Emilie Rossi

N Thuy Tran

Christophe Hirtz

Sylvain Lehmann

Myriam Taverna

Efficient extraction of intact HSA-Aβ peptide complexes from sera: toward albuminome biomarker identification

Keywords:

The performances of three commercial albumin extraction methods for the isolation of intact albumin-amyloid beta peptide (HSA-Aβ) complexes from serum were compared using different analytical approaches. To determine the extraction yield, the repeatability and the selectivity of the extraction procedures, a capillary electrophoresis coupled to UV detection method was developed. For the evaluation of the specificity and integrity of the extracted HSA-Aβ complexes, SDS-PAGE, hybrid and ultra-sensitive ELISA experiments were conducted. All the extraction methods showed different characteristics depending on their chemical binding affinities toward albumin. The ProteoExtract Albumin Depletion kit extracted albumin with a high repeatability but was not efficient for the extraction of intact HSA-Aβ complexes. The PureProteome Albumin magnetic beads showed a high specificity toward HSA thanks to the grafting of anti-HSA antibodies on their surface but tended to dissociate HSA from Aβ peptides. The Pierce Albumin depletion kit showed a high extraction yield, no selectivity towards the different albumin proteoforms and proved to be the most efficient method for the extraction of intact HSA-Aβ complexes from serum.

Introduction

Human Serum Albumin (HSA) is the most abundant protein in human serum with a concentration of 640 µM [START_REF] Carter | Structure of serum albumin[END_REF]. HSA is well known to regulate the osmotic pressure in blood but also to bind and transport a large variety of endogenous (like fatty acids for example) and exogenous compounds (drugs) [START_REF] Fasano | The extraordinary ligand binding properties of human serum albumin[END_REF][START_REF] Hansen | Practical Aspects of the Ligand-Binding and Enzymatic Properties of human serum albumin[END_REF].

HSA can also bind small metabolites, peptides and proteins [START_REF] Weisiger | Affinity of human serum albumin for bilirubin varies with albumin concentration and buffer composition. Results of a novel ultrafiltration method[END_REF][START_REF] Gay | Characterization of peptides and proteins in commercial HSA solutions[END_REF]. Proteins and peptides bound to HSA have been called "albuminome" for the first time by Gundry's group in 2005 [START_REF] Cotter | Tandem Time-of-Flight ( TOF / TOF ) Mass Spectrometry and Proteomics[END_REF]. Since that, more and more studies have been conducted on albumin and its bound proteins and peptides [START_REF] Gundry | Investigation of an albuminenriched fraction of human serum and its albuminome[END_REF][START_REF] Holewinski | A fast and reproducible method for albumin isolation and depletion from serum and cerebrospinal fluid[END_REF][START_REF] Liu | Proteomic and network analysis of human serum albuminome by integrated use of quick crosslinking and twostep precipitation[END_REF]. Because HSA has a serum half-life of 16-20 days, the albuminome can be protected from the renal clearance and directly reflect the actual physiological state of a patient but also disease stages, which is not always the case for other "free" biomarkers that have shorter half-life [START_REF] Dennis | Albumin binding as a general strategy for improving the pharmacokinetics of proteins[END_REF]. The HSA bound proteins and peptides have indeed been demonstrated as a source of biomarkers and have been used through their proteomic signatures for the diagnosis of different diseases such as cancers [START_REF] Camaggi | Serum albumin-bound proteomic signature for early detection and staging of hepatocarcinoma: Sample variability and data classification[END_REF] or heart diseases [START_REF] Stanley | Heart disease, clinical proteomics and mass spectrometry[END_REF][START_REF] Lopez | Proteomic signatures of serum albumin-bound proteins from stroke patients with and without endovascular closure of PFO are significantly different and suggest a novel mechanism for cholesterol efflux[END_REF]. Among the proteins bound to HSA, we find the amyloid beta peptide 1-42 (Aβ 1-42), one of the three validated biomarkers of Alzheimer Disease (AD) in the cerebrospinal fluid (CSF) [START_REF] Motter | Reduction of β-Amyloid Peptide 42 in the Cerebrospinal Fluid of Patients with Alzheimer's Disease[END_REF][START_REF] Grasso | Mass spectrometry is a multifaceted weapon to be used in the battle against Alzheimer ' s disease : Amyloid beta peptides and beyond[END_REF][START_REF] Zetterberg | Update on biomarkers for amyloid pathology in Alzheimer's disease[END_REF]. The interaction of HSA with Aβ peptides has been first demonstrated in 1996 by Biere et al [START_REF] Biere | Amyloid βpeptide is transported on lipoproteins and albumin in human plasma[END_REF]. Since then, different groups have further characterized this interaction and studied the influence of HSA on Aβ peptides aggregation in the case of AD [START_REF] Kuo | Amyloid-β peptides interact with plasma proteins and erythrocytes: Implications for their quantitation in plasma[END_REF][START_REF] Choi | Molecular Insights into Human Serum Albumin as a Receptor of Amyloid-β in the Extracellular Region[END_REF][START_REF] Bode | Serum Albumin's Protective Inhibition of Amyloid-β Fiber Formation Is Suppressed by Cholesterol, Fatty Acids and Warfarin[END_REF].

