Jens Koch

Christian Neusüss

Herve Cottet
email: herve.cottet@umontpellier.fr

Laurent Leclercq

Marine Morvan

Christian Neusüß

Hervé Cottet

Modulation of the electroosmotic mobility using polyelectrolyte multilayer coatings for protein analysis by capillary electrophoresis

Keywords: capillary coatings, polyelectrolyte multilayers, protein separation, polymer coatings

come

Modulation of the electroosmotic mobility using polyelectrolyte multilayer coatings for protein analysis by capillary electrophoresis

HIGHLIGHTS

• Electroosmotic mobility varies from -43 to -63 Tiselius units depending on polycation • Electroosmotic flow magnitude is in the order of PB>-PLL>DEAEDq • Coatings with poly(L-lysine citramide) gave the best separation efficiencies • Average RSD on migration times (resp. separation efficiency) was 1.7% (resp. 9.6%)

• CE-MS separation of positional isomers of the glycoprotein ribonuclease B

Introduction

Capillary coatings are frequently used to limit adsorption phenomena onto the capillary wall in capillary electrophoresis (CE), and to increase repeatability / reproducibility of CE separations, either for protein or small ions analysis [START_REF] Pattky | Advantages and limitations of a new cationic coating inducing a slow electroosmotic flow for CE-MS peptide analysis: a comparative study with commercial coatings[END_REF]. Capillary coatings should thus ensure high separation efficiency and good repeatability, which is particularly important for CE coupled to mass spectrometry (CE-MS). Different approaches have already been explored and reviewed [START_REF] Huhn | Relevance and use of capillary coatings in capillary electrophoresis-mass spectrometry[END_REF][START_REF] Lucy | Non-covalent capillary coatings for protein separations in capillary electrophoresis[END_REF][START_REF] Horváth | Polymer wall coatings for capillary electrophoresis[END_REF][START_REF] Doherty | Microchannel wall coatings for protein separations by capillary and chip electrophoresis[END_REF][START_REF] Stutz | Protein attachment onto silica surfaces-a survey of molecular fundamentals, resulting effects and novel preventive strategies in CE[END_REF][START_REF] Garza | Simplifying capillary electrophoresis-mass spectrometry operation: Eliminating capillary derivatization by using self-coating background electrolytes[END_REF], such as covalently bonded polymer coating, dynamic coating or physically adsorbed polymers, including successive multiple ionic-polymer layers (SMIL).

Compared to the first category, SMIL coatings are very easy to prepare and to regenerate.

