Marine Morvan
email: marine.morvan@fgu.cas.cz

Ivan Mikšík

The chiral proteomic analysis applied to aging collagens by LC-MS: amino acid racemization, post-translational modifications, and sequence degradations during the aging process

Keywords: aging, chiral separation, collagen, D-amino acids, liquid chromatography, mass spectrometry

Collagen is the most abundant protein in the animal and human bodies, and it is not exempt from this aging phenomenon. Some age-related changes may appear on collagen sequences, such as increased surface hydrophobicity, the appearance of post-translational modifications, and amino acids racemization. This study has shown that the protein hydrolysis under deuterium conditions is privileged to limit the natural racemization during the hydrolysis. Indeed, under the deuterium condition, the homochirality of recent collagens is preserved whose amino acids are found in their L-form. However, in aging collagen, a natural amino acid racemization was observed. These results confirmed that the % D-amino acids are progressive according to age. The collagen sequence is degraded over time, and a fifth of the sequence information is lost during aging. Post-translational modifications (PTMs) in aging collagens can be a hypothesis to explain the modification of the hydrophobicity of the protein with the decrease of hydrophilic groups and the increase of hydrophobic groups. Finally, the exact positions of Damino acids and PTMs have been correlated and elucidated.

Introduction

Aging is a natural and uncontrolled phenomenon that the animal body, including human, undergoes. In vivo, molecules are not exempt from this aging process. Indeed, some age-related protein changes may appear like a loss of proteolytic capacity [START_REF] Levine | Oxidative modification of proteins during aging[END_REF]- [START_REF] Silzel | Influence of Asp Isomerization on Trypsin and Trypsin-like Proteolysis[END_REF], increased surface hydrophobicity [START_REF] Levine | Oxidative modification of proteins during aging[END_REF], appearance of post-translational modifications such as oxidation [START_REF] Levine | Oxidative modification of proteins during aging[END_REF], [START_REF] Santos | Protein Posttranslational Modifications: Roles in Aging and Age-Related Disease[END_REF], [START_REF] Mikšík | Proteins and their modifications in a medieval mummy: Proteomic Analysis of Mummy[END_REF], phosphorylation [START_REF] Santos | Protein Posttranslational Modifications: Roles in Aging and Age-Related Disease[END_REF], methylation [START_REF] Santos | Protein Posttranslational Modifications: Roles in Aging and Age-Related Disease[END_REF], [START_REF] Truscott | Is protein methylation in the human lens a result of nonenzymatic methylation by S-adenosylmethionine?[END_REF], deamination [START_REF] Mikšík | Proteins and their modifications in a medieval mummy: Proteomic Analysis of Mummy[END_REF], [START_REF] Sh | Paleoproteomics studies of ancient caprinae: a review[END_REF], and acylation (particularly acetylation [START_REF] Santos | Protein Posttranslational Modifications: Roles in Aging and Age-Related Disease[END_REF], carbonylation [START_REF] Mikšík | Proteins and their modifications in a medieval mummy: Proteomic Analysis of Mummy[END_REF], carboxymethylation [START_REF] Mikšík | Proteins and their modifications in a medieval mummy: Proteomic Analysis of Mummy[END_REF]) [START_REF] Levine | Oxidative modification of proteins during aging[END_REF], and racemization of amino acids [START_REF] Fujii | Simultaneous Stereoinversion and Isomerization at the Asp-4 Residue in βB2-Crystallin from the Aged Human Eye Lenses[END_REF]- [START_REF] Ha | Age-related isomerization of Asp in human immunoglobulin G kappa chain[END_REF].

Recent animal and human proteins are found in their L-amino acid forms. However, during the natural aging process, an amino acid racemization can take place in proteins via amino acids racemases (enzymatic process) or/and via a succinimidyl intermediate (non-enzymatic process) [START_REF] Morvan | Recent Advances in Chiral Analysis of Proteins and Peptides[END_REF]. This amino acid racemization affects the three-dimensional protein conformation and can induce some aggregation [START_REF] Fujii | Asp isomerization increases aggregation of α-crystallin and decreases its chaperone activity in human lens of various ages[END_REF]- [START_REF] Ageeva | Chiral Linked Systems as a Model for Understanding D-Amino Acids Influence on the Structure and Properties of Amyloid Peptides[END_REF], disorder [START_REF] Dyakin | Racemization in Post-Translational Modifications Relevance to Protein Aging, Aggregation and Neurodegeneration: Tip of the Iceberg[END_REF], malfunctions [START_REF] Dyakin | Racemization in Post-Translational Modifications Relevance to Protein Aging, Aggregation and Neurodegeneration: Tip of the Iceberg[END_REF], misfolding [START_REF] Dyakin | Racemization in Post-Translational Modifications Relevance to Protein Aging, Aggregation and Neurodegeneration: Tip of the Iceberg[END_REF], and toxicity [START_REF] Dyakin | Racemization in Post-Translational Modifications Relevance to Protein Aging, Aggregation and Neurodegeneration: Tip of the Iceberg[END_REF]. In addition, the amount of D-amino acids in proteins is progressive according to age. In 1975, D-Asp enrichment in human tooth enamel proteins via natural racemization was measured as a content of approximately 0,1 % per year [START_REF] Helfman | Aspartic acid racemization in tooth enamel from living humans[END_REF]. Furthermore, some D-amino acids were related to aging human proteins and can be linked to aging diseases. It is the case of D-Asp in elastin linked to arteriosclerosis [START_REF] Powell | On the accumulation of d-aspartate in elastin and other proteins of the ageing aorta[END_REF], [START_REF] Ritz-Timme | Aspartic acid racemization: evidence for marked longevity of elastin in human skin[END_REF], D-Asp in β-amyloid linked to Alzheimer's disease [START_REF] Lee | Evidence for D-Aspartyl-β-Amyloid Secretase Activity in Human Brain[END_REF], D-Asp, D-Asn, D-Ser, and D-Thr in α-crystallin linked to cataract [START_REF] Hooi | Racemisation and human cataract. d-Ser, d-Asp/Asn and d-Thr are higher in the lifelong proteins of cataract lenses than in age-matched normal lenses[END_REF]. The complete list and the exact position of these D-amino acids in till now described protein sequences are recently summarized [START_REF] Morvan | Recent Advances in Chiral Analysis of Proteins and Peptides[END_REF]. D-amino acids are also found in aging and/or ancient proteins and can be used for the ageestimation. Indeed, different studies used the quantification of D-Asp from bone [START_REF] Ritz | Identification of osteocalcin as a permanent aging constituent of the bone matrix: basis for an accurate age at death determination[END_REF], lens [START_REF] Yasunaga | Aspartic acid enantiomer quantification using ultraperformance liquid chromatography-tandem mass spectroscopy combined with deuterium-chloride hydrolysis to improve age estimation in Antarctic minke whale Balaenoptera bonaerensis[END_REF], and tooth [START_REF] Mahlke | Molecular clocks in ancient proteins: Do they reflect the age at death even after millennia?[END_REF], [START_REF] Matteussi | Age estimation in humans through the analysis of aspartic acid racemization from teeth: A scoping review of methods, outcomes, and open research questions[END_REF] for age-estimation, using a HCl/H2O protein hydrolysis. However, in a hydrogenated environment, a natural racemization of L-amino acids to its enantiomer can take place. Different kinetics were observed according to the nature of amino acids [START_REF] Frank | Determination of the rate of acidic catalyzed racemization of protein amino acids[END_REF], [START_REF] Csapá | Hydrolysis of proteins performed at high temperatures and for short times with reduced racemization, in order to determine the enantiomers of d-and l-amino acids[END_REF]. To prevent this amino acids racemization during the acidic hydrolysis, a DCl/D2O condition is privileged [START_REF] Morvan | Recent Advances in Chiral Analysis of Proteins and Peptides[END_REF], [START_REF] Yasunaga | Aspartic acid enantiomer quantification using ultraperformance liquid chromatography-tandem mass spectroscopy combined with deuterium-chloride hydrolysis to improve age estimation in Antarctic minke whale Balaenoptera bonaerensis[END_REF], [START_REF] Miyamoto | Detection and quantification of d -amino acid residues in peptides and proteins using acid hydrolysis[END_REF], [START_REF] Danielsen | Simultaneous Determination of L-and D-Amino Acids in Proteins: A Sensitive Method Using Hydrolysis in Deuterated Acid and Liquid Chromatography-Tandem Mass Spectrometry Analysis[END_REF]. Indeed, in a deuterium environment, the hydrogen on the alpha carbon was exchanged with a deuterium atom and decreases considerably the racemization. Yasunaga et al. compared the racemization of D-Asp for age-estimation under HCl/H2O and DCl/D2O hydrolysis conditions. Errors in age estimations were decreased by at least one-half compared with that of the HCl/H2O hydrolysis method [START_REF] Yasunaga | Aspartic acid enantiomer quantification using ultraperformance liquid chromatography-tandem mass spectroscopy combined with deuterium-chloride hydrolysis to improve age estimation in Antarctic minke whale Balaenoptera bonaerensis[END_REF].

