maenas (intertidal zone) and Segonzacia mesatlantica - Station ...

More documents

Recommendations

Info

114 CHAPITRE 3. STRUCTURE DE L’HC DE C. MAENAS PAR ESI-MS and the purified complexes were issued from another single individual ; those samples were withdrawn one day after catching the crabs. Sample preparation for ESI-MS All samples used for ESI-MS were desalted through concentration-dilution cycles using 10 kDa Microcon (Millipore) prior to analysis. Typically, 30 µl of native hemolymph diluted in 470 µl 10 mM ammonium acetate (AcNH4), pH 6.8 or 250 µl of purified sample (protein concentration about 1- 3 µg/µl) were first concentrated to almost dryness and then resuspended in 400 µl 10 mM AcNH4. At least 10 successive similar concentration-dilution steps were performed in order to ensure that the samples were correctly desalted prior to ESI-MS analysis. The samples were stored at 4°C in 10 mM AcNH4 until analysis. The desalting was realised just prior to MS analysis and desalted samples for non-covalent analysis were not kept more than 2 days at 4°C because of dissociation occurring upon longer storage. Non-covalent and denaturing ESI-MS Mass spectrometry experiences were performed on a MicroTOF instrument (BRUKER Daltonics, Bremen, Germany) equipped with an ESI source. For non-covalent analysis, desalted samples were diluted in 10 mM AcNH4 at a final concentration of approximately 1.5 µM (for 900 kDa dodecamers). The injection rate for the samples was 4 µl/min. The nebulization voltage was set to 5 kV, nebulization gas (N2) pressure was 1 bar, drying gas flow was 4 l/min, source temperature was 200°C and the capillary exit voltage was set to 400 V. The calibration was made using 1 mg/ml CsI in water/isopropanol (50 :50 volume). The acquisition range was 500 to 20000 m/z. Spectra were smoothed using a Savitzky