A 2-layers anionic terminating coating of polybrene (PB) and dextran sulfate (DS), developed by Katayama et al., was the first SMIL system applied for CE separation [START_REF] Katayama | Stable Capillary Coating with Successive Multiple Ionic Polymer Layers[END_REF] and permitted over 100 runs of acidic proteins separations in neutral phosphate buffer. This SMIL system was next extended to a 3-layer cationic terminating coating for the separation of basic proteins in acidic conditions (phosphate buffer, pH 3) and for CE/MS analysis of blockers in formate buffer [START_REF] Katayama | Stable Cationic Capillary Coating with Successive Multiple Ionic Polymer Layers for Capillary Electrophoresis[END_REF]. Catai et al. further investigated SMIL systems composed of PB/poly(vinyl sulfate) (PVS) bilayers for the separation of proteins at pH 7 in phosphatebased buffers of various concentrations [START_REF] Catai | Noncovalently bilayer-coated capillaries for efficient and reproducible analysis of proteins by capillary electrophoresis[END_REF]. They showed that the optimal separation efficiency is obtained in 300 mM Tris-phosphate buffer. The same group also analyzed intact proteins using a cationic 3-layers SMIL based on PB and DS in Tris-phosphate buffer by CE and acetic acid buffer by CE/MS [START_REF] Haselberg | Capillary electrophoresis of intact basic proteins using noncovalently triple-layer coated capillaries[END_REF]. A CE/MS method was developed for the characterization and integrity assessment of the antigens TB10.4 and Ag85B and their chemically produced glycoconjugates, which are glycovaccine candidates against tuberculosis [START_REF] Tengattini | Monitoring antigenic protein integrity during glycoconjugate vaccine synthesis using capillary electrophoresis-mass spectrometry[END_REF]. In order to prevent protein adsorption to the inner capillary wall and to achieve efficient separation of the antigen proteoforms, a 3-layers coating of PB-DS-PB was used in combination with 1.5 M acetic acid as background electrolyte [START_REF] Tengattini | Monitoring antigenic protein integrity during glycoconjugate vaccine synthesis using capillary electrophoresis-mass spectrometry[END_REF]. CE/MS analysis revealed the presence of several closely related degradation products, including truncated, oxidized and conformational variants. Both the affinity and composition of the therapeutic protein EGa1, an antagonist of the epidermal growth factor receptor which is overexpressed on the surface of tumor cells, were determined by CE and CE/MS, using PB/PVS and PB/DS/PB capillary coatings and various background electrolytes, showing the heterogeneity of EGa1 [START_REF] Haselberg | Capillary electrophoresis-based assessment of nanobody affinity and purity[END_REF]. Efficient protein separations have been achieved by Schlenoff et al. using multilayers of poly(diallyldimethylammonium) chloride (PDADMA) and poly(styrene sulfonate) (PSS) [START_REF] Graul | Capillaries Modified by Polyelectrolyte Multilayers for Electrophoretic Separations[END_REF]. Similar SMIL system was further studied by Nehmé et al. who investigated the influence of various experimental parameters (polymer concentration, number of layers, ionic strength, rinsing time, storage and regeneration conditions) on separation efficiency and repeatability for the separation of peptides [START_REF] Nehme | Influence of polyelectrolyte coating conditions on capillary coating stability and separation efficiency in capillary electrophoresis[END_REF][START_REF] Nehme | Influence of polyelectrolyte capillary coating conditions on protein analysis in CE[END_REF] and proteins [START_REF] Nehme | Influence of polyelectrolyte capillary coating conditions on protein analysis in CE[END_REF] in Tris-phosphate buffer at pH 2.5. The authors found that the best separations are obtained using 1.5 M ionic strength coating solutions, 5-layers coatings and ''sufficient'' polyelectrolyte (PE) concentrations (0.2% w/v) [START_REF] Nehme | Influence of polyelectrolyte coating conditions on capillary coating stability and separation efficiency in capillary electrophoresis[END_REF][START_REF] Nehme | Influence of polyelectrolyte capillary coating conditions on protein analysis in CE[END_REF]. The influence of background electrolyte (BGE) pH on EOF has been investigated on SMIL coatings and was found substantial in the case of weak polyelectrolytes but not significant in the case of strong polyelectrolytes [START_REF] Swords | Assessment of polyelectrolyte coating stability under dynamic buffer conditions in CE[END_REF]. Thickness and morphology of SMIL coatings has been investigated by atomic force microscopy on PB, PB/DS and PB/DS/PB layer coatings [START_REF] Haselberg | Thickness and morphology of polyelectrolyte coatings on silica surfaces before and after protein exposure studied by atomic force microscopy[END_REF]. Protein adsorption was observed on fused silica capillary and on PB/DS but not on PB and on PB/DS/PB coatings [START_REF] Haselberg | Thickness and morphology of polyelectrolyte coatings on silica surfaces before and after protein exposure studied by atomic force microscopy[END_REF]. SMIL coatings based on PDADMA/DS have been used for the separation of recombinant allergen variants in ammonium bicarbonate buffer at pH 6.7 with excellent repeatability/reproducibility [START_REF] Weinbauer | Successive multiple ionic polymer layer coated capillaries in the separation of proteins -Recombinant allergen variants as a case study[END_REF]. Recently, topographic and recognition imaging (TREC) was performed by Leitner et al. [START_REF] Leitner | Mapping molecular adhesion sites inside SMIL coated capillaries using atomic force microscopy recognition imaging[END_REF] using atomic force microscopy on 4-layers SMIL coated capillaries based on PDADMA and DS (or ending with a moderately charged statistical poly(acrylamide-co-2-acrylamido-2-methyl-1-propane sulfonate), 55% PAMAMPS), showing different topologies (roughness) and charge distributions (adhesion map). Due to lower charge density, the 55% PAMAMPS coating decelerated the EOF compared to the DS in the last layer, and displayed lower charge heterogeneity, while the topography was very similar [START_REF] Leitner | Mapping molecular adhesion sites inside SMIL coated capillaries using atomic force microscopy recognition imaging[END_REF][START_REF] Stock | Advanced portrayal of SMIL coating by allying CZE performance with in-capillary topographic and charge-related surface characterization[END_REF].

Twenty-three different 5-layers SMIL coatings were investigated, combining different polyanions (poly(L-glutamic acid), poly(acrylic acid) (PAA), poly(acrylic acid-co-acrylamide), hyaluronic acid (HA), dextran sulfate (DS), poly(styrene sulfonate) (PSS), poly(methacrylic acid) (PMA), poly(L-lysine citramide) (PLC)) and different polycations (poly(L-lysine) (-PLL) and copolymers of lysine with serine and tyrosine, non quaternized diethylaminoethyl dextran, polyallylamine (PAAM), PDADMA, polyethyleneimine (PEI), poly(dimethylamino ethyl methacrylate) (PDMAEMA), and systematically compared regarding the separation performances (separation efficiency, analysis time and repeatability/reproducibility on migration times) of proteins in BGE conditions compatible with MS (0.5 M acetic acid) [START_REF] Bekri | Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking[END_REF]. 5-layers SMIL coating was chosen to get positively charged coating for separation in acidic conditions and as good compromise between the number of layers and the performances of the coating [START_REF] Bekri | Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking[END_REF]. It was shown that not only the nature of the polycation constituting the last layer, but also the nature of the polyanion in the intermediate layers of the SMIL, played an important role in the separation performances. The best results were obtained with 5layer PDADMA/PLC SMIL with excellent run-to-run and capillary-to-capillary repeatability/reproducibility on migration times (RSD = 0.1%) and peak efficiencies (RSD = 1.2%). However, we did not find any systematic correlation between PE characteristics such as linear charge density (ξ), weak / strong acid-base moieties, PE backbone nature and the separation performances. ξ is defined as the ratio of the Bjerrum length (0.71 nm in pure water at room temperature) to the average distance b between two adjacent charges:

ξ = l b /b (1)
The present work aims at studying 5-layers SMIL capillary coatings in acetic acid BGE using new polycations that were not previously investigated: polybrene (PB), quaternized diethylaminoethyl dextran (DEAEDq) and -poly(lysine) (-PLL), in combination with PAA, DS, PSS, PMA and PLC as polyanions. The influence of the nature of the polyelectrolytes used in the SMIL and of the ionic strength of the construction buffer on the performances of separation of proteins is discussed. To identify the best polyelectrolyte couples, a test mixture of 5 standard proteins was used to evaluate separation performances based on separation efficiency and repeatability of the separations in 0.5 M acetic acid BGE. Intra and inter capillary repeatability / reproducibility were investigated based on 7 repetitions per capillary and 3 different capillaries for the most promising PE couples. The most promising coatings were applied for the separation of glycoproteins by CE-MS.

