and LOCOPROL [Mertens, 2003] but one can mention tens of others in the past decade until the recent Satloc [START_REF] Gradinariu | SATLOC -An innovative train control system for low trafficdensity lines[END_REF], GaLoROI [Manz, 2014] or 3inSat [Rispoli, 2014] projects. The concept of "virtual Balises" has been investigated in lots of projects since the beginning of the studies such as in RUNE [START_REF] Albanese | The RUNE project: navigation performance of GNSS-based railway user navigation equipment[END_REF] or recently in 3InSat [START_REF] Rispoli | Recent Progress in Application of GNSS and Advanced Communications for Railway Signaling[END_REF] or ERSAT [START_REF] Facchinetti | Trends in GNSS Italian application scenarios in transportation[END_REF]. On-going projects are STARS [START_REF] Gurnik | STARS presentation[END_REF] and Rhinos [START_REF] Neri | A Method for Multipath Detection and Mitigation in Railway Control Applications[END_REF] focusing respectively on GNSS signal reception in railway environments and integrity concepts for railways. A complementary national project was DemoOrt in Germany [ GNSS provides a means of absolute positioning with achievable accuracies at the order of metres using basic consumer-level devices to the order of centimetres based on more sophisticated solutions. It is today used in an extremely wide range of applications from smartphone navigation to network timing synchronisation in critical infrastructure to safetycritical aircraft operations. In the railways, GNSS is today widely used for applications where its use is not critical such as basic tracking and synchronisation of onboard systems. The industry is also starting to recognise the potential for using GNSS for more advanced applications, from those that are regulated and include an element of liability to train control and operations. For example, GNSS is currently used for Selective Door Opening systems which have an associated safety-criticality. Many of the more advanced applications have demanding requirements which are not straightforward to meet with GNSS due to the challenges presented by the railway environment in terms of GNSS signal availability and quality. Features such as tunnels, cuttings, trackside buildings and vegetation can block signals as well as reflect them, inducing multipath effects in GNSS receivers. There is a general consensus that GNSS must be integrated with other sensors and sources of information in order to meet the very demanding safety requirements of train control applications, which can be more demanding than in civil aviation applications. GNSS is also vulnerable to interference and spoofing threats and solutions must be identified to mitigate this.

In recent years there has been significant research and development undertaken into the Virtual Balise application. This uses an onboard GNSS-based positioning system to detect when a train passes well-defined ERTMS 'Balise' locations stored in a database onboard, enabling the odometer calibration to be performed. This allows physical Balises to be removed from the track, making savings in terms of CAPEX and OPEX (maintenance). The current focus for this application is on local and regional lines, largely since it represents an economically justifiable way of introducing ERTMS onto such lines which will improve safety and/or capacity. Other advanced developments include the mandated Positive Train Control concept in the USA, for which GNSS is an optional source of positioning, and the research into the use of GNSS for Degraded Mode Working in the UK.

The Role of On-Board Localisation in Future Train Control Systems 3.1 The SmartRaCon approach in Shift2Rail

The European research for Railways will be done for the next coming years mainly in the Shift2Rail Joint Undertaking. The European Union, two railways and six industry members founded the Joint Undertaking.

The Joint Undertaking Shift2Rail comprises five Innovation Programs (IP) and five Cross Cutting Activities (CCA) of railway research and development for the near future. More than 40 Technical Demonstrators will be developed in the field of rolling stock, traffic management and control systems, infrastructure, IT solutions and rail freight.

Four Partners with a strong expertise in research and development in the area of train control, communication and localisation formed the consortium "Smart Rail Control -SmartRaCon":

 German Aerospace Centre (Deutsches Zentrum für Luft-und Raumfahrt -DLR), Germany  Centro de Estudios e Investigaciones Técnicas (CEIT), San Sebastián (Gipuzkoa), Spain  Fondation de Cooperation scientific RAILENIUM, Famars, France  Nottingham Scientific Ltd. (NSL), Nottingham, England

The vision of SmartRaCon is to realise a fail-safe, multi-sensor onboard positioning system at minimal cost which not only provides train positioning to the ETCS kernel but also acts as an enabler for multiple areas within the area of signalling, potentially including Traffic Management, Train Integrity and Virtual Coupling. The objective of the consortium is to develop the constituents of the on-board positioning system based on multi-constellation GNSS, complemented by other positioning technologies. The activities will also include the definition of the expected performance thanks to field-testing, certification process definition and testing tools.