Recently, chiral chromatographic and electrophoretic separation methods for the enantioseparation of (un-)derivatized D-and L-amino acids were described [START_REF] Morvan | Recent Advances in Chiral Analysis of Proteins and Peptides[END_REF]. Mass spectrometry optimization can also enhance the discrimination of derivatized D-and L-amino acids, specifically using ionmobility mass spectrometry [START_REF] Pérez-Míguez | Chiral Discrimination of DL-Amino Acids by Trapped Ion Mobility Spectrometry after Derivatization with (+)-1-(9-Fluorenyl)ethyl Chloroformate[END_REF], [START_REF] Furuhashi | Isomer analysis by mass spectrometry in clinical science[END_REF].

Collagen is the most abundant protein in animal bodies, including humans, and is the major structural component of the extracellular matrix present in tissues and organs [START_REF] Blum | The Collagen Family[END_REF], [START_REF] Köster | An In Situ Study of Collagen Self-Assembly Processes[END_REF]. To date, 28 different types of collagens were described [START_REF] Blum | The Collagen Family[END_REF]. However, the most common of them is collagen type I which is found in 90 % [START_REF] Köster | An In Situ Study of Collagen Self-Assembly Processes[END_REF]. Its three-dimensional structure is a triple-helix form made up of polypeptide chains α 1 and 2 (COL1A1 and COL1A2). The appearance of D-amino acids in the collagen protein reverses the rotation of the triple helix and decreases the helix-helix interactions until the overall destabilization of the three-dimensional structure [START_REF] Shah | Structural consequences ofDamino acids in collagen triple-helical peptides[END_REF], [START_REF] Punitha | Molecular Dynamics Investigations on the Effect of D Amino Acid Substitution in a Triple-Helix Structure and the Stability of Collagen[END_REF]. However, D-amino acids can also be involved in the cross-linking of assembly molecular material. Indeed, in aging collagen, the presence of D-Ala and D-Glu destabilized the structure while D-Lys stabilized it [START_REF] Krishnamoorthy | Effect of d-amino acids on collagen fibrillar assembly and stability: Experimental and modelling studies[END_REF].

For this work, the amino acids racemization in collagen samples (type I alpha 1 and 2) from different organisms (bovine and rat) and at different ages (recent, 4-months and 4-years) were studied. Artificial aging was applied to standard bovine collagens to mimic natural aging. A comparison of HCl/H2O and DCl/D2O effects on collagen hydrolysis was studied. The amount of D-amino acids in bovine collagen after artificial aging was compared to that obtained in rat collagen all along the natural aging at the same age. After enzymatic treatment, arising peptides at different ages were compared. Common peptides between recent and aging collagen were identified as peptides from enzymatic digestions, and non-common peptides come from aging degradations. The evolution of post-translational modifications on the collagen sequences was also studied according to age. Finally, the exact position of D-amino acids and amino acids modified by post-translational modifications were elucidated.

Materials and methods

Chemicals

Acetic acid (AcOH, ≥99,8%), acetic acid-d4 (DOAc, ≥99,9 atom %D), acetonitrile (ACN, ≥99,9%), ammonium hydrogen carbonate (NH4HCO3, ≥99%), chloroform (99,8%), αchymotrypsin from bovine pancrease, type II (≥40 units/mg protein), collagen from bovine achilles tendon, type I (bovine collagens), deuterium chloride solution (DCl, ≥99,9 atom %D), deuterium oxide (D2O, 99.9 atom %D), dimethyl sulfoxide (DMSO, ≥99,9%), ethyl acetate (EtOAc, ≥99,7%), formic acid (FA, ≥98%), heptafluorobutyric acid (HFBA, ≥99,5%), methanol (MeOH), Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA, Marfey's reagent, ≥99%), pepsin from porcine stomach mucosa (≥2,500 units/mg protein (E1%/280)), phosphate buffer solution (1M, pH 7,4), proteinase K from Tritirachium album (≥30 units/mg protein), sodium bicarbonate (NaHCO3, ≥99,5%), tris(hydroxymethyl)aminomethane (Tris, ≥99,8%), trypsin from porcine pancreas (13,000-20,000 BAEE units/mg protein) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Acetone (≥99,5%) and sodium chloride (NaCl) were from Penta (Chrudim, Czech Republic). Ultrapure water was obtained using a Milli-Q system from Millipore (Bedford, MA, USA)

Hereditary hypertriglyceridemic (HTG) rats were originally selected from Wistar rats as previously described [START_REF] Vrána | The hereditary hypertriglyceridemic nonobese rat: an experimental model of human hypertriglyceridemia[END_REF]. HTG rats aged 4-months (bred in the Institute of Physiology, Academy of Sciences of the Czech Republic, Prague) were studied. Age-matched normotensive Wistarderived Lewis rats were used as controls. Rats were housed under controlled conditions (temperature 23±1 °C, 12 h : 12 h light-dark cycle). They were fed a standard rat chow (Velaz, ST-l) containing 0.4% sodium chloride. Water and food were available ad libitum.

Amino acid: D-α-alanine (D-Ala, ≥98%), D-arginine (D-Arg, ≥98%), L-arginine (L-Arg, ≥98%), D-aspartic acid (D-Asp, 99%), D-cysteine hydrochloride monohydrate (D-Cys, ≥98%), L-cysteine (L-Cys, 97%), D-glutamic acid (D-Glu, ≥99%), glycine (Gly, ≥99%), D-histidine monohydrochloride monohydrate (D-His, ≥98%), DL-5-hydroxylysine hydrochloride (DL-Hyl, ≥98%), trans-4-hydroxy-D-proline (D-Hyp, 97%), DL-isoleucine (DL-Ile, 99%), D-leucine (D-Leu, 99%), D-lysine (D-Lys, ≥98%), DL-methionine sulfoxide (DL-Met sulfoxide, ≥98.5%), Lmethionine sulfoxide (L-Met sulfoxide, ≥98%), D-phenylalanine (D-Phe, ≥98%), D-proline (D-Pro, ≥99%), L-proline (L-Pro, ≥99%), DL-serine (DL-Ser, ≥98%), D-serine (D-Ser, ≥98%), Dthreonine (D-Thr, ≥98%), L-threonine (L-Thr, ≥98%), D-tryptophan (D-Trp, ≥98%), L-tryptophan (L-Trp, ≥98%), D-tyrosine (D-Tyr, 99%), L-tyrosine (L-Tyr, ≥98%), and D-valine (D-Val, ≥98%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). DL-α-alanine (DL-Ala), L-glutamic acid (L-Glu), DL-leucine (DL-Leu), DL-methionine (DL-Met), and DL-β-phenyl-α-alanine (DL-Phe) were obtained from Reachem (Mississauga, Canada). L-aspartic acid (L-Asp, 98%), Lhistidine (L-His, 98%), L-methionine (L-Met, 98%) and L-valine (L-Val, 98%) were obtained from Roana (Budapest, Hungary). L-isoleucine (L-Ile, allo-isoleucine free), L-lysine monohydrochloride (L-Lys) were obtained from Calbiochem (San Diego, USA). Lhydroxyproline NH.CH2.CH(OH).CH2 (L-Hyp, ≥98,5%) was obtained from BDH (BDH Chemicals Ltd., Poole, UK).