The Aβ 1-42 peptide can be quantified in CSF for AD diagnosis thanks to immune enzymatic techniques (ELISA) [START_REF] Zetterberg | Update on biomarkers for amyloid pathology in Alzheimer's disease[END_REF][START_REF] Mo | Cerebrospinal fluid β-amyloid1-42 levels in the differential diagnosis of Alzheimer's disease -Systematic review and metaanalysis[END_REF][START_REF] Olsson | CSF and blood biomarkers for the diagnosis of Alzheimer's disease: a systematic review and metaanalysis[END_REF] or mass spectrometry (MS) [START_REF] Grasso | Mass spectrometry is a multifaceted weapon to be used in the battle against Alzheimer ' s disease : Amyloid beta peptides and beyond[END_REF][START_REF] Kuhlmann | Working, CSF Aβ 1-42 -an excellent but complicated Alzheimer's biomarker -a route to standardisation[END_REF][START_REF] Bros | Quantitative detection of amyloid-β peptides by mass spectrometry: State of the art and clinical applications[END_REF] and it has been shown that AD patients have lower concentrations of this peptide in their CSF than controls [START_REF] Olsson | CSF and blood biomarkers for the diagnosis of Alzheimer's disease: a systematic review and metaanalysis[END_REF]. However, the CSF sampling is invasive and is performed only at the hospital, making it difficult to perform at regular intervals of time to follow the evolution of AD. In addition, it precludes the early detection of this disease as only patients suffering already from cognitive impairment or memory loss attend memory centers or go to a neurologist consultation at the hospital.

In this way, scientists have shown more and more interest in blood as an alternative matrix for AD diagnosis. They tried to quantify the peptide Aβ 1-42 in plasma and serum, but this peptide has not been clinically validated yet [START_REF] Lewczuk | Amyloid β peptides in plasma in early diagnosis of Alzheimer's disease: A multicenter study with multiplexing[END_REF][START_REF] Tzen | Plasma Aβ but not tau is related to brain PiB retention in early Alzheimer's disease[END_REF][START_REF] Graff-Radford | Association of low plasma Aβ42/Aβ40 ratios with increased imminent risk for mild cognitive impairment and Alzheimer disease[END_REF]. Possible reasons are the low concentration of this peptide in plasma (<0.5 nM) and the presence of components in the blood that can cause interferences in the immunoassays. But another explanation is that 90-95% of amyloid peptides are bound to HSA [START_REF] Biere | Amyloid βpeptide is transported on lipoproteins and albumin in human plasma[END_REF][START_REF] Kuo | Amyloid-β peptides interact with plasma proteins and erythrocytes: Implications for their quantitation in plasma[END_REF], so would not be considered in those quantifications. To solve this problem and increase the accuracy and reliability of blood tests, several studies emerged during the last few years. First, to overcome the issues related to the low Aβ peptides concentration in plasma, different studies have developed methods to preconcentrate it before its quantification [START_REF] Ovod | Amyloid β concentrations and stable isotope labeling kinetics of human plasma specific to central nervous system amyloidosis[END_REF][START_REF] Nakamura | High performance plasma amyloid-β biomarkers for Alzheimer's disease[END_REF][START_REF] Schindler | High-precision plasma β-amyloid 42/40 predicts current and future brain amyloidosis[END_REF] or have proposed ultra-sensitive immunoassays able to detect it [START_REF] Vergallo | Plasma amyloid β 40/42 ratio predicts cerebral amyloidosis in cognitively normal individuals at risk for Alzheimer's disease[END_REF]. To lower the interferences of other plasma components, different studies have treated the plasma before analysis [START_REF] Janelidze | Plasma β-amyloid in Alzheimer's disease and vascular disease[END_REF][START_REF] Park | Chemically treated plasma Aβ is a potential bloodbased biomarker for screening cerebral amyloid deposition[END_REF]. Finally, to also consider the amyloid beta bound to other plasma proteins in the quantification, recent works have put some efforts on the treatment of the plasma before analysis by immunoassays [START_REF] Risacher | Plasma amyloid beta levels are associated with cerebral amyloid and tau deposition, Alzheimer's Dement. Diagnosis[END_REF][START_REF] Shahpasand-Kroner | A two-step immunoassay for the simultaneous assessment of Aβ38, Aβ40 and Aβ42 in human blood plasma supports the Aβ42/Aβ40 ratio as a promising biomarker candidate of Alzheimer's disease[END_REF]. However, these studies did not focus on the fraction of Aβ 1-42 peptides specifically bound to HSA. Yamamoto et al. [START_REF] Yamamoto | Serum levels of albumin-amyloid beta complexes are decreased in Alzheimer's disease[END_REF] focused for the first time on the complex formed between Aβ 1-42 and HSA in serum by developing a homemade hybrid ELISA. They showed that low serum Aβ 1-42-HSA complexes level was associated with a higher AD prevalence and this could be used to monitor its progression.