Materials and Methods

Chemicals

2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane sulfonic acid (HEPES) and acetic acid were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Lysozyme (Lys, purity ≥ 90%), β-lactoglobulin (Lac, purity ≥ 90%), Ribonuclease A (RNAse A, purity ≥ 60%), myoglobin (Myo, purity ≥ 95%) and trypsin inhibitor (TI, purity not indicated by the supplier)

were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Ultrapure water was obtained using a MilliQ system from Millipore (Molsheim, France). Analytical grade NaCl was purchased from Merck (Fontenay-sous-Bois, France).

Poly(acrylic acid) (PAA, M w 30,000 g/mol, PDI = 2.5, pK a = 3.9-4. 50,000 g/mol, PDI = 2.5, pK a = 4.2-4.8, ξ = 2.88) was synthesized as previously described [START_REF] Leclercq | Conformation of water soluble copolymers of methacrylic acid and benzyl methacrylate[END_REF].

Quaternized diethylaminoethyl dextran (DEAEDq, M w 500,000 g/mol, ξ = 0.35) was obtained from Pharmacia (Uppsala, Sweden). -poly(lysine) (-PLL, M n 5,000 g/mol, pK a = 9.0, ξ = 0.85) was obtained from Streptomyces Albulus and was supplied by Chisso (Tokyo, Japan). It is an homopolymeric compound of ~30 lysine monomers linked at the ε-amino groups [START_REF] Shima | Polylysine produced by Streptomyces[END_REF]. All the polyacids are under their sodium form and all the polybases are under their hydrochloride form.

Capillary coatings

Capillaries were first conditioned with 1 M NaOH for 20 min, water for 5 min and 20 mM HEPES + NaCl (varying from 0 to 1 M) buffer at pH 7.4 for 10 min. The polyelectrolyte multilayer systems are built within the capillaries using successive flushes of polycation and polyanion solutions at 3 g/L in the 20 mM HEPES buffer pH 7.4 (+ possibly NaCl as indicated on the graphs). Each polyelectrolyte layer was deposited by a 7 min flush (starting with the polycation) and with a 3 min rinsing with the HEPES/NaCl buffer between two polyelectrolyte layers. Five layers were built finishing with a polycation layer. Before separation, the capillaries were then flushed with the background electrolyte (0.5 M acetic acid, pH 2.5) for 10 min.

CE separations

Bare fused silica capillaries were purchased from Composite Metal Services (Photonlines, France). CE experiments were carried out using a 3D-CE Agilent technologies system (Waldbronn, Germany) using 50 μm i.d. × 40 cm (31.75 cm to the detector). The protein test mixture was prepared by dissolving lysozyme, β-lactoglobulin, RNAse A, myoglobin and trypsin inhibitor at 1 g/L in pure water [START_REF] Taichrib | Toward a screening method for the analysis ofsmall intact proteins by CE-ESI-TOF MS[END_REF]. Protein mixture was injected hydrodynamically at 30 mbar for 4 s. MS-compatible background electrolyte was 0.5 M acetic acid, pH 2.5.

0.01% DMF in the BGE was used as EOF marker and was co-injected hydrodynamically at 30 mbar for 4 s. The capillary was rinsed with buffer for 3 min between two successive runs.

The temperature of the capillary cartridge was set at 25°C. CE experiments were performed by applying a negative polarity voltage of -30 kV. UV detection was performed at 214 nm.

All the separations were repeated seven times. Separation efficiency was determined using the following equations:

2 5 54 m t N.      (1)
where t m is the migration time and is the half-height peak width determined using CEval 0.6 software [START_REF] Dubský | CEval: All-in-One Software for Data Processing and Statistical Evaluations in Affinity Capillary Electrophoresis[END_REF] available at the following address

[https://echmet.natur.cuni.cz/software/ceval].