Sample preparations

Standard and proteinogenic amino acids preparation

Standard amino acids preparation method: standard amino acids were dissolved in D2O at 50 mM.

Amino acids derivatization method:

To standard and proteinogenic amino acid solutions, 100 µL of NaHCO3 (1 M) in D2O was added, and 200 µL of 38.7 mM Nα-(2,4-dinitro-5-fluorophenyl)-Lalaninamide (FDAA) in acetone. Solutions were vortexed and incubated at 40°C for 60 min. Reactions were quenched by the addition of 50 µL of DCl (6 M) in D2O. Compounds of interest are extracted by ethyl acetate (0.5 mL). A saturated sodium chloride solution (300 μL) was added in the aqueous phase to increase the difference in density between the aqueous and organic phases and facilitate the extraction. Undesired hydrogen fluoride formed during the derivatization reaction stays in the aqueous phase and is not injected to limit the HPLC and LC column degradations.

From extracted organic phases, 20 µL of these solutions in 20 µL of DMSO were analyzed by HPLC at 340 nm or LC-MS.

Recent and aging collagens preparation

Artificial aging of bovine collagens

Bovine collagen samples (n=6) in phosphate buffer (100 mM, pH 7,4) at 2 mg.mL -1 in glass tubes, were placed outside under natural meteorological conditions for 4-months and 4-years.

Extraction of natural aging HTG rat collagens

Tendons from the tails of HTG rats (n=2) were scraped with a scalpel, then rinsed three times with 1M NaCl solution, each for 24h at 4°C. Extracted collagens were rinsed with ultrapure water and lyophilized.

Enzymatic digestion

Recent and aging bovine and rat collagens type I alpha 1 and 2 were digested successively by pepsin (0.1 mg.mL -1 in 3 % AcOH, 50/1 sample/enzyme, w/w) and trypsin (0.02 mg.mL -1 in 50 mM NH4HCO3, 50/1 sample/enzyme, w/w). Collagen residues were lyophilized after both enzymatic treatments. Bovine and rat 4-months aging collagens produce an insoluble material after these treatments while recent and 4-years aging collagens do not. These insoluble parts were then digested by proteinase K (0.05 mg.mL -1 in 10 mM Tris, 50/1 sample/enzyme, w/w), and resulting insoluble parts were digested by chymotrypsin (0.04 mg.mL -1 in 50 mM NH4HCO3, 50/1 sample/enzyme, w/w). The totality of bovine and rat 4-months aging collagens was soluble after chymotrypsin enzymatic treatment. Each enzymatic digestion was performed overnight at 37°C in the darkroom.

Hydrolysis

Proteins and peptides hydrolysis method: intact proteins and each peptide fraction were hydrolyzed in individual glass tubes placed into 10-mL Pierce Reacti-vials (Kimble Chase, Mexico) that had been purged with nitrogen thrice. After 18 h at 130°C in 400 µL 1:1 DCI/(2H4)acetic acid (DCl/DOAc) on the bottom of the vial, 200 µL of D2O was added to the proteinogenic amino acid solution obtained in each tube. Then, these proteinogenic amino acids were derivatized following the amino acids derivatization method described in section 2.2.1.

HPLC parameters

HPLC for amino acids analysis

Analyses were performed in a HPLC system from Agilent (Agilent Technologies, Santa Clara, USA). Mobile phase A constituted to 0.1 % of FA in ultrapure water (v/v) and mobile phase B constituted to 0.1 % of FA in ACN (v/v). The chromatographic column was the Phenomenex Aeris 3.6 µm peptide XB-C18, 250 x 2.1 mm (Phenomenex Inc., Torrance, USA). The mobile phase operated in gradient mode, starting at 2 % mobile phase B and increasing for 30 min to reach 8 % B, 110 min to reach 40 %, and then increasing to 100 % B at 125 min, remaining for 20 min, and returning to 2 % B for 10 min maintaining constant until the end of the analysis. The flow rate was set at 0.250 mL.min -1 , the column was held at ambient temperature (25 °C), the injection volume was 5 µL, and UV detection was performed at 340 nm. The Agilent HPLC system was connected to a maXis quadrupole time-of-flight (Q-TOF) mass spectrometer with ultrahigh resolution (Bruker Daltonics, Bremen, Germany).

HPLC for peptides analysis

Analyses were performed on an HPLC system from Agilent (Agilent Technologies, Santa Clara, USA) consisting of a degasser, binary pump, autosampler, thermostatted column compartment and multi-wavelength detector. Mobile phase A constituted to 0.1 % of HFBA in ultrapure water (v/v) and mobile phase B constituted to 0.1 % of HFBA in ACN (v/v). Elution was achieved using a linear gradient when separation was started by running the system isocratically for two minutes with 2 % of mobile phase B, followed by a gradient elution to 35 % B at 40. min, Next gradient was 10 minutes to 100 % B. Finally, the column was eluted with 100 % B for 10 min. Equilibration before the next run was achieved by washing with buffer A for 10 min. The chromatographic column was the Jupiter 4 μm Proteo 90Å column (250 x 2 mm ID, Phenomenex Inc., Torrance, USA). The flow-rate was 0.250 mL.min -1 , the column temperature was held at 25°C and UV absorbance detection was done at 214 nm, injection volume was 20 µL.

Bovine and rat collagen peptides obtained after enzymatic treatments were separated and collected in several fractions, e.g. 31 fractions for collagen sample treated by pepsin and trypsin, 41 fractions after proteinase K treatment, and 37 fractions after chymotrypsin treatment. Chromatograms with fraction delimitations are presented in supplementary materials (Figures S1, S2, andS3).

NanoLC-MS parameters

The nanoHPLC apparatus used for protein digest analysis was a Proxeon Easy-nLC (Proxeon, Odense, Denmark). It was coupled to an ultrahigh resolution MaXis Q-TOF (quadrupoletime of flight) mass spectrometer (Bruker Daltonics, Bremen, Germany) by nanoelectrosprayer. The nLC-MS/MS instruments were controlled with the software packages HyStar 3.2 and micrOTOFcontrol 3.0. The data were collected and manipulated with the software packages ProteinScape 3.0 and DataAnalysis 4.0 (Bruker Daltonics).

Five microliters of the peptide mixture were injected into an NS-AC-12dp3-C18 Biosphere C18 column (particle size: 3 µm, pore size: 12 nm, length: 200 mm, inner diameter: 75 µm) with an NS-MP-10 Biosphere C18 precolumn (particle size: 5 µm, pore size: 12 nm, length: 20 mm, inner diameter: 100 µm), both manufactured by NanoSeparations (Nieuwkoop, Holland).

The separation of peptides was achieved via a linear gradient between mobile phase A (ultrapure water) and B (ACN), both containing 0.1 % FA (v/v). Separation was started by running the system with 5 % mobile phase B, followed by a gradient elution to 7 % B at 5 min, and 30 % B at 180 min. The next step was a gradient elution to 50 % B in 10 min and then a gradient to 100 % B in 10 min. Finally, the column was eluted with 100 % B for 20 min. Equilibration between the runs was achieved by washing the column with 5 % mobile phase B for 10 min. The flow rate was 0.200 µL.min -1 and the column was held at ambient temperature (25 °C).

MS parameters

Online nano-electrospray ionization (easy nano-ESI) was used in positive mode. The ESI voltage was set to +4.5 kV, scan time: 3 Hz. Operating conditions: drying gas (N2): 4 L.min -1 ; drying gas temperature: 180 °C; nebulizer pressure: 100 kPa. Experiments were performed by scanning from 50 to 2200 m/z. The reference ion used (internal mass lock, chip cube high mass reference (HP-1221), Agilent Technologies, Waldbronn, Germany) was a monocharged ion of C24H19F36N3O6P3 (m/z 1221.9906). Mass spectra corresponding to each signal from the total ion current chromatogram were averaged, enabling an accurate molecular mass determination. All HPLC-MS and nanoLC-MS analyses were done in duplicate.