To perform a relevant albuminome investigation, the HSA with its partners has first to be extracted while preserving the interaction between them. The extraction of HSA can be performed using different treatment methods. The most common one is the precipitation with ethanol [START_REF] Colantonio | Effective removal of albumin from serum[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], which enables the isolation of HSA without effective cost, but the precipitation is time consuming and requires the use of chemicals that can break the interaction between HSA and its bound proteins. In 2017, Liu et al [START_REF] Liu | Proteomic and network analysis of human serum albuminome by integrated use of quick crosslinking and twostep precipitation[END_REF] proposed a two-steps precipitation method to extract HSA with its albuminome through the use of formaldehyde as a crosslinker to maintain the interaction between HSA and its bound proteins. However, the release of the covalently bound molecules requires a boiling step that is not adapted in our case for Aβ peptides. A common method to enrich HSA is the use of immunoaffinity or dye-based depletion kits. Since few years, different works have exploited these different depletion procedures to extract the albuminome from plasma [START_REF] Gundry | Investigation of an albuminenriched fraction of human serum and its albuminome[END_REF][START_REF] Liu | Proteomic and network analysis of human serum albuminome by integrated use of quick crosslinking and twostep precipitation[END_REF][START_REF] Lopez | Proteomic signatures of serum albumin-bound proteins from stroke patients with and without endovascular closure of PFO are significantly different and suggest a novel mechanism for cholesterol efflux[END_REF][START_REF] Zhou | An investigation into the human serum "interactome[END_REF]. In 2013, Holewinski [START_REF] Holewinski | A fast and reproducible method for albumin isolation and depletion from serum and cerebrospinal fluid[END_REF] et al compared several methods to isolate albumin and its bound proteins from serum. Their goal was to select the most interesting one in terms of efficiency and repeatability. They then characterized the isolated HSA bound proteins by proteomic approaches (LC-MS/MS) without releasing a priori the bound proteins. So, no evidence was provided in this study on the stability of the isolated HSA-proteins complexes during the extraction procedures.

More recently, Pisanu et al [START_REF] Pisanu | Comparative evaluation of seven commercial products for human serum enrichment/depletion by shotgun proteomics[END_REF] and De Jesus et al [START_REF] De Jesus | Depleting high-abundant and enriching low-abundant proteins in human serum: An evaluation of sample preparation methods using magnetic nanoparticle, chemical depletion and immunoaffinity techniques[END_REF] have compared different depletion procedures but this time to remove the highest abundant proteins for biomarker discovery studies. In conclusion, none of these studies has focused on the isolation of intact HSA complexes and on maintaining as much as possible the interaction between HSA and its partners.

The objective of our study was therefore to investigate commercial HSA depletion methods as new tools for intact HSA complexes extraction to quantify bound Aβ peptides in blood. Our goal was to select the one allowing an efficient extraction of HSA, maintaining as much as possible the HSA-Aβ complexes and producing a good repeatability and specificity toward HSA. In addition, as HSA exists as a mixture of several proteoforms (native, cysteinylated, glycated, carbamylated,…) [START_REF] Marie | Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin[END_REF][START_REF] Taverna | Specific antioxidant properties of human serum albumin[END_REF] and considering that the glycated HSA proteoform has been demonstrated to bind molecules with different affinities compared to native HSA [START_REF] Neelofar | An overview of in vitro and in vivo glycation of albumin: a potential disease marker in diabetes mellitus[END_REF], a particular attention was paid to evaluate the ability of these extraction methods to preserve the natural abundance of each of them during the depletion process. To allow this evaluation, we developed a quantitative capillary electrophoresis method to determine not only the extracted HSA amount but also its proteoform profile after extraction. This is the first time that albumin proteoform abundances were quantified and compared before and after an extraction process. To ensure that the selected extraction methods did not alter the HSA-Aβ complexation, two different approaches were employed to analyse the extraction fraction. The first one consists in the quantification of the complex by the hybrid ELISA developed by Yamamoto et al [START_REF] Yamamoto | Serum levels of albumin-amyloid beta complexes are decreased in Alzheimer's disease[END_REF]. The second consists in the quantification of Aβ 1-42 peptides released from HSA during the extraction procedure. Brilliant Blue R-250 were acquired from Bio-Rad (Hercules, CA, USA). Pre-stain proteins ladder was obtained from Euromedex (Souffelweyersheim, France). The Human β amyloid (1-42) high sensitive Elisa kit was obtained from Wako (Osaka, Japan). The Neurology 3-Plex A from Quanterix (Lexington, MA, USA) was used as well as the following reagents: Simoa Discs, Simoa Cuvettes, Simoa Sealing Oil and SIMOA Buffer (1&2). Deionized water was prepared using a Direct-QR 3 Water Purification System from Millipore (Billerica, MA, USA). All buffers were prepared using deionized water and filtered through a 0.22 µm nylon membrane (VWR) before use.

Experimental

Biological samples

Horseradish peroxidase (HRP) conjugated goat anti human albumin antibody was obtained from Euromedex (Souffelweyersheim, France). For the specificity and integrity with the hybrid ELISA studies, serum samples were obtained from Zen-Bio. They were collected from 25 consented healthy adult donors in a United States FDA licenced Blood Bank, aliquoted upon reception and stored at -20°C before use. For the integrity study with the SIMOA ELISA, serum samples originate from the ethically approved Montpellier Neurobank #DC-2008-417 at the certified NFS 96-900 CHU resource center BB-0033-00031, www.biobanques.eu. Authorization to handle personal data was granted by the French Data Protection Authority (CNIL) under the number 1709743 v0.d.