CE-MS

CZE-ESI-MS was performed on an HP 3DCE electrophoresis instrument from

Results and discussion

Three polycations, namely polybrene (PB), quaternized diethylaminoethyl dextran (DEAEDq) and -poly(lysine) (-PLL), were selected for the present study to complete the previous study based on -PLL, copolymers of lysine with serine and tyrosine, diethylaminoethyl dextran, PAAM, PDADMA, PDMAEMA and PEI [START_REF] Bekri | Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking[END_REF]. DEAEDq is a strong polybase as PDADMA, the latter of which was previously found to be the best polycation in terms of separation performances [START_REF] Bekri | Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking[END_REF]. -PLL has low charge density (compared to -PLL)

similarly to PLC polyanion which was previously found the best polyanion in terms of separation performances [START_REF] Bekri | Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking[END_REF]. PB is often used in the literature and should thus be compared to other polycations [START_REF] Katayama | Stable Capillary Coating with Successive Multiple Ionic Polymer Layers[END_REF][START_REF] Katayama | Stable Cationic Capillary Coating with Successive Multiple Ionic Polymer Layers for Capillary Electrophoresis[END_REF][START_REF] Catai | Noncovalently bilayer-coated capillaries for efficient and reproducible analysis of proteins by capillary electrophoresis[END_REF][START_REF] Haselberg | Capillary electrophoresis of intact basic proteins using noncovalently triple-layer coated capillaries[END_REF][START_REF] Tengattini | Monitoring antigenic protein integrity during glycoconjugate vaccine synthesis using capillary electrophoresis-mass spectrometry[END_REF][START_REF] Haselberg | Capillary electrophoresis-based assessment of nanobody affinity and purity[END_REF][START_REF] Haselberg | Thickness and morphology of polyelectrolyte coatings on silica surfaces before and after protein exposure studied by atomic force microscopy[END_REF]. The five polyanions, namely PAA, PMA, PSS (acrylic polymers), PLC (polyamide) and DS (polysaccharide), were selected to be associated with the three polycations. This work investigates 5-layers SMIL coatings for two reasons: (i) an odd number of layers was chosen to get positively charged coatings for separation in acidic conditions; and (ii) 5-layers was found to be a good compromise in terms of protein separation efficiency and coating stability [START_REF] Bekri | Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking[END_REF]. As coating solutions, PE were dissolved at 3 g/L in HEPES buffer at pH 7.4 to ensure the ionization of all PE, including weak polybases / polyacids.

Influence of the nature of the PE and intra-capillary repeatability

Figure 1A (resp. Figure 1B) displays the electropherograms obtained for 5-layers SMIL coatings using PB and -PLL (resp. DEAEDq) polycations. Surprisingly, the range of migration times are very different for PB (total analysis time as low as 3 min), -PLL (total analysis time up to 6 min), or DEAEDq (up to 20 min and even more when combined to PAA or PSS). This is due to the broad range of electroosmotic mobility (from -39.5 Tiselius units (TU) in the case of the DEAEDq/PAA to -62.6 TU for PB/PSS). Electroosmotic mobility values are given by decreasing order in Table 1 together with the separation efficiency (N/l) and RSD on the migration times of proteins and of the electroosmotic marker. EOF magnitude (absolute value of the electroosmotic mobility) was found to decrease in the order order PB>-PLL>DEAEDq, whatever the polyanion associated. The modulation of the EOF according the nature of the polycation (and polyanion) on a wide range of mobilities (about 23 TU) is of great practical interest for applications, either to speed up the analysis, or to increase the resolution by decreasing the EOF magnitude. In the case of DEAEDq, the EOF was so slow with PAA, PSS or PMA that all the proteins were not detected in about 20 min. RSD on migration times are usually lower than 3-4%, with excellent results for PLC polyanion. The lowest repeatability was obtained with PAA polyanion.

In agreement with the previous study [START_REF] Bekri | Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking[END_REF], coatings with PLC gave the best results in terms of separation efficiencies (around 300,000 plates/m for PB/PLC and -PLL/PLC couples). Such high separation efficiency can be also directly observed on the electropherograms in Figure 1A when looking at the fine resolution of small protein peaks (close to the main proteins). Surprisingly, PLC, which is the polyanion with the lowest linear charge density among those studied in this work, can lead to the lowest EOF magnitude (when combined with highly charged PB) or to the highest (when combined with lower charged -PLL or with DEAEDq), showing the complexity of the physicochemical parameters involved in the regulation of the EOF magnitude (thickness and compactness of the SMIL layers, charge repartition [START_REF] Leitner | Mapping molecular adhesion sites inside SMIL coated capillaries using atomic force microscopy recognition imaging[END_REF][START_REF] Stock | Advanced portrayal of SMIL coating by allying CZE performance with in-capillary topographic and charge-related surface characterization[END_REF], penetration of the electrostatic double layer in the SMIL [START_REF] Zimmermann | Electrokinetics of a poly(N-isopropylacrylamid-co-carboxyacrylamid) soft thin film: Evidence of diffuse segment distribution in the swollen state[END_REF], charge stoichiometry of complexation between oppositely charged PE layers [START_REF] Lounis | Prediction of polyelectrolyte complex stoichiometry for highly hydrophilic polyelectrolytes[END_REF][START_REF] Lounis | Modelling and predicting the interactions between oppositely and variously charged polyelectrolytes by frontal analysis continuous capillary electrophoresis[END_REF]).

As already reported in [START_REF] Bekri | Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking[END_REF], we confirm here that the nature of the polyanion is of primary importance for the separation efficiency, even if the polyanion is not the last layer of the SMIL coating, and thus not in direct contact with the proteins. Regarding the choice of polycation leading to the best performances, the best polycations are PLL, PB, DEAEDq and PDADMAC [START_REF] Bekri | Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking[END_REF]; the worth polycations are PLL, PAAM, PDMAEMA, PEI and nonquaternized DEAED. From this study and from [START_REF] Bekri | Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking[END_REF], we can conclude that the best SMIL coatings were obtained with: (i) polycations having low to moderate linear charge densities (ξ between 0.35 and 1.44) and (ii) with PLC polyanion (low charge density and amide structure) ahead from vinylic polyanions (in the decreasing order of PMA, PSS and PAA), and far beyond polysaccharides (DS et HA). It is however difficult to find a general rational explanation for the better performances of PLC, since its main chemical characteristics (low charge density and high hydroxyl groups content) are not present in the second best polyanions (PMA and PSS [START_REF] Bekri | Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking[END_REF]), or are present in one of the worst (HA).