Peptide identifications

Peptide data were processed using ProteinScape software (Bruker Daltonics, Bremen, Germany). Collagen type I alpha 1 and 2 chains were identified by correlating tandem mass spectra to the NCBI, IPI, and SwissProt databases, using the MASCOT search engine (http://www.matrixscience.com). The taxonomy was limited to Bos taurus and Rattus norvegius due to the provenance of the samples. SemiTrypsin was chosen as the enzyme parameter. Three missed cleavages were allowed, and an initial peptide mass tolerance of ±15.0 ppm was used for MS and ±0.03 Da for MS/MS analysis. Arginine, asparagine, and glutamine were assumed to be deamidated, methionine to be dioxidized, lysine, methionine, and proline to be oxidized, lysine, serine and threonine to be formylated, lysine to be acetylated, carbamylated, carboxylated and carboxymethylated, arginine, aspartic acid, cysteine, histidine, lysine, serine, threonine, and tyrosine were allowed to be phosphorylated. All these possible modifications were set to be variable. Monoisotopic peptide charge was set at 1+, 2+, and 3+. The Peptide Decoy option was selected during the data search process to remove false-positive results. Only significant hits (accepted as identified on ProteinScape) were selected.

Results and discussion

Effect of hydrolysis conditions

Figure 1 shows the amino acid racemization of recent bovine collagens under HCl/H2O (Figure 1A) and DCl/D2O (Figure 1B) hydrolysis conditions. Amino acid racemization takes place only under HCl/H2O hydrolysis conditions for all amino acids except Ser and Thr which were not racemized. Methionine, phenylalanine, tryptophan, and tyrosine were not detected. For this work, a DCl/D2O hydrolysis is privileged to limit the racemization during the hydrolysis and not distort the results when determining the % D-amino acids.

D-amino acids in aging collagens

This chiral analysis method, described in section 2.3.1 and newly developed for this work using standard amino acids, was then used to confirm the presence of D-amino acids in bovine collagen after in vitro artificial aging, at different ages. In addition, this analytical method was also used for the detection of D-amino acids in rat collagen with in vivo natural aging. Preliminary study was performed to confirm the presence of amino acid only in their L-form in recent bovine collagen (Table S5). During the aging process, the artificial amino acid racemization, applied to bovine aging collagens, mimes the natural amino acid racemization. Most of the different amino acids were racemized over time. It is the case of D-Arg, D-Asp, D-Leu, D-Pro, D-Ser, D-Thr, and D-Val most frequently racemized in 4-years than in 4-months collagen samples. In addition, the percentage of Arg, His, Thr, and Val in their D-form was higher in the older sample (Figures 2 and3, Figures S4 andS5, Table S1 -S5). S1. The total % RSD of %D-amino acids was 4.37 %. S2. The total % RSD of %D-amino acids was 10.97 %.

Most interesting amino acid fractions of bovine collagens were selected and compared to the same amino acid fractions of rat collagens at the same age. The selection of fractions was made based on the high percentage of amino acid racemization, and the diversity of the nature of amino acids racemized. The comparison of the percentage of D-amino acids is shown in Figures S6 andS7 and table S2 and S6. Into selected fractions of soluble part of collagen after pepsin and trypsin enzymatic treatment, some D-amino acids, e.g. D-Arg, D-Ile, D-Leu, D-Phe, D-Pro, and D-Tyr, were present in bovine collagen after artificial aging and not in rat collagen with natural aging. Other amino acids were less frequently present in rat collagen with natural aging, such as D-Asp, D-His, D-Ser, and D-Hyp. Conversely, D-Thr and D-Val were more frequently present in rat collagen with natural in vivo aging. Concerning the % D-amino acids, D-Glu, D-His, D-Thr, and D-Val were most racemized in their D-form in rat collagen with natural aging. The same trend was observed in selected fractions of the insoluble part of bovine and rat collagens after pepsin, trypsin, and proteinase K enzymatic treatment (Figure 4 and Table S3 andS7 S3 and S7. The total % RSD of %D-amino acids from rat collagen was 7.07 %.

Peptide analysis

Peptides in each fraction from recent and aging collagens were analyzed by nanoLC-MS (sections 2.4 and 2.5) and identified (section 2.6)

Position of D-amino acids

The identification of peptides in each collected fraction (for the method see section 2.6) allowed us to determine their position on the bovine and rat collagen sequences (type I alpha 1 and 2). Regrouping the racemization rate of each amino acid in each fraction (section 3.2) and the peptide identification in these same fractions, the exact position of amino acids totally racemized in their D-form have been determined on the bovine and rat collagen sequences (Figure 5, 6, S8 and S9, Table 1 andS8).

In bovine collagens, 21 specific sites of Thr were identified as totally racemized in the D-form for 4-months aging sample and 18 specific sites for 4-years aging sample in the COL1A1 collagen sequence. Their exact positions have shown that 70 % of them were common at both ages. For COL1A2, 14 and 15 Thr specific sites were found in the 4-months and 4-years aging samples respectively, and 45 % were common at both ages. These specific sites of D-Thr totally racemized represent 48 % (4-months) and 41 % (4-years) of total Thr on the bovine COL1A1 sequence. For bovine COL1A2, these D-Thr specific sites represent 32 % (4-months) and 34 % (4-years) of total Thr on the protein sequence. D-Val was exclusively totally racemized in 4-months aging collagens and represent 19 % of total Val on bovine COL1A1 and 4 % on bovine COL1A2 sequences. D-Arg was entirely racemized in 4-years aging collagens and represented 75 % of total Arg on bovine COL1A1 and 63 % on bovine COL1A2 sequences.

For amino acids totally racemized in fractions of 4-month aging rat collagens selected according to most promising bovine fractions at the same age, 12 specific sites for D-Glu (17 %) and 6 for D-Val (14 %) were identified in COL1A1. In COL1A2, 10 specific sites for D-Glu (16 %) and 6 for D-Val (12 %) were identified.

Peptides from enzymatic digestion and aging degradation

After successively enzymatic digestions by pepsin and trypsin, all peptides and residues from recent and aging bovine collagens (COL1A1 and COL1A2) were soluble and identified by nanoLC-MS (section 2.4). Peptides from the insoluble part of collagen protein after pepsin and trypsin treatment at 4-months of aging were not added in the study. Arising peptides in aging soluble collagen samples common to arising peptides in recent collagen were identified as peptides from enzymatic digestions. Other peptides were identified as peptides from aging degradation (Figure 7). For aging bovine collagens type I alpha 1 and 2 (COL1A1 and COL1A2), the number of peptides from aging degradation is higher than arising peptides from enzymatic digestions. Indeed at 4-months, 46 % (COL1A1) and 48 % (COL1A2) of peptides come from aging degradations for both collagen types, and 55 % (COL1A1) and 46 % (COL1A2) at 4-years. The total of arising peptides from enzymatic digestion and aging degradation did not reach 100 % for both collagen chains and at both ages. Approximately, a fifth of the sequence information was lost during aging.

Post-translational modifications during the aging process

Hydrophilic (e.g. oxidation, dioxidation, deamidation, phospho, sulfo) and hydrophobic (e.g. acetyl, carbamyl, carboxy, carboxymethyl, carboxyethyl, formyl, methyl) post-translational modifications were studied on the bovine and rat collagen sequences (COL1A1 and COL1A2). Differences according to the organism, age, and artificial/natural aging were observed. The number of modifications and the corresponding percentage according to the total number of possible modified sites are summarized in Table 2 and S9-11 for each sample at different ages.

Based on previous results (see section 3.3.2), about a fifth of bovine sequence information was lost during aging. The post-translational modifications studies show that the percentage of hydrophilic modifications (deamidation, phospho and sulfo) in COL1A1 decreases considerably. Exception for oxidation modifications on lysines, methionines, and prolines (exact positions in Figures S10 -S12, Tables S9, and S10), which increase in 4-months samples, before decreasing in older samples in correlation with the degradation and the loss of sequence information. On the other hand, the polypeptide chain alpha 2 seems more subject to hydrophobic (acetyl, carbamyl, carboxymethyl, formyl, and methyl) modifications than the COL1A1, during the aging process. Exception for oxidation and phospho modifications which increase (Table 2). In sum, the decrease of hydrophilic groups and the increase of hydrophobic groups during aging may be a hypothesis to explain the evolution of the insolubility of collagen along life, to add to the protein degradations. The comparison between artificial (bovine) and natural (rat) aging on post-translational modifications on collagen sequences is summarized in Table 3. The percentages of modifications were slightly higher under artificial aging.