Albumin extraction methods

A commercial plasma derived HSA solution at 40 mg/mL in water, called the "HSA standard solution" was first prepared. HSA was then extracted from human serum or from the HSA standard solution according to the manufacturer's instructions with little modifications for the three albumin extraction kits.

Extraction of HSA with Pierce Albumin Depletion Kit

First, the depletion column was prepared by adding 400 µL of the slurry resin into a spin column. The column was then centrifuged with a micro star 17 R centrifuge (VWR) for 1 min at 12,000 g to remove the excess liquid. 200 µL of binding/wash solution (included in the kit) was then added to activate the resin after centrifugation (12,000 g for 1min). 15 µL of serum or HSA standard solution was applied onto the column, incubated for 2 minutes at room temperature and the column was then centrifugated (7000 g for 1 minute). The flow through was reapplied onto the column. After a second incubation and centrifugation in the same conditions than before, the column was washed 4 times with 50 µL of binding/wash solution each. To elute the extracted albumin and its bound partners, the resin was washed 4 times with 200 µL of a solution composed of 20 mM sodium phosphate and 250 mM sodium thiocyanate, pH 7.2. The elution fractions were collected separately in 2mL Eppendorf tubes before being pooled for further analysis.

Extraction of HSA with ProteoExtract Albumin Depletion Kit

The column was first prepared by removing the storage solution and then activated by adding 1 mL of the ProteoExtract Albumin Binding Buffer using a gravity flow. 30 µL of serum or HSA standard solution was diluted in the binding buffer to have a total volume of 350 µL which was then applied onto the column. The column was then washed twice with 600 µL of binding buffer. To elute the extracted albumin, the column was washed twice with 600 µL of a solution containing 20 mM sodium phosphate and 250 mM sodium thiocyanate, pH 7.2. The elution fraction was then analysed directly by CZE-UV or SDS-PAGE.

Extraction of HSA with PureProteome Albumin Magnetic Beads

The beads were first resuspended by vortexing the tube for 10 seconds. 750 µL of the resuspended beads were then pipetted and added in a 2 mL Eppendorf tube. The tube was then placed in a magnetic stand to allow the beads migrating to the magnet, then the storage buffer was removed. This step was performed every time to remove any liquid from the beads. The beads were then washed twice with 500 µL of PBS. For each step, PBS was added to the beads and the tube was vortexed for 10 seconds before removing the PBS solution. Then, 30 µL of serum or HSA standard solution diluted in 70 µL of PBS was applied on the beads and incubated for one hour with a continuous end-over-end rotation. The tube was then placed in the magnetic stand to remove the HSA-depleted serum. The beads were then washed three times with 500 µL PBS. To recover the extracted HSA, the beads were resuspended three times with 120 µL of 200 mM Glycine-HCl, pH 2.0. All the elution fractions were then neutralized with 80 µL of 1M Tris, pH 8.0 (to get a final volume of 200 µL for each elution fraction) before being pooled for further analysis.

Capillary zone electrophoresis coupled to UV detection (CZE-UV) 2.4.1 Instrumentation

Capillary electrophoresis experiments were conducted on a P/ACE MDQ Instrument (Sciex, Framingham, MA, USA), coupled to a diode array detection.

Method

UV detection was set at 214 nm. Polyvinyl alcohol (PVA) capillaries, purchased from Sciex with an internal diameter of 50 µm and a total length of 60.2 cm (effective length 50 cm) were employed.

Software 32 KaratTM version 7.0 (Sciex) was used to control the instrument and to collect the data. The background electrolyte (BGE) consisting of 75 mM HEPES at pH 8.0, containing 0.5 mM SDS was prepared by adding 0.894 g of HEPES and 7.21 mg of SDS to deionized water. The pH was then adjusted to 8.0 with 1 M NaOH before adding water to a final volume of 50 mL. Before the first use, the capillary was rinsed at 30 psi during 10 minutes with water and BGE. Between each analysis, the capillary was flushed at 20 psi during 5 minutes with water and BGE. The sample was hydrodynamically injected from the inlet by applying a pressure of 0.5 psi for 15 seconds. The separation was then conducted at -25 kV at 25°C. At the end of the day, the capillary was rinsed with water (3 min, 30 psi) and BGE (3min, 30 psi) before being stored at 4°C with the ends in water.

Validation

The developed CZE-UV method for the separation of the HSA proteoforms was validated using a plasma derived HSA standard sample at 1 mg/mL in water. For the intraday study, the sample was analysed six consecutive times, the same day and using the same capillary. For the intermediate precision study, the sample was analysed over three days using the same capillary. For the inter-capillary study, the sample was analysed twice on three PVA capillaries over different days (n=6). The relative standard deviation (RSD) of the migration times (tm), total peak area (tPA) (sum of the peak areas of all HSA proteoforms) and relative peak area (rPA) (percentage of each HSA proteoform peak, related to the total peak area) were then calculated. For the linearity study, plasma-derived HSA standard solutions at 5 concentrations were prepared (1.0;1.5;2.0;4.0;5.0 mg/mL) and analysed three times with the developed CZE-UV method. The tPA was then calculated for each albumin concentration. Fisher's tests were then performed to validate the linear model between the HSA concentration and the tPA. The limit of detection (LOD) and limit of quantification (LOQ) were determined using the slope of the calibration curve [START_REF] Shrivastava | Methods for the determination of limit of detection and limit of quantitation of the analytical methods[END_REF] .