Among The electrophoretic conditions (capillary dimensions, voltage, electroosmotic mobility) and the solutes (different proteins and one peptide (oxytocine)) reported in Table 2 are generally different, which makes the comparison difficult. Nevertheless, we can emphasize that the average N/l values on all data presented in Table 2 is 160,000 plates/m; with significantly higher performances for PLL-PLC, PB-PLC and PDGMI [START_REF] Haselberg | Capillary electrophoresis-mass spectrometry using noncovalently coated capillaries for the analysis of biopharmaceuticals[END_REF]. It should be also noted that separation efficiency is generally higher for the highly basic lysozyme, probably because of strong electrostatic repulsion between the positively charged coating and the protein.

Study of intra-capillary and inter-capillary reproducibility on the best capillary coatings

Inter-capillary reproducibility was tested on three different capillaries for the 6 previously selected SMIL coatings. Such studies are important in practice to increase the reproducibility of CE in terms of migration times and separation efficiency. However, the inter-capillary reproducibility is rarely investigated in the literature, most likely because such studies are time consuming. Examples of electropherograms obtained on three different capillaries are presented in Figure 2. All the numerical values relative to EOF mobility, separation efficiency and RSD on migration times are gathered in Table 3, for each of the 18 tested capillaries, showing the reproducibility for each polyelectrolyte system. Basically, the intra-capillary RSD (calculated on 7 runs and averaged for the 5 test proteins) on protein migration times are very low: typically lower than 2.5%, with an average value on the 18 studied capillaries of 1.3%. The same was observed for the migration time of the EOF marker: RSD lower than 1.1% with an average of 0.6% for all the capillaries. For the 6 selected couples of polyelectrolytes, the intra-capillary average <N/l 5P > 7 runs calculated on 7 runs and averaged on the 5 test proteins was systematically found higher than 100,000 plates/m (except for one PB-PSS capillary). Average RSD on N/l values was obviously higher than for migration times, but still reasonable for such experimental parameter: RSD ~ 9.6% on the 18 tested capillaries. The detailed figures of merit obtained for the separation efficiency of each protein are given in Table SI1 (in Supporting Information).

From this study, we can conclude that the 6 investigated PE couples can be used for repeatable and reasonably reproducible separations with a broad range of electroosmotic mobility (from to -43.7 to -62.9 TU) and separation efficiency higher than 10 5 plates/meter.

Nevertheless, there is still place for some possible improvement in the inter-capillaries reproducibility. ANOVA statistical tests performed on µ eof , <N/l 5P > and the migration times of proteins (not shown) clearly demonstrate that the inter-capillary dispersions on these parameters are significantly higher than the intra-capillary dispersions. This can be for instance illustrated by the third PB-PSS capillary (third trace from the top in Figure 2A) which provided significantly higher separation efficiencies than the two others. For PB-PMA, the first capillary (first trace from the top in Figure 2A) lead to lower separation efficiencies. By the way, this study already gives important hints about the nature of the PE couples that are the most adequate to improve coating performances and reproducibility. One possible Table 3. Inter-capillary study on PB-PLC, -PLL-PLC, PB-PMA, PB-PSS, DEAEDq-PLC and DEAEDq/PMA 5-layers SMIL coatings. Average electroosmotic mobility, average separation efficiency <N/l 5P > 7 runs and relative standard deviations on separation efficiency and migration times obtained for 3 different capillaries of each coating. <N/l 5P > 7 runs and RSD% on migration times are calculated on the average of the five proteins and on 7 repetitions. Experimental conditions as in Figure 1.

Couples

μ eof (10 -9 m -2 V -1 s - 1 )
<N/l 5P > 7 runs (10

Influence of the ionic strength of the SMIL construction electrolyte

The impact of the ionic strength of the coating solution is known to be a crucial parameter in the building of SMIL coating [START_REF] Nehme | Influence of polyelectrolyte coating conditions on capillary coating stability and separation efficiency in capillary electrophoresis[END_REF][START_REF] Nehme | Influence of polyelectrolyte capillary coating conditions on protein analysis in CE[END_REF][START_REF] Schlenoff | Charge and Mass Balance in Polyelectrolyte Multilayers[END_REF] and polyelectrolyte complexes [START_REF] Leclercq | A physico-chemical approach of polyanion-polycation interactions aimed at better understanding the in vivo behaviour of polyelectrolyte-based drug delivery and gene transfection[END_REF]. It notably set the thickness of the PE layer. Higher ionic strength leads to thicker deposited PE layer with the formation of loops [START_REF] Schlenoff | Charge and Mass Balance in Polyelectrolyte Multilayers[END_REF]. On the contrary, low ionic strength leads to thin layers due to electrostatic repulsions [START_REF] Schlenoff | Charge and Mass Balance in Polyelectrolyte Multilayers[END_REF]. In this work, different concentrations (0, 0. 4. From Figures 3A to 3C and Table 4, it can be concluded that increasing the ionic strength of the coating tends to decrease the coating performances (lower N/l values) and stability (higher RSD on migration times). This conclusion is different to what was obtained for the separation of peptides and proteins in acidic phosphate buffer on a PSS/PDADMA SMIL coating [START_REF] Nehme | Influence of polyelectrolyte coating conditions on capillary coating stability and separation efficiency in capillary electrophoresis[END_REF][START_REF] Nehme | Influence of polyelectrolyte capillary coating conditions on protein analysis in CE[END_REF], where the optimum ionic strength was found at 1 M for the coating buffer.