Conclusion

Artificial aging was successfully applied to standard collagen to mimic the natural aging in biological samples. Several collagen samples from different organisms (bovine and rat), were studied at different ages (recent, 4-months, 4-years). DCl/D2O hydrolysis was privileged to HCl/H2O to limit the natural racemization of amino acids during the protein hydrolysis. This new chiral analysis method allowed us to determine the percentage of D-amino acids in collagen according to age. Results presented show a % D-amino acidsage correlation. Peptide analysis shows that the amount of peptides from aging degradation is higher than arising peptides from enzymatic digestions. The collagen protein sequence is increasingly degraded over time. The posttranslational modifications study showed a decrease of hydrophilic groups and an increase of hydrophobic groups during aging. These sequence modifications may be a hypothesis to explain the evolution of the insolubility of collagen throughout life. The percentage of post-translational modifications was not significantly different between artificial and natural aging in both organisms. The combination of these results allowed us to determine the exact positions of Damino acids and PTMs.

Figure 1 :

 1 Figure 1: Effect of hydrolysis conditions on proteinogenic amino acid racemization from standard bovine collagens. Hydrolysis conditions: HCl/H2O (A) and DCl/D2O (B). Peak 1 identifies amino acids in their L-form and Peak 2 in their D-form. Exact masses of derivatized amino acids: 342,104420 (Ala), 427,168420 (Arg), 386,094250 (Asp), 400,109900 (Glu), 408,126220 (His), 384,151370 (Ile/Leu), 651,211740 (Lys), 368,120070 (Pro), 358,099340 (Ser), 372,114990 (Thr), 370,135720 (Val) and 384,114990 (Hyp).

Figure 2 :

 2 Figure 2: Representation of % D-amino acids in 4-months aging bovine collagen fractions (n=2) after pepsin and trypsin enzymatic treatment. Percentages of D-amino acids superior to 50 % are indicated on the graph. All percentages of D-amino acids are summarized in TableS1. The total % RSD of %D-amino acids was 4.37 %.

Figure 3 :

 3 Figure 3: Representation of % D-amino acids in 4-years aging bovine collagen fractions (n=3) after pepsin and trypsin enzymatic treatment. Percentages of D-amino acids superior to 50 % are indicated on the graph. All percentages of D-amino acids are summarized in TableS2. The total % RSD of %D-amino acids was 10.97 %.

  ). Some D-amino acids, e.g. D-Arg, D-Lys, D-Phe, and D-Pro, were present in bovine collagen and not in rat collagen. D-Asp, D-His, D-Ser, and D-Hyp were less frequently present in rats. Conversely, D-Thr was more frequently present in rat collagen. Concerning the % D-amino acids, D-Glu and D-Thr were most racemized in their D-form in rat collagen with the natural aging, and D-Val was most racemized in bovine collagen after the artificial aging.

Figure 4 :

 4 Figure 4: Representation of % D-amino acids in selected fractions of bovine and rat aging collagen after pepsin, trypsin, and proteinase K enzymatic treatment. Percentages of D-amino acids superior to 50 % are indicated on the graph. All percentages of D-amino acids are summarized in TableS3and S7. The total % RSD of %D-amino acids from rat collagen was 7.07 %.

Figure 5 :

 5 Figure 5: Exact position of D-amino acids totally racemized in their D-form on the bovine collagen COL1A1 sequence at 4-months (left) and 4-years (right).

Figure 6 :

 6 Figure 6: Exact position of D-amino acids totally racemized in their D-form on the bovine collagen COL1A1 sequence at 4-months (left) and on the rat collagen COL1A1 sequence at 4-months (right).

Figure 7 :

 7 Figure 7: Number of arising peptides from enzymatic digestion and aging degradation in recent, 4-month, 4-year bovine artificial aging collagens.

3. 3 . 4 .

 34 Comparison of exact positions of D-amino acids and posttranslational modificationsBoth amino acid racemization and post-translational modifications can appear, simultaneously or not, during aging. Figures8 and S14-18 regroup the exact position of D-amino acids totally racemized and the exact position of post-translational modifications.

Figure 8 : 4 -

 84 Figure 8: 4-years bovine collagen COL1A1 sequence modifications: post-translational modifications (red), amino acids in their Dform totally racemized (blue), and both modifications on the same amino acid (green).

Table 1 :

 1 Exact position of D-amino acids totally racemized in their D-form on the bovine collagen sequences at different ages.

			COL1A1 COL1A1			COL1A2 COL1A2
	AA	Position	4-months 4-years	AA	Position	4-months 4-years
	R	131	✓	R	31	✓
		186	✓		38	✓
		219	✓		41	✓
		239	✓		130	✓
		243	✓		154	✓
		252	✓		163	✓
		267	✓		178	✓
		303	✓		232	✓
		309	✓		280	✓
		311	✓		325	✓
		321	✓		340	✓
		360	✓		379	✓
		369	✓		397	✓
		414	✓		411	✓
		468	✓		421	✓
		471	✓		430	✓
		486	✓		438	✓
		510	✓		448	✓
		527	✓		474	✓
		563	✓		484	✓
		573	✓		571	✓
		597	✓		586	✓
		630	✓		607	✓
		675	✓		643	✓
		684	✓		661	✓
		696	✓		669	✓
		732	✓		673	✓
		744	✓		691	✓
		762	✓		706	✓
		795	✓		739	✓
		801	✓		775	✓
		864	✓		792	✓
		881	✓		828	✓

Table 2 :

 2 Total number and percentage of post-translational modifications on bovine collagen COL1A1 and COL1A2 sequences at different artificial aging.

				COL1A1
	Modifications	Amino acids Recent	%	4-	%	4-	%
			(n=3)	recovery	months	recovery	years	recovery
					(n=2)		(n=3)
	Oxidation	KPM	79	22,8%	98	28,2%	65	18,7%
	Dioxidation	M	0	0%	0	0%	0	0%
	Deamidation	NQR	5	3,4%	4	2,7%	3	2,0%
	Phospho	CDKHRSTY 27	8,0%	20	5,9%	9	2,7%
	Acetyl	K	0	0%	1	1,8%	0	0%
	Carbamyl	K	0	0%	0	0%	0	0%
	Carboxy	K	0	0%	0	0%	0	0%
	Carboxymethyl	K	0	0%	0	0%	0	0%
	Carboxyethyl	K	0	0%	0	0%	0	0%
	Formyl	KST	9	5,3%	12	7,1%	9	5,3%
	Methyl	DE	4	3,0%	3	2,2%	2	1,5%
	Sulfo	STY	4	3,2%	4	3,2%	1	0,8%
				COL1A2
	Modifications	Amino acids Recent	%	4-	%	4-	%
			(n=3)	recovery	months	recovery	years	recovery
					(n=2)		(n=3)
	Oxidation	KPM	40	13,8%	56	19,4%	40	13,8%
	Dioxidation	M	0	0%	0	0%	0	0%
	Deamidation	NQR	4	2,6%	3	1,9%	4	2,6%
	Phospho	CDKHRSTY 9	2,9%	16	5,2%	17	5,6%
	Acetyl	K	0	0%	0	0%	1	2,0%
	Carbamyl	K	0	0%	1	2,0%	0	0%
	Carboxy	K	0	0%	0	0%	0	0%
	Carboxymethyl	K	0	0%	0	0%	1	2,0%
	Carboxyethyl	K	0	0%	0	0%	0	0%
	Formyl	KST	4	2,5%	6	3,8%	7	4,5%
	Methyl	DE	2	1,9%	3	2,9%	3	2,9%
	Sulfo	STY	1	0,8%	1	0,8%	0	0%

Table 3 :

 3 Number and percentage of post-translational modifications on bovine and rat collagens COL1A1 and COL1A2 sequences at 4-months after pepsin and trypsin enzymatic treatment.