Concept and Approach

The overall concept is based on the need to ensure that the safety levels provided by existing signalling and control systems are not compromised in the movement towards an on-board positioning system. SmartRaCon will provide various contributions to ensure that this is achieved. This includes setting up and undertaking test campaigns, analysing the data from such campaigns, improving specifications, providing various inputs to the development of a safety case, as well as other more specific contributions which build on the positioning technology expertise within the consortium such as simulation based KPI evaluation, multiconstellation or sensor integration, etc.

Testing processes and the route to acceptance of GNSS and associated technology will be enhanced such that standardised methods are put in place in terms of the equipment used, measurements made, and analysis tools and results delivery. This includes a Route Clearance service, simulation tools for railway KPIs evaluation, Digital Route Maps (DRM) as an input to on-board positioning systems, in terms of their utilisation, distribution and management. Contributions will be made to the formation of a consolidated set of specifications, critically including methodology for testing the capabilities of common-off-the-shelf equipment against these specifications. There will be a focus on DRM technology and the Virtual Balise concept. The need for lab simulations will be identified here and it is proposed to develop a Local Environment Model as a specific task. Based on the outcomes of initial testing, solutions for performance optimisation will be proposed through in-depth knowledge in the hybridisation of GNSS with inertial sensors, odometry, dead reckoning, DRM and Wireless Communications Technology (WCT). Further specific tasks proposed are the development of a GNSS Railway Integrity Concept, input to the design of a Safety-Critical Railway GNSS Receiver, a Failure Modes and Effects Analysis (FMEA), formation of a GNSS Railway Threat Model, and demonstration of a Route Clearance service.

Expected Impact

In terms of impacts there is a target for the work to generate future business in various ways. Work will lead to an established core of safety expertise within the consortium concerning the use of on-board positioning technology for railway applications and this will be important as the technology grows and improves. There is an ambition to develop a Route Clearance service model for use in safely introducing the technology to specific new lines and for new applications. The partners will also gain a holding in the supply chain(s) involved in developing and certifying dedicated hardware, algorithms and the infrastructure required to deliver the services e.g. local integrity monitoring stations.

1. Improved services and customer quality: the use of enhanced communication technologies will derive in the following outcomes: trains will be managed more efficiently providing improved reliability, enhanced capacity of the lines and providing communication capabilities for real-time data transmission for improved passenger information, offering a better customer experience. 2. Reduced system costs: the design and implementation of the on-board gateway will be based on mass-market technologies and open interfaces, which will enable flexible architectures and application of engineering standards, allowing a correct system design adapted to the requirements of different market segments. 3. Enhanced interoperability: remaining technical "open points" related to electromagnetic compatibility will be closed. 4. Simplified business processes as modular architectures are introduced to divide the validation effort and improve standardization. The interface from the core system to the new technologies to be incorporated to the gateway will be defined as a seamless continuous upgrade possibility.

Implementation

Regarding the on-board positioning, the priority tasks are:

 to contribute to the test set-up, data collection and analysis according to specifications,  to assist with further development of those specifications,  to provide input to the safety case,  to build a DRM prototype.

SmartRaCon expect the contribution to data collection/test campaigns to be part of a wider effort including founder members to analyse and validate positioning system performance, throughout the various stages of the testing outlined in the Scoping Paper. The same may be said regarding progression of the specifications output from the NGTC project. The specific tasks proposed to contribute to the safety case will be led by SmartRaCon but will in certain aspects require input and cooperation with founder members. Other proposed tasks are seen as generally internal to the consortium requiring a low level of interaction with the founder members in order to set requirements and gain feedback on results. This is the case for example with the proposed Route Clearance service. In all cases a close level of cooperation with the founder members is important in order to take into account the specific characteristics of the proposed positioning system solution.