One explanation is that the polyelectrolyte couples selected in the present work are composed of two weak polyelectrolytes or one weak and one strong polyelectrolyte, whereas PSS and PDADMA used in [START_REF] Nehme | Influence of polyelectrolyte coating conditions on capillary coating stability and separation efficiency in capillary electrophoresis[END_REF][START_REF] Nehme | Influence of polyelectrolyte capillary coating conditions on protein analysis in CE[END_REF] are both strong polyelectrolytes, and thus lead to very stable polyelectrolyte complexes towards salt addition even at high concentration. The low stability of the coating and poor separation efficiency can be due to a lower homogeneity of the layer exposed to the solution in the capillary due to the destabilization of the coating with increasing ionic strength of the construction electrolyte. As a general trend, the electroosmotic mobility decreased (in absolute value) with increasing ionic strength of the coating solution from typically about 5 to 10 TU when passing from 0 to 1M added NaCl. The loss in separation efficiency with increasing ionic strength is not similar for all coatings: PB-PSS seems more robust than those with PLC or DEAEDq.

b 4 proteins: Lac (2), RNAse (3), Myo (4), TI (5). Protein (1) was not detected (EOF was too slow). c 3 proteins: RNAse (3), Myo (4), TI [START_REF] Doherty | Microchannel wall coatings for protein separations by capillary and chip electrophoresis[END_REF]. Protein (1) and (2) were not detected (EOF was too slow). Interestingly it is possible not only to separate according to the number of mannose, but also to separate several positional isomers of the same mass, which was not possible applying a neutral coating (data not shown). To the best of our knowledge this is the first time that such a separation is shown for intact glycoproteins. For the 5-mannose variant of ribonuclease up to 4 positional isomers have been determined on the glycan level [START_REF] Prien | The high mannose glycans from bovine ribonuclease B isomer characterization by ion trap MS[END_REF]. Here one wider and one more narrow peaks are observed. Similarly, for the 6 mannose glycoform, two peaks and for the 7 mannose glycoform, three peaks can be distinguished.

CE-MS experiments

For the higher masses, the situation is not clear due to weak intensity and potentially due to the high number of possible isomers.

Conclusions

In this work, up to 43 different 5-layers SMIL coated capillaries were evaluated for CE separation of proteins in a 0.5M acetic acid BGE. It has been shown that the nature of the polycation used in the SMIL coating allows modulating the electroosmotic mobility on a wide range of mobilities (about 23 TU interval): from -39.5 TU in the case of the DEAEDq/PAA, up to -62.6 TU for PB/PSS. EOF magnitude (absolute value of the electroosmotic mobility) was found to decrease in the order PB>-PLL>DEAEDq, whatever the polyanion associated. Coatings with PLC gave the best results in terms of separation efficiencies (around 300,000 plates/m for PB/PLC and -PLL/PLC couples). The second best polyanion after PLC was PMA, closely followed by PSS. From this study and from [START_REF] Bekri | Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking[END_REF], we can conclude that the best SMIL coatings were obtained with: (i) polycations having low to moderate linear charge densities (ξ between 0.35 and 1.44) and (ii) with PLC polyanion (low charge density and amide structure) ahead from vinylic polyanions (in the decreasing order of PMA, PSS and PAA), and far beyond polysaccharides (DS et HA). Excellent average intracapillary RSD on protein migration times was obtained (1.3%). The time of preparation of the 5-layers SMIL coating is about 1.5 h, including the activation of the capillary (35 min), but the coating is robust and doesn't require regeneration between runs. The whole procedure is simple and easy to implement and is fully automatized using commercial CE equipment. Surprisingly, increasing the ionic strength of the coating solution tends to decrease the separation efficiency of the proteins, at least in the 0.5 M acetic acid BGE. The presented novel coatings have been shown to be compatible with CE-MS (no interferences have been observed). The separation of glycoforms of the same mass (positional or structural isomers) of ribonuclease B shows the potential of the set-up.

  AgilentTechnology (Waldbronn, Germany) coupled to a compact quadrupole -quadrupole time of flight (QqTOF) -MS instrument from Bruker Daltonik (Bremen, Germany). The CZE was performed using the same parameter (BGE, temperature, voltage) as given above. The CZE-ESI-MS coupling was performed with a commercial triple-tube sheath liquid interface (Agilent Technologies, Waldbronn, Germany). The sheath liquid (SL) was composed of isopropanol (IPA) / H 2 O (1:1) with 0.5% (v/v)) of formic acid (FAc). The SL was delivered at a flow rate of 4 µL/min by a syringe pump (Cole-Parmer®, IL, USA) equipped with a 5-mL syringe (5MDF-LL-GT, SGE Analytical Science, Melbourne, Australia). The nebulizer was set to 0.3 bar. Electrospray ionization was performed in positive mode (U=4500V). Mass Spectra were acquired at m/z=500-2500 using 1Hz data acquisition. Data processing was carried out by Bruker Compass Data Analysis software (Version 4.3, Bruker Daltonik, Bremen, Germany).