			COL1A1
	Modifications	Amino acids	4-months	% recovery 4-months	% recovery
			bovine		rat (n=2)
			(n=2)
	Oxidation	KPM	89	25,5%	57	16,4%
	Dioxidation	M	0	0%	0	0%
	Deamidation	NQR	3	2%	2	1,4%
	Phospho	CDKHRSTY	18	5,6%	4	1,2%
	Acetyl	K	1	1,8%	0	0%
	Carbamyl	K	0	0%	0	0%
	Carboxy	K	0	0%	0	0%
	Carboxymethyl	K	0	0%	0	0%
	Carboxyethyl	K	0	0%	0	0%
	Formyl	KST	8	5,0%	3	1,8%
	Methyl	DE	3	2,1%	1	0,7%
	Sulfo	STY	2	1,7%	0	0%
			COL1A2
	Modifications	Amino acids	4-months	% recovery 4-months	% recovery
			bovine		rat (n=2)
			(n=2)
	Oxidation	KPM	39	13,2%	31	10,7%
	Dioxidation	M	0	0%	0	0%
	Deamidation	NQR	2	1,3%	2	1,3%
	Phospho	CDKHRSTY	12	4,0%	5	1,6%
	Acetyl	K	0	0%	0	0%
	Carbamyl	K	0	0%	0	0%
	Carboxy	K	0	0%	0	0%
	Carboxymethyl	K	0	0%	0	0%
	Carboxyethyl	K	0	0%	0	0%
	Formyl	KST	4	2,7%	4	2,5%
	Methyl	DE	3	2,8%	1	1,0%
	Sulfo	STY	1	0,9%	1	0,8%

Acknowledgements

This work was supported by the Czech Science Foundation, grant number 20-03899S.

CRediT authorship contribution statement

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


François H Guiziou

0. The Corridors of Landlocked Ethiopia

Keywords: Ethiopia, Djibouti, Horn of Africa, landlocked states, transport systems

Following the loss of access to its Eritrean ports (Assab and Massawa), Ethiopia has been a landlocked state. The large majority of its economic activities are concentrated around the capital area, which benefits from strong road and rail links to the port of Djibouti and, to a lesser extent, to the port of Berbera (Somaliland). In an effort to reduce its dependence on intermediary states, Ethiopia is striving to develop alternative routes with neighbouring countries that have costal access, such as Sudan and Kenya. Thanks to massive public investment and support from external actors-particularly the People's Republic of China-Ethiopia's access-diversification strategy is making headway. However, progress is hampered considerably by its internal and regional security situation.

or less directly to African states and African regions (including [START_REF] Hoyle | Inter-port competition in developing countries: an East African case study[END_REF][START_REF] Debrie | L'enclavement, réévaluation théorique et application à l'Afrique de l'Ouest[END_REF][START_REF] Debrie | From colonization to national territories in continental West Africa: The historical geography of a transport infrastructure network[END_REF]Oliete & Magrinyà, 2022;[START_REF] Ducruet | Inland cities, maritime gateways, and international trade[END_REF], none of these works focus specifically on the transportation system in Ethiopia and the Horn of Africa region 1 . With a population of 115 million inhabitants (World Bank, 2020), Ethiopia has featured on the list of landlocked countries since its separation from Eritrea in 1993. The situation worsened in the wake of the conflict between the two countries from 1998-2000, resulting in the loss of the two major Eritrean ports of Massawa and Assab (along with the latter's refinery), previously serving as the major port for the corridor leading to Ethiopia's capital region, Addis Ababa. Following the closure of the Assab corridor, traffic was diverted to Djibouti, which seized the opportunity to invest in its harbour facilities and position itself as the principal access point to Ethiopia, particularly the capital area, which is the main urban-industrial zone in demographic terms (4 million inhabitants, and over 1,000 companies, according to the United Nations Development Programme [UNDP] [2018]). Since the late 1990s, the Ethiopian authorities have sought to reduce their reliance on Djibouti, while also working to preserve and modernize this critical transportation route. As a result, alternative regional corridor projects have emerged. However, doubts have been raised over the capacity, both financial and political, of Ethiopian authorities to succeed in this diversification strategy. This case study aims to illustrate the challenges faced by landlocked countries when developing multiple entry routes to avoid territorial isolation, particularly in the face of recurring and major security issues in neighbouring states. Although this study does not provide an exhaustive list of all the corridors and projects involved, it has four main objectives: (1) to provide a characterization of the Ethiopian hinterland served by its principal corridor, the port of Djibouti; (2) to describe the situations in the neighbouring seaports of Berbera and Assab, which are also major parts of Ethiopia's transportation network; (3) to examine alternative projects aimed at opening up the region; (4) to discuss the sustainability of this strategy, with a particular focus on security concerns 2 . Ethiopia's main hinterland: The capital region The capital region and its neighbouring territories are the lifeblood of present-day Ethiopia. Addis Ababa, a relatively recent city (established in 1886), owes much of its rapid development to its historic connection to the port of Djibouti and the French railway (a service that ran between 1901 and 1917). As a result, three states-Eritrea, Djibouti, and Somaliland (a proto-state born out of the Somali civil war)-share 450 km of coastline. In general, three ports provide effective access to landlocked Ethiopia and its capital: Assab (900 km from Addis Ababa), Djibouti (at an 850 km-distance), and Berbera (at a 950 kmdistance). The location of these ports is highly advantageous from a geomorphological standpoint: (1) because they provide access to the hinterland, which is limited at certain grades because of the natural barriers of the Great Rift and the Awash Valley, as well as to the Ethiopian highlands in general; (2) with regard to the tectonic foreland, because these ports are located in close proximity to the international Bab el-Mandeb Strait, which is an extension of the rift and a site of intense global traffic (in which these ports only participate to a marginal extent) and, at the regional scale, where these ports are very well integrated.

The vast majority of the traffic in this main corridor is by road 3 . The axes of these major ports towards the capital region converge at Awash at the A1 junction, the A10 junction, and the reinforced track passing through Dire Dawa (central-eastern corridor), as shown in Figure 10.1. An alternative route is the B11 road (Mile) then the A2 road (Kombolcha), which offer lighter traffic volumes at the cost of a steeper road. The Ethio-Djibouti railway takes a more southerly route through Dire Dawa, joining the A10 road coming from the Somali border and the Berbera road (eastern corridor). Finally, an important road connection with the major Djibouti/Assab corridor in the Amhara region is located in the east (via the B21, B31, and B22 roads). These routes combine to form a corridor, with Djibouti serving as the undisputed central hub, Assab taking on a marginalized status, and Berbera positioned as the newcomer. However, they share a comparable hinterland: the capital area (for the three routes); the Afar region and the Amhara region (for Assab and Djibouti); and the northern Somali region (for Djibouti and Berbera). Furthermore, these routes ensure, by means of irrigation systems from the capital region, the supply of a significant part of the country.

The centre-periphery relation is heavily marked by Addis Ababa's political and demographic weight, as well as its attractive power 4 . In addition to the central-eastern corridor (also known as the "Black Ribbon" 5 ) and the eastern corridor mentioned above, the other road corridors serving the majority of the population from Addis Ababa are distributed as follows: the northern corridor towards Mekele and Zalambessa-Kokobay (Eritrean border); the northwestern corridor by Gondar towards Aksum; the western corridor towards Nekemte, Asosa, and Kurmuk (Sudanese border); finally, an axis extending southward to join the Kenyan border with Moyale, following the Rift Valley (A7-A8 highways). All these roads and railways are subject to both arid and tropical climates. Because of the risks associated with these climatic characteristics, such as floods, landslides, and fires, costly maintenance is required for these corridors to operate at full capacity. < Figure 10.1 here > Figure 10.1: the Ethio-Djibouti corridor, Berbera, and Assab. Map created by the author.