Integrated Assessment

A technology and impact evaluation is an essential element within the Shift2Rail Joint Undertaking in order to show the effect that this initiative will have on its key target KPIs: to double the capacity and availability and to reduce the costs of the railway system by 50%. The Shift2Rail Project "IMPACT-1" is producing a comprehensive bottom-up KPI model with the aim to show the interdependencies between the technological or procedural developments and the high level KPIs of the railway system. The overall objective is to prove the achievement of the objectives of Shift2Rail by determining to which extend the aims of reducing costs and improving availability and capacity will be reached. In a first step, these interdependencies will be analysed as cause-and-effect chains in order to obtain a qualitative model. Subsequently, the qualitative relations will be replaced by mathematical and logical descriptions. This is necessary in order to apply the model to data of the different market segments like high speed, regional, urban / suburban and freight rail. The analysis of the interdependencies as well as the application of the model is done in close collaboration between industry, infrastructure managers, railway operators and scientific institutions. Thus a KPI model will be generated which covers all aspects of the entire railway system. The presentation will cover the approach that has been chosen to develop the qualitative and quantitative model, share the experience made during this process and show the first results of the impact assessment of the Joint Undertaking Shift2Rail.

Impact on the Competitiveness of the European railway systems

The introduction of GNSS in the rail domain is now recognized as a powerful tool for ERTMS deployment, old system renewal. A study performed by Bocconi University for the ERSAT EAV projects shows that GNSS-based ERTMS proves to be especially convenient because of relevant savings in operating expenses: -67% each year compared to the traditional ERTMS [Galileo Services, 2016]. Moreover, the cost/benefit ratio will be maximized if satcoms are integrated in the global system.

This introduction is supported both by the GNSS domain through the GSA that identified rail as a real future market (figure 7), and by the railway sector in order to facilitate ERTMS diffusion, reduce costs and enhance safety on local and regional lines. As shown on figures 7 and 8, if asset management applications are currently driving shipments of GNSS devices, safetyrelevant applications (signalling and train control) based on GNSS will be increasingly developed in the next 15 years.

First application is through the Virtual Balise Concept in ERTMS L2 or 3 but next steps to be prepared are also Moving block operation and automatic train operation.

Figure 3: Future market evolution in rail applications

The NGTC project, that gathered together the main industries involved in ETCS and CBTC, consolidated the use of GNSS in the Virtual Balise concept and has developed and validated Generic Moving Block principles applicable to different railway types. Designed as a lighthouse project for Shift2Rail IP2, the outputs of NGTC regarding the analyses of the Satellite Positioning Signal Receiver Parameters that are relevant for the signalling applications and the process of their qualification and validation and, Initial Safety Analyses in regards to use of satellite positioning in ERTMS, will be major inputs for several Shift2Rail projects.

Conclusions and Perspective

Safe on-Board localisation is -besides reliable and safe communication -one of the key elements of future train control systems. Four partners from the science and innovation sector have founded the consortium SmartRaCon to perform research and innovation activities in the context of the European Joint Undertaking Shift2Rail. The aim is to develop and demonstrate technologies for safety-critical positioning applications in the railway sector where it is mandatory to guarantee a high safety and reliability the positioning result.

Several Shift2Rail projects will start in the next years to develop the technologies which will be demonstrated to show the results of the innovation. The aim of these projects is to demonstrate innovative onboard localisation technologies for future train control systems.

Figure 1 :

 1 Figure 1: Overview of current setup of the localisation in the on-board train control

  Meyer zu Hörste, 2009] One European operated line embeds a light GNSS solution today. It is a single track line of 150 km length with a system developed by the University of Applied Sciences Upper Austria (FH OO) and operated by Stern & Hafferl, respective Salzburg AG. An on-board unit communicates the GNSS-based train position to the trackside train controller which is responsible for the movement authority of the train [Stadlmann; 2006]. The system is implemented in SIL2.In April 2012, a big recognition step has occurred when the European Commission, the European Railway Agency and the European Rail Sector Associations signed together an ERTMS Memorandum of Understanding concerning the strengthening of cooperation for the management of ERTMS. It mentions in particular that GNSS can play a major role in the rail sector[ERTMS, 2012].In the same period, new initiatives have been launched such as the NGTC project (Next Generation Train Control) that aims to study how the new developments for ERTMS/ETCS (for interoperable networks) and those for CBTC (Communication-Based Train Control) systems (for urban networks) can be mutualized. Satellite positioning is one of the NGTC focuses.