Figure 1 .

 1 Figure 1. Electrophoretic separation of 5 test proteins in acetic acid BGE on 5-layers SMIL coatings based on -PLL or PB polycations (A) or on DEAEDq polycation (B) (polyanion as indicated on the graph). Experimental conditions: 5-layers SMIL coated capillary terminating with the polycation. Capillary: 40 cm (31.75 cm to the detector) x 50 μm I.D. Electrolyte: 0.5 M acetic acid, pH 2.5. Applied voltage: -30 kV. Hydrodynamic injection: 30 mbar, 4 s. Sample mixture: 1 g/L of each protein in water. Peak identification: Lyz (1), Lac (2), RNAse A (3), Myo (4), TI (5) at 1 g/L each in water. Hydrodynamic co-injection of 0.01 % DMF: 30 mbar, 4 s. Temperature: 25 °C. For the coating procedure, see the experimental section.

  15 M, 0.5 M and 1 M) of NaCl were added into the 20 mM HEPES solution buffer pH 7.4 used as a background electrolyte for the SMIL construction. The corresponding electrophoregrams are given in Figure 3A to 3C for the 6 couples of PE (PB/PSS, PB/PMA, PB/PLC, -PLL/PLC, DEAEDq/PMA and DEAEDq/PLC). Three different Figures are used because the migration times are very different from one coating to the other due to differences in EOF. Numerical data about the repeatability of the separation performances are given in Table

Figure 3 .

 3 Figure 3. Influence of the NaCl concentration added to the construction electrolyte on the electrophoretic separation of 5 test proteins in 0.5 M acetic acid. Experimental conditions: 5-layers SMIL coatings (PB/PSS, PB/PMA, PB/PLC, -PLL/PLC, DEAEDq/PMA and DEAEDq/PLC, as indicated on the graphs), 40 cm (31.75 cm to detector) x 50 m I.D. capillary. Electrolyte: 0.5 M acetic acid, pH 2.5. Applied voltage: -30 kV. Hydrodynamic injection: 30 mbar, 4 s (0.74% of the total capillary volume). Sample mixture: 1 g/L of each protein in water. Peak identification: Lyz (1), Lac (2), RNAse (3), Myo (4), TI (5). Hydrodynamic co-injection of 0.01 % DMF: 30 mbar, 4 s. Temperature: 25 °C. Coating solutions: 3g/L PE in 20 mM HEPES pH 7.4 containing NaCl at a concentration indicated on the graphs. See the experimental section for other experimental conditions.

  Various CE-MS experiments have been performed applying several of the SMIL coatings to both the standard proteins and some glycoproteins. It turns out that the coated capillaries can be used for CE-MS experiments without any interferences in the ionization or in the resulting mass spectra. Based on the results for the standard proteins, it can be concluded that the separation efficiency can be maintained beside some minor impact of the interface (open capillary rather than a pressure balanced system as in the CZE-UV experiments). To illustrate the possibilities, the basic glycoprotein ribonuclease B was measured with DEAEDq-PLC coating. Extracted ion electropherograms of the four most abundant charge states of the various glycoforms (5-9 mannose from the N-glycosylation of the highmannose type) are shown in Figure4.

Figure 4 .

 4 Figure 4. CZE-MS of ribonuclease B applying a DEAEDq-PLC coating. Extracted Ion Electropherogramm of glycoforms with 5 mannose (m/z=1355.5; 1490.9; 1656.5; 1863.4 corresponding to 14899.3u) (A), 6 mannose (1370.2; 1507.1; 1674.5; 1883.7 corresponding to 15061.5u) (B), 7 mannose (1385.0; 1523.4; 1692.5; 1904.0 corresponding to 15223.6u) (C), 8 mannose (1399.7; 1539.6; 1710.5; 1924.2 corresponding to 15385.8u) (D), 9 mannose (1414.4; 1555.8; 1728.5; 1944.5 corresponding to 15547.9u) (E). The vertical line through the maximum of (B) is drawn to illustrate the separation.

  the 13 new SMIL coatings studied in this work and presented in Figures1A and 1B, six coatings with the highest separation performances were selected for the intercapillary reproducibility study (see section 3.2): namely PB/PSS, PB/PMA, PB/PLC, -PLL/PLC, DEAEDq/PMA and DEAEDq/PLC. The six polyelectrolyte couples were selected to get both the best separation efficiencies and to cover a wide range of electroosmotic mobilities.As a matter of comparison, Table2gathers some figures of merit about the separation efficiency, and the repeatability on migration times, using SMIL coatings and volatile electrolytes (mostly in acidic conditions), taken in the literature and the 6 best coatings studied in this work. Compared to Table1, Table2also gives the detailed performances for each protein, and the N/l values averaged on n repetitions when available (n=7 in this work).