The Djibouti corridor

The port city of Djibouti, a product of French colonization established in 1883, is currently the principal port serving Ethiopia. Despite a lack of any statistical backing, it is often claimed that more than 90 % of goods officially entering the Ethiopian territory transits through its harbour facilities (United Nations Conference, 2013). The port comprises several installations, including the old port, (the Port authority of Djibouti, which is now dedicated to bulk cargo and warships calling); the Doraleh container terminal managed by the Société de gestion du terminal à conteneurs de Doraleh (SGTD) (Doraleh Container Terminal Management Company), established in 2009; eight gantry cranes, initially developed and operated by Dubai Port World (DPW) until their expulsion in 2018, (cf. infra); the oil port of Doraleh (Horizon Terminal Djibouti, established in 2005); and the Doraleh Multipurpose Port (DMP), a general port built by a Chinese company in 2017 comprising four gantry cranes. The current capacity of the port of Djibouti is around 1.2 million TEU (20-ft equivalent units). Two other, smaller, export-focused facilities, the Tadjourah terminal (dedicated mainly to Ethiopian potash, established in 2017) and the port of Goubet (salts, since 2017), complete the port landscape. Since 2003, all these facilities have been under the supervision of the Djibouti Ports and Free Zones Authority (DPFZA), a government agency that manages and coordinates the development of new terminals and infrastructure. The port infrastructure of Djibouti meets international standards, and the Djiboutian authorities are currently discussing new developments such as the ambitious industrial park project of Damerjog, which would include the installation of a refinery (while putting original plans for a livestock terminal, which is currently functioning as a quarantine yard, on hold). The initial works, started in 2020, are still in the early stages, and currently limited to the construction of a jetty and a terreplein.

Although a very large part of traffic between Djibouti and its hinterland is by road (the RN-1 National Highway and then the A1 road), it is hoped that the construction of an electric railway will increase the amount of traffic bound for Adama in Addis Ababa, as well as industrial facilities in Dire Dawa and Awash. Just a stone's throw away from the route of the first road laid out by French engineers at the beginning of the twentieth century, this Chinesebuilt railway is the primary argument put forward by the Djiboutian authorities' in demonstrating their competitive edge over rival ports. Container capacity could offer a maximum capacity of 100,000 TEU 6 per year-assuming that three freight trains were to operate at full capacity every day-which would amount to approximately 10 % of SGTD and DMP's transit per year. The route provides several benefits, including shorter distance, enhanced safety, as well as smoother traffic flow, achieved by minimizing load breaks caused by the prevalence of roads and the time-consuming process of emptying and loading containers onto trailers (United Nations Conference, 2013). Among the eight Ethiopian dry ports, those of Modjo and Indode have rail-mounted stacker cranes, and so are well-equipped for container management and enable efficient exports to the manufacturing zones around Addis Ababa (Akaki, Kality, Sebeta, Feri-Lebu, etc.). It is worth noting that this dry-port strategy is also utilized for major roads, albeit in a more rudimentary way, that is, without dedicated multimodal infrastructure. This illustrates the synergy between the A1 and the railways, which have played a significant role in creating a thriving industrial corridor between Sebeta and Adama, comparable in scale to those found in the Horn of Africa. Furthermore, the importance of Awash, with its gasoline reserves, and Dire Dawa, known for its cement production, both situated along the railway, cannot be overstated.

The frequent meetings held between the Ethiopian and Djiboutian authorities demonstrate their intention to proceed with caution in their fledgling partnership, despite Ethiopia's clear desire to diversify access to the sea. For Djibouti, the stakes are very high, since the state budget is heavily dependent on the port revenue generated by Ethiopian trade (20-25 %, World Bank, 2013) and the security provided by the foreign detachments stationed on its territory (France, USA, China, etc.). Despite being a small state, Djibouti's strategic location has attracted the accumulation of forces of allied and competing states in the international arena, making it a curious case. The reasons behind the presence of these foreign actors are manifold: antipiracy, counterterrorism, strategic positioning in the vicinity of the Bab el-Mandeb strait, and so on. Except for China-which is a crucial partner for Ethiopia-and France-which has a long-standing relationship with Djibouti-these foreign actors have no direct and significant interest in the politics of the Horn of Africa.

The emergence of Berbera and the Assab hypothesis

In recent years, Djibouti has seen the emergence of a potential competitor: the port of Berbera (Figure 10.2), Somaliland's unique port. The port of Berbera has a long history, with ships first throwing anchor there back in ancient times. Its first major dock was developed by the Soviets in 1967, to be expanded by the Americans in the late 1980s, to then thrive in the modern commercial landscape thanks to the help of DPW. However, the Emirati company moved its operations to Somaliland in 2016 because of the political and financial difficulties gripping Djibouti in 2014. These difficulties led to the company's expulsion from Djibouti in 2018, despite the commercial success of the Doraleh terminal, of which DPW was manager and remains joint owner. In favour of DPW, the London Court of International Arbitration ordered the Djiboutian state to pay US $500 million 7 . The challenges faced in Djibouti prompted DPW to seek solutions elsewhere. Back in 2016, the company obtained a 30-year management contract for the Berbera port facilities from the Somaliland government. DPW then embarked on a $442 million project to modernize the port, significantly improving harbour management and almost doubling the wharf line (1 km in total). DWP also developed a container terminal spanning 33 ha. This terminal is equipped with three gantry cranes, with an estimated capacity of more than 350,000 TEU counters at startup in 2021, up from the previous capacity of 100,000. Long confined to being a bulk and cattle port, Berbera is now the only port in Somalia to have a dedicated container terminal built to international standards.

The Djiboutian conflict offered Berbera the opportunity to welcome one of the major players in international maritime trade. Indeed, DPW is present in 40 countries worldwide and handles around 10 % of global container traffic. Prior to this, Berbera was already an important regional port, offering deep-water access, an ISPS certification obtained in 2006 (International Ship and Port Security Code), and facilities for the Global Food Programme, including two silos and a small oil terminal. The addition of a competent container terminal modelled on the Doraleh facility, boasting a free zone and a focus on regional industry development, harks back to the rise of Djibouti. Berbera now stands as a key competitor that could disrupt the Djiboutian hegemony. The recent agreements with Ethiopia in 2000 and 2016, the official docking of the Ethiopian Shipping and Logistics Services Enterprise ships since 2021, the new free zone law enacted in Somaliland in 2021 8 , as well as the DPW's tariff policy (Table 10.1), are all factors contributing to the port's immense success, with support from Ethiopia also playing a crucial role in its rise to prominence. Ethiopia not only sees Berbera as a complementary option to the Djibouti axis, but also as a necessary step forward for the industrial development of its eastern regions. What the Ethiopian government and logistics actors see as complementarity is in fact a power struggle between Djibouti and Berbera to capture the other's market share. While Djibouti is striving to maintain its lead, Berbera has the advantage of opening up a previously neglected hinterland. This hinterland includes Somaliland, with its population of around 5-6 million, and its capital Hargeysa, which has over one million inhabitants. A bypass currently under construction will help to streamline the dense traffic linked to the corridor. The main hinterland of this corridor is the Ethiopian ethno-federal region populated by Somalis (6.4 million inhabitants 9 ), mainly up to the town of Dire Dawa and encompassing the entire Ogaden. The main choke point of traffic flows is the "border city" of Jigjiga, the capital of the ethno-federal region. This city is a busy transit zone located 70 km from the Tog Wajale border post. This corridor handles the majority of export livestock destined for the countries of the Arabian Peninsula, the majority of food aid destined for Ethiopia (65 % according to widely cited figures), as well as an increasing share of bulk and container cargo bound for Dire Dawa and the capital region via the A10. The secondary corridor's road network has been continuously strengthened since the end of the 2000s, which bodes well for its change of status and the possible capture of traffic in the Djibouti area. For Somaliland, there is also a major political question at stake, since asserting itself as a state and partner of Ethiopia allows it to partially guarantee its sovereignty in the face of a still non-functional federal Somalia, as well as to further assert its status on the international scene [START_REF] Stepputat | Politics of circulation: The makings of the Berbera corridor in Somali East Africa[END_REF][START_REF] Tahir | Critical infrastructures as sites of conflict over state legitimacy: The case of Hargeisa Airport in Somaliland, Northern Somalia[END_REF]. Finally, the position of the port of Assab remains open to speculation. Boasting high-quality infrastructure developed by the Soviets, and located in the immediate vicinity of the Bab el-Mandeb Strait, the port of Assab was recently used by the United Arab Emirates for military operations in Yemen (2015Yemen ( -2021)). This port offered the immense advantage of housing Ethiopia's one and only refinery whose operations were shut down in 1997. Following the reestablishment of relations between Ethiopia and Eritrea in 2018 ("Ethiopia and Eritrea," 2018), plans were made for Arab pipeline projects in Assab, but none have seen completion 10 . Though the port remains limited in capacity (seven berths, no gantry cranes) it does offer scope for improvement and a quality anchorage. In the short or medium term, however, it does not seem likely that Assab will reclaim its position as a major port facility.