Quantification

To quantify HSA, a calibration curve was created by measuring tPA for different HSA concentrations (1.0-5.0 mg/mL) using the developed CZE-UV method. The HSA from the HSA standard solution was then extracted using one of each depletion kit and the extraction yield could be determined using the calibration curve. By knowing the amount of HSA employed for each extraction method and the resulting amount of HSA in each elution fraction, the HSA extraction yield (HSA EY) could be easily determined using eqn (1): After this, the solutions were discarded from the wells and the microplate was washed five times with 300 µL of washing buffer. After this washing step, 100 µL of HRP-conjugated anti human albumin polyclonal antibody, diluted 1:30000 in a solution of 1:25 diluted Blocking Reagent, was added in the different wells. The microplate was covered with a plate seal and was then incubated at 4°C for one hour. The antibody solutions were then removed from the wells and the microplate was washed five times with the washing solution. After washing, 100 µL of 3,3',5,5'-tetramethylbenzidine substrate (TMB) were added to the wells. The microplate was then covered with an aluminium paper and left in the dark for 30 min. To stop the reaction, 100 µL of acidic stop solution was added into each well.

∶ % = amount of
Absorbances were read at 450 nm with an EL800 instrument from Biotek (Winooski, Vermont, USA).

Measurements of all samples were carried out in triplicates. For the HSA-Aβ complex quantification, a 2.6.2 SIMOA (Quanterix) for amyloid beta peptide 1-42 quantification

Sample preparation method

After extraction with the Pierce Albumin Depletion kit or PureProteome Albumin Magnetic Beads, the elution fractions were filtered and dissolved in PBS in order to concentrate and quantify the free amyloid-beta peptide 1-42. For that, after having washed a 3 kDa Nanosep membrane with 2x500 µL of deionized water, the elution fraction was applied onto the membrane and centrifuged at 4,000 g until approximately 20 µL of sample was left in the retentate. Then 500 µL of PBS was added onto the membrane and the obtained sample was centrifuged at 4,000 g until 70 µL was left onto the membrane.

This fraction was then analysed. Two duplicates per extraction methods were prepared.

Analysis of the free amyloid-beta peptide 1-42

Concentration of human amyloid beta peptide 1-42 was measured with the Single Molecule Array (SIMOA ) technology (Quanterix, Lexington, MA, USA), comprising a SIMOA HD-1 analyzer [START_REF] Rissin | Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations[END_REF].

This method is approximately 1000-fold more sensitive than common ELISA methods. The monoclonal anti Aβ capture antibody was attached to the surface of paramagnetic beads. The presence of the antigen was recognized by biotinylated monoclonal anti Aβ 1-42 detection antibodies. After the formation of sandwich complexes on magnetic micro beads, streptavidin-β-galactosidase was added to bind to the biotinylated detection antibody, when present. Resorufin ß-d-galactopyranoside, which acts as a substrate, becomes fluorescent when it comes into contact with the streptavidin-β-galactosidase fixed on the beads that had captured the antigen. The beads were then loaded into arrays of 216,000 femtolitersized wells, which held only one bead per well. The wells were sealed with oil. The number of fluorescent wells was counted. The ratio of wells containing a fluorescent bead to the total number of wells is proportional to the concentration of the antigen. The free Aβ 1-42 peptide samples were diluted 4-fold with the Quanterix SIMOA sample diluent buffer before analysis. Controls for SIMOA performance were obtained with serial measurement of quality control (QC) samples; this analysis confirmed the low intra-assay variation (CV ranged from 1 to 6%) and the good inter-assay variation (CV ranged from 4 to 18%).

Results and discussion

Optimization and validation of the CZE-UV method for the quantification of human serum albumin

In order to compare the performances of different HSA depletion approaches aimed at extracting HSA complexed to its partners, we developed first a reliable quantitative method for HSA analysis. Most current methods for HSA quantification measure the total HSA concentration. However, as this plasma protein exhibits several proteoforms, we searched for a method allowing not only the total concentration determination but also the relative abundance of each proteoform. In order to avoid HSA adsorption onto the capillary wall, several neutral coated capillaries were tested. According to our previous work on CE-UV of HSA [START_REF] Marie | Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin[END_REF], homemade PEO coated capillary was first used with a BGE of 75 mM HEPES, pH 7.4 containing 0.5 mM SDS. As expected, a separation of three HSA proteoforms into glycated (peak1), native (peak 2) and cysteinylated forms (peak 3), was obtained (figure 1A). However, the regeneration of this semi-permanent coating each day and between runs was necessary to obtain a good reproducibility, thus increasing the total analysis time. A commercial permanent PVA coated capillary avoiding tedious regeneration steps, was then tested under the same electrophoretic conditions. A similar and well-resolved separation of the three HSA proteoforms was achieved (figure 1B) with that alternative coating. The resolution between peaks 1 and 2 (Rs1,2), or peaks 2 and 3 (Rs2,3) and the asymmetry factors (0.80-1.2) were similar with both capillaries. Moreover, the analysis duration with the PVA capillary was significantly shortened. The BGE was also optimized before the method validation to improve resolution between peaks 2 and 3 for better quantification accuracy. By increasing the pH of the BGE from 7.4 to 8.0, this resolution was greatly improved. At pH 8.0, both resolutions (Rs1,2 and Rs2,3) (figure 1C) were satisfactory. Considering the significant reduction of the total analysis time, the PVA capillary has been selected for the rest of our studies.

Before applying the CZE-UV method to real samples after HSA extraction from serum, the validation of the method was carried out in terms of intra-day, inter-day and inter-capillary repeatability and linearity. The RSD obtained for tm, tPA and rPA of the three main peaks (native, glycated and cysteinylated proteoforms) are reported in Table 1. The intra-day study showed the excellent repeatability for tm and very satisfactory repeatabilities for tPA and rPA. For the inter-day experiments, the RSD of the different parameters slightly increased but remained satisfactory with values less than 5% for tPA and rPA. The RSD of the tPA and rPA of the inter-capillary study were very satisfactory, especially when considering it was conducted on different days. All these results showed the excellent reproducibility of the method. The quantification performances of the method were also evaluated by analysing plasma derived HSA standard solutions in water at five concentrations and by plotting the total peak area against the concentration. It showed that the method was linear in the range of 1.0 to 5.0 mg/mL (y= 77321x-5616) with a good correlation coefficient (0.98). A linearity test was performed and validated the linearity of the developed method (Fexp = 0.044 < Flimit = F5% [START_REF] Hansen | Practical Aspects of the Ligand-Binding and Enzymatic Properties of human serum albumin[END_REF][START_REF] Dennis | Albumin binding as a general strategy for improving the pharmacokinetics of proteins[END_REF]). The LOD and the LOQ were determined to be 0.31 and 1.0 mg/mL respectively [START_REF] Shrivastava | Methods for the determination of limit of detection and limit of quantitation of the analytical methods[END_REF]. These results indicated that the method is suitable for the quantification of HSA in the elution fractions from the different enrichment methods.

Comparison of the performances of the different extraction methods

To select the most efficient HSA extraction method to enrich HSA and its partners from plasma, three HSA extraction methods based on different affinity mechanisms were compared in terms of yield and repeatability of extraction, selectivity toward the different HSA proteoforms, specificity regarding other plasma proteins and integrity of the isolated complex. The HSA standard solution at 40 mg/mL was treated to assess the three first criteria whereas serum was rather used for specificity and integrity of the methods.

Extraction Yield

The extraction yield of each HSA extraction method was determined by analysing the elution fractions resulting from the treatment of the HSA standard solution. The analysis was performed with the developed CZE-UV method. Six replicates of each extraction methods were prepared, and the six corresponding elution fractions were analysed the same day to determine the mean yield of extraction and its standard deviation. The extraction yields obtained were 83 + 17%, 71 + 5% and 77 + 10 % for Pierce Albumin Depletion kit, ProteoExtract Albumin Depletion kit and PureProteome Albumin Magnetic Beads, respectively. Pierce Albumin Depletion kit is thus the method extracting the highest amount of HSA but also showing the less satisfactory repeatability. On the contrary, the ProteoExtract Albumin Depletion kit extracts slightly less HSA than the two others but exhibits the highest repeatability. The high extraction yield of Pierce extraction method could be explained by the fact that centrifugation is used to recover the different elution fractions from the resin so that sample loss is less likely to occur. On the contrary, for ProteoExtract extraction method, a gravity flow is employed to recover the elution fraction, so it is possible that some sample stays stuck onto the resin.

Selectivity for a particular HSA proteoform

The selectivity of each HSA extraction method was evaluated by comparing the abundance of the three HSA proteoforms in the HSA standard solution before and after 6 treatments performed using each enrichment method. The initial solution and elution fractions were analysed by the CZE-UV method.

By comparing the abundances and the associated standard deviation of the three main HSA proteoforms in the elution fractions and in the HSA standard solution, the Pierce Albumin Depletion kit and the PureProteome magnetic beads did not exhibit selectivity for a particular HSA proteoform (Figure 2). On the contrary, after treatment with the ProteoExtract Albumin Depletion kit, the glycated form was slightly enriched whereas the cysteinylated form was depleted. To ascertain the observed selectivity of the ProteoExtract Depletion kit for these two proteoforms, a Mann-Whitney test was performed. The glycated form abundance in the HSA standard was significantly different from that in the ProteoExtract elution fractions (p value < 0.05). The same result was obtained for the cysteinylated form. Thus, this statistical test confirmed that the ProteoExtract led to an enrichment of the glycated at the expense of the cysteinylated HSA proteoforms. The selectivity tendency of ProteoExtract could be explained by the interaction mode involved between the solid support and HSA. Indeed, ProteoExtract is composed of a resin which captures HSA through electrostatic interactions. HSA proteoforms have different hydrodynamic volumes, and do not expose thus the same charges at their surfaces. Their resulting electrostatic interaction with the resin can therefore be different. Thus, it is possible that the resin of ProteoExtract interacts preferentially with the glycated form. Although Pierce extraction method is also based on a resin substrate, this specificity could not be observed due to its low repeatability (see 3.2.3).

HSA extraction repeatability

The repeatability of each extraction method was evaluated the same day by extracting six times the same batch of HSA standard solution. The RSD of the extraction yield and proteoform abundances in the elution fractions for the different procedures are reported in Table 2. The Pierce Albumin Depletion kit and PureProteome Albumin magnetic beads led to less repeatability. For Pierce, several elution fractions were collected separately before being pooled together for analysis. Thus, it is possible that HSA was not fully recovered from the different Eppendorf tubes and that the volume of the pooled elution fraction was not exactly the same between replicates. For PureProteome Albumin magnetic beads, the elution fractions were collected through the capture of the beads by a magnetic stand, so recovered volume variation may also occur between replicates. The ProteoExtract Albumin Depletion kit showed the most interesting results in terms of repeatability as elution fractions were recovered once in one tube, without any centrifugation or magnetic stand.

HSA extraction specificity

The specificity of each HSA isolation procedure was assessed by analysing the extracted proteins present in the elution fraction by SDS-PAGE with coomassie staining (Figure 3) and calculating the percentage of albumin in the corresponding elution fraction. PureProteome Albumin Magnetic Beads and ProteoExtract Albumin Depletion kits were the most specific methods, extracting 93.5% and 92.2% of albumin respectively. For the Pierce Albumin Depletion kit, other bands at 150 kDa and ~ 80 kDa for instance which may correspond to Immunoglobulin G and transferrin respectively were present in the elution fraction, which led to a less satisfactory specificity (60.3%). The specificity difference between the HSA isolation methods studied is mainly due to the different binding mechanisms involved. Indeed, the PureProteome Albumin Magnetic Beads kit isolates HSA through an antibody ligand, specific to this protein whereas Pierce and ProteoExtract Depletion kits rely rather on electrostatic interaction of HSA with resins, which are known to be less specific than antibodies. The new resin proposed by ProteoExtract appeared however more specific than Cibacron Blue (Pierce).

Integrity of the HSA-Aβ complex

The interaction between albumin and its protein/peptide partners must be preserved during the extraction procedure as our objective is to study the HSA bound Aβ peptides. Thus, the preservation of the HSA-Aβ complex is the most important criteria for our purpose. To evaluate the capacity of the three extraction methods to maintain intact the complex after extraction, two different approaches were used.

The first one consisted in the quantification of the HSA-Aβ complexes in the elution fractions by a hybrid ELISA recently reported [START_REF] Yamamoto | Serum levels of albumin-amyloid beta complexes are decreased in Alzheimer's disease[END_REF] which combines a capture anti-Aβ and a detection anti-HSA antibodies. The second approach relied on the determination of the free Aβ 1-42 peptide in the elution fraction by an ultra-sensitive SIMOA ELISA to ensure that the HSA bound Aβ 1-42 peptide was not released from HSA during the elution step. For the first approach, we used the calibration curve resulting from the analysis of diluted sera. By applying serum diluted from 1:4000 to 1:128000, a linear decreasing of the signal was observed (y= 1575x + 0.0469, r²=0.9938). The elution fractions resulting from the three albumin extraction methods and the HSA standard solution were then diluted the same way in PBS for hybrid ELISA analysis. The HSA standard solution signal was slightly higher than the PBS blank one (by 1.2) but lower than that of the three different elution fractions (by ~1.5). This proves that all the elution fractions contained HSA-Aβ complexes. We can however not exclude that some extra HSA could be non-specifically adsorbed on the surface of the ELISA microplate, leading to an overestimation of the signal. However, since the aim of this experiment was only to compare the amount of HSA-Aβ complexes in different elution fractions which were expected to contain the same quantity of HSA (similar extraction yields, see 3.2.1), the hybrid assay could still be used for a comparative study of HSA-Aβ complexes integrity. The extraction yields of total HSA (which includes also HSA-Aβ complexes) calculated based on the calibration curve were 85 + 16%, 18 + 5% and 48 + 9% (n=3)

for Pierce, ProteoExtract and PureProteome kits, respectively. Considering that the yield achieved with ProteoExtract was too low, this extraction method was not retained for the rest of the experiments. For Pierce and PureProteome methods, the second approach was then applied to investigate if the difference in the complex extraction yields was due to a different affinity toward the HSA-Aβ complex or a tendency of PureProteome to dissociate more the extracted complex during the elution step. The concentration of free Aβ 1-42 peptide in the initial serum, determined by SIMOA, was 16.2 pg/mL.

After its extraction with Pierce and PureProteome methods, Aβ 1-42 peptide found in the elution fractions was concentrated and diluted in PBS before SIMOA analysis. Its concentration in the elution fraction after extraction with the Pierce method was 3.2. pg/mL (representing 20% of the initial one).

Using the PureProteome method, the concentration reached 34.9 pg/mL (representing 215% of the initial concentration). This much higher amount of Aβ 1-42 peptide found in the PureProteome elution fraction

indicates that this extraction method partly dissociates the interaction between this peptide and HSA during the elution step. The complexes dissociation tendency of PureProteome method can be explained by the rather acidic conditions (pH 2) required to elute albumin and its partners from the magnetic beads.

On the contrary the elution step of Pierce method is performed under milder conditions (pH 7.2), leading likely to less complexes dissociation. The Aβ 1-42 peptide detected in both elution fractions could however originate also to a certain extend from its nonspecific binding to the resin/beads, especially for Pierce Albumin Depletion kit which showed the lowest specificity towards albumin. However, the Aβ 1-42 peptide nonspecific adsorption on Pierce resin was assumed to be very low considering that its concentration in the elution fraction was five times lower than its initial serum one. The overall results

proved that the HSA-Aβ complexes can be extracted from serum and that the Pierce Albumin depletion kit appears as the most efficient method for this purpose.

Conclusions

Extraction of albumin and its bound partners represents a real challenge and also the most critical step of an albuminome study. In this work, three albumin extraction methods were compared in order to isolate and study specifically the HSA-Aβ complexes. Based on the five criteria investigated (extraction yield, selectivity, repeatability, specificity, and integrity), all the three methods presented interesting but different characteristics. The Proteoextract method presented the most interesting results in terms of repeatability but this method tended to change the natural abundances of HSA proteoforms during the process and it extracted only a small quantity of HSA-Aβ complex. The PureProteome Albumin magnetic beads method extracted more specifically HSA with a high extraction yield but tended to disrupt the interaction between albumin and its bound Aβ peptides. Finally, the best extraction efficiency of intact HSA-Aβ complexes was obtained using the Pierce Albumin Depletion Kit. Indeed, if the extraction method used dissociates even partly the interaction between albumin and its partners, a specific albuminome study is then made impossible. Thus, Pierce Albumin Depletion kit can be considered as the best serum sample preparation method to extract HSA bound Aβ peptides for the study of AD biomarkers.

Illustrations and tables captions

  HSA in the elution fraction x 100 amount of HSA treated2.5 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)SDS-PAGE was used to analyse the elution fractions resulting from the depletion treatments of serum with the three kits. 10 µL of elution fraction was mixed with 2 µL of a sample buffer, composed of 60% glycerol, 300 mM Tris pH 6.8; 6mM EDTA, 12% SDS, 864 mM mercaptoethanol-2 and 0.05% of bromophenol blue. The obtained 12 µL of sample were mixed and heated at 95°C for 10 minutes. The samples were then loaded on the 10% Precast TGX gel. Protein weight markers were also analysed. The electrophoretic separation was performed at 100 V for 5 min and then 130 V for 50 min. The gels were then washed with deionized water for 15 minutes, stained with Coomassie Brilliant Blue for 45 minutes and bleached overnight in a solution of 7% acetic acid in 23% ethanol. Gels were read on a Chemic doc imaging system from BioRad and the images were processed with ImageLab software (BioRad, Hercules, CA, USA). The specificity of each extraction method was estimated by dividing the HSA band intensity in the elution fraction lane by the total band intensities.2.6 Elisa2.6.1 Hybrid ELISATo detect specifically the HSA-Aβ complex, we used the Human β amyloid (1-42) high sensitive Elisa kit and the HRP conjugated anti-HSA antibody. 100 µL of samples (diluted serum, PBS or diluted elution fractions) were dispensed in the wells of the anti-human Aβ 1-42 antibody (BAN50) coated microplate, which was previously blocked for one hour at room temperature with 200 µL of 1:10 diluted Blocking Reagent. The microplate was then covered with a plate seal and refrigerated overnight (4°C).

  calibration curve was created by serial dilution of the initial serum in PBS. The calibration curve was performed in duplicates with serum dilution factor ranging from 1:4000 to 1:128000. The HSA-Aβ complex extraction yields (HSA-Aβ EY) were then calculated thanks to eqn (2): $%& ' ∶ ()* -, % = estimated dilution factor of the treated serum x 100 theoretical dilution factor of the treated serum where the estimated dilution factor of the treated serum is the one determined by using the standard calibration curve, and the theoretical one is determined by considering the dilutions made during the different steps of the extraction procedure.

Figure 1 :

 1 Figure 1: Comparison of CZE-UV profiles obtained for plasma derived HSA standard using PEO and PVA capillaries with different BGE pHs: A) PEO capillary with BGE at pH 7.4; B) PVA capillary with BGE at pH 7.4; C) PVA capillary with BGE at pH 8.0. Peaks assignment: 1: glycated HSA; 2: native HSA; 3: cysteinylated HSA Conditions: Sample: 1mg.ml -1 HSA in water, BGE: 75 mM HEPES with 0.5 mM SDS, capillary: 50 µm, 50 cm (effective length), voltage: -25kV, temperature: 25°C, injection: 0.5 psi for 15 s.

Figure 2 :Figure 3 :Table 1 :

 231 Figure 2: Comparison of the main HSA proteoform abundances in the elution fractions resulting from the treatment of HSA standard solution (40 mg/mL) by three different enrichment methods and in a non-treated HSA standard solution. Plotted values represent the mean+SD for 5 replicates (HSA standard) and 6 replicates (elution fractions). Asterisks * indicate statistically significant difference between HSA standard solution and ProteoExtract elution fraction according to Mann-Whitney test (p value <0.05)

Fig1

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Acknowledgements

This work was supported by the Ecole doctorale « Molécules, Matériaux, Instrumentation et Biosystèmes », which provided the financial support of Emilie Rossi as fellowship. This work was also supported by the « Rotary-Espoir en Tête FR Neurodon » program which financed the SIMOA equipment. The authors would like to thank X. Labouze, S. Karolak and V.Richard for discussions about the statistical tests.