Table 1 .

 1 Electroosmotic mobility and separation performances (separation efficiency and repeatability) obtained for the 5-layers polyelectrolyte coatings studied in the present work. Experimental conditions as in Figure1. +/-one SD based on n=7 repetitions b average on all detected proteins for the first run c RSD (%) on migration times are calculated by the average of the RSD obtained for the five proteins on n=7 repetitions.

	Polycation Polyanion	μ eof (10 -9 m -2 V -1 s -1 ) a	N/l (10 3 plates/m) b	RSD t m (%) c	RSD t eof (%) c
		PSS	-62.6 +/-1.1	103	2.5	1.1
	PB	PMA DS	-55.3 +/-0.5 -54.3 +/-1.5	145 67	1.1 4.5	0.5 1.5
		PLC	-50.5 +/-0.8	293	2.3	0.8
		PLC	-53.9 +/-0.4	336	1.0	0.4
	ɛ-PLL	PSS PMA	-51.2 +/-1.4 -50.3 +/-1.0	58 102	4.9 3.4	1.3 1.0
		PAA	-48.0 +/-2.3	115	8.1	2.2
		PMA	-45.6 +/-0.8	139	3.4	0.8
		PLC	-43.3 +/-0.3	136	0.5	0.3
	DEAEDq	DS	-43.0 +/-0.8	87	4.4	0.8
		PSS	-42.8 +/-0.4	125	3.3	0.4
		PAA	-39.5 +/-1.2	94	5.6	1.2

a

Table 2 .

 2 Figures of merit of different SMIL coatings reported in the literature for the separation of proteins and one peptide (oxytocine) in volatile electrolytes and comparison with the experimental data obtained in this work.

	Nature of the SMIL coating	Solute (injected concentration)	Experimental conditions a	on t m 2 V -1 s -1 ), RSD μ eof (10 -9 m -	plates/m) b N/l (10 3	n b	Ref
	PB-PVS	rhGH	75 mM ammonium	Not	150	5	[31]
		(1.5 g/L)	formate, pH 8.5 ; 80	mentioned
			cm × 70 cm × 50
			µm ; + 30 kV ; 20 °C
	PB-DS-PB	Oxytocin	50 mM acetic acid,	Not	143	5	[31]
		(50 mg/L)	pH 3 ; 80 cm × 70	mentioned
			cm × 50 µm ; -30
			kV ; 20 °C
	PB-DS-PB	Oxytocin	525 mM acetic acid,		429	5	[31]
		(50 mg/L)	pH 3 ; 80 cm × 70	1.4 %
			cm × 50 µm ; -30
			kV ; 20 °C
	PDADMA-	Lys	0.5 M acetic acid,	-54.6	135	4	[22]
	PLC-	Lac	pH 2.5 ; 40 cm × 30	0.8 %	170
	PDADMA-	RNAse A	cm × 50 m ; -30		62
	PLC-PDADMA	Myo TI c	kV ; 25 °C		122 65

a BGE, capillary dimensions, voltage, temperature

Table 4 .

 4 Electroosmotic mobility and separation performances (separation efficiency and RSD on migration times) obtained for 5-layers polyelectrolyte coating at different concentrations of NaCl in the construction electrolyte. μ eof and <N/l> 7 runs are calculated on the average of 7 repetitions. RSD% on migration times are calculated by the average of the RSD on all detected proteins. See experimental section for experimental conditions.

	NaCl (M)	Polycation Polyanion	μ eof (10 -9 m -2 V -1 s -1 )		<N/l> 7 runs (10 3 plates/m)	RSD% t m	RSD% t eof
					5 p a	4 p b	3 p c
	0	PB	PSS	-62.0	321			3.4	1.3
	0.15			-57.9	216			1.9	0.4
	0.5			-54.9	193			2.3	0.5
	1			-49.7	123			7.7	1.9
	0	PB	PMA	-55.3	209	204		0.1	0.1
	0.15			-54.4	128	132		6.7	3.1
	0.5			-53.1	122	110		2.9	0.6
	1			-50.1	-	86		3.8	0.8
	0	PB	PLC	-50.5	266	250		2.3	0.8
	0.15			-50.8	191	196		3.7	0.4
	0.5			-47.3	-	61		5.2	1.4
	1			-42.0	45	46		6.7	0.8
	0	-PLL	PLC	-53.9	297			1.0	0.4
	0.15			-47.2	264			5.8	1.0
	0.5			-45.1	174			5.6	0.7
	1			-43.2	82			4.6	1.0
	0	DEAEDq	PMA	-43.3	105		115	1.4	0.5
	0.15			-39.9	-	-	65	6.8	1.2
	0.5			-39.2	-	-	45	8.6	0.1
	1			-35.8	-	-	82	5.3	0.7
	0	DEAEDq	PSS	-43.1	-	107		3.3	0.4
	0.15			-41.4	-	96		8.6	1.0
	0.5			-39.4	-	85		8.0	0.8
	1			-39.2	-	76		9.0	0.8
	a 5 proteins: Lyz (1), Lac (2), RNAse (3), Myo (4), TI

b n is the number of repetitions for the calculation of N/l

c Each protein at 1 g/L

important parameter to optimize SMIL coating performances is the ionic strength of the coating solutions.

LIST OF CAPTIONS