<

Other corridors

The opening of corridors to Ethiopia via Port Sudan and Suakin (Sudan); Massawa (Eritrea); and Mombasa and Lamu (Kenya) also open up other, albeit more marginal, flows (Figure 10.3), which represent alternatives to the eastern axis in terms of serving the peripheral regions of Ethiopia:

-The Sudanese corridor starts from a road that leads to Metemma (the principal border post on the high plateaus); the Amhara area and to the towns of Gondar (400 000 inhab.); and Bahir Dar (300 000 inhab.). Despite operations being hindered by Sudanese political difficulties, the ports remain functional. They are however distant from the Ethiopian border (950 km), which significantly limits the volume of flows.

-The Massawa corridor, which primarily serves the Eritrean capital, Asmara, and to a lesser extent, the northern region of Ethiopia via Kokobay the border post (320 km), has the potential to facilitate transport access in Ethiopia. In this vein, the construction of a motorway on the Mekele-Kokobay stretch in Ethiopia has been completed. However, due to the ongoing conflict in the region, this corridor currently offers little value in terms of supporting the Ethiopian national diversification strategy.

-The Kenyan corridors, comprising the Mombasa-Isiolo-Moyale and Lamu-Isiolo-Moyale axes (spanning 1,260 km and 1,200 km respectively), serve as gateways towards the south. Though the former offers limited flow, the latter (currently still under construction), is a large-scale Chinese project: the Lamu Port and Lamu-Southern Sudan-Ethiopia Transport (LAPSSET) Corridor. Its first quays became operational, after a decade-long delay, at the beginning of 2022. The purpose of these quays is to facilitate the movement of materials needed to complete the corridor, including roads, railways, and power grids that will connect the southern part of Ethiopia. The first phase of the project, which has already been completed, had a preliminary budget of US $400 million dollars and will pave the way for subsequent new phases, culminating a total cost of US $25 billion dollars upon completion. By 2065, the colossal project is expected to interconnect on a regional and continental scale, extending all the way to the north-eastern region of Kenya, which has a low population density of Somali Kenyans. This project will therefore serve as a crucial international corridor bound for the South of Ethiopia and South Sudan, which represents the most significant market in the region 11 . < Figure 10.3 here> Figure 10.3: current and potential corridors in the Addis Abeba Region. Map created by the author.

In summary, the ports of Somalia-excluding Berbera-such as the ports of Boosaaso, Muqdisho, and Kismaayo, cannot claim to be anything more than weak supply lines for the Somali territory. This applies to the informally organized Somali areas that extend from Djibouti to Kenya via Ethiopia. Despite the diverse nature and spatial organization of these flows, they are all limited in terms of tonnage and financial volumes.

Conclusion

Industry development in Ethiopia, whose added value exceeded US $21 billion in 2020-a five-fold increase from a decade ago (World Bank, 2021)-has resulted in a considerable reduction in the cost of importing and exporting containers. The corridors in Ethiopia could therefore potentially provide a leverage effect. However, two key issues remain:

-First, there is the heavy dependence on external funding and construction, and regional-level competition to attract traffic, as illustrated by the ongoing rivalry with Berbera Port in Djibouti. The corridors clearly operate within a strained diplomatic environment, marked by a complex interplay of economic and diplomatic factors that often extends beyond the borders of Ethiopia. It should be remembered that Djibouti and Berbera enjoy the advantage of guaranteed security by international external actors: China, the USA, and France for Djibouti; UAE for the Berbera region [START_REF] Larsen | Gulf state rivalries in the Horn of Africa: Time for a Red Sea policy? DIIS Policy Brief[END_REF]. Though successful, this added layer of maritime security may come at a cost, as it entails a strong reliance on local political actors. In the event of changes to international or regional strategic (and economic) objectives involved on a local level-such as the intervention of UAE in Yemen; Eritrea's involvement in the Ethiopian civil conflict; or fierce competition between the United States and China in the Indo-Pacific sphere-there is every chance that existing strategies will change rapidly.

-Second, there is the issue of regional security. Excluding Ethio-Eritrean relations, which shook the transport landscape at the turn of the century, the Tigray rebellion placed enormous pressure on the Djibouti corridor when the front line came near Mile on the RN1-A1. The capture of this city would have resulted in the disruption of the supply to the capital region, and its probable collapse. Furthermore, the Somali and the Oromo regions are still plagued by conflictual trends that lead to a considerable slowdown in transportation and project development. Similarly, the buffer zone established by Kenyan forces during Operation Linda Nchi (2011-2012) and its subsequent developments, particularly in support of the frontier Somali area of Jubbaland, aimed to prevent the spread of Somali Islamist insurrection promoted by the militant organization Al-Shabaab in the area where the Lamu-Moyale corridor will be constructed.

Thus, in order to bridge the gap between marginal and central regions and mitigate Ethiopia's extreme centre-periphery divide, significant public investment is required, and even then, at least a minimal level of security must be in place for ventures to prove profitable. However, it is difficult to envision the feasibility of this strategy for the heavily indebted Ethiopian state, which lacks the resources to simultaneously handle internal, high-intensity armed conflicts and pursue major infrastructure plans. This represents a major obstacle to Ethiopia's development. All things considered, the case study of Ethiopia presented in this chapter could provide an excellent illustration of the concept of "inversion territoriale" ("territorial inversion"), a term coined by [START_REF] Debrie | L'enclavement, réévaluation théorique et application à l'Afrique de l'Ouest[END_REF]. The authors used this term in the context of West Africa to describe the development of transport routes by inland countries as a means of reversing their landlocked status. As with their description, we can see that strong inter-port competition is driven by the desire of the Ethiopian giant, with its huge population and economic potential, to expand its market reach and free itself from its dependence on the port of Djibouti by diversifying its access routes. Though this shift is still in its early stages because of the limits mentioned above, it underscores the need to re-evaluate Ethiopia's current status as a landlocked country.

Table 10 .

 10 1: Tariffs (one movement) for a normal container (USD) at Djibouti and Berbera container terminals (DPW Berbera Tariff Book 2021; Société de Gestion du Terminal de Doraleh (SGTD) (Doraleh Container Terminal Management Company) Tariff Book 2021).

  Figure 10.2 here > Dubai Port World Berbera Masterplan. Image sourced from Wikicommons.

	Figure 10.2:
		Djibouti		Berbera
		up to 1 TEU	over 1 TEU	up to 1 TEU over 1 TEU
	Discharging/charging full	133	166	130	195
	Discharging/charging empty	94	121	80	120
	Transhipment full	141	201	141	175
	Transhipment empty	112	137	124	159

Only Hoyle & Charlier's article (1995) deals specifically with East Africa (Kenya and Tanzania), and it only makes passing references to Ethiopia.

The research method was based on: (1) the study of satellite imagery; (2) internet sources related to the topic (mainly official reports and specialized websites); (3) a network of local informants; (4) two interviews with academics from the region (non-specialists in the field of transportation). A literature review on corridors was conducted prior to the study. Due to the security situation in Ethiopia, it was not possible to conduct fieldwork.

Ethiopia's climate resilient transport sector strategy (2020). Retrieved from https://www.mofed.gov.et/media/filer_public/15/31/153174c3-b472-4339-b3bb-fb2c48cad629/transport_cr.pdf

 4 It should be noted that, in addition to Ethiopia's current landlocked status, there are two other landlocked situations at the infra-national level: one from the highlands to the lowlands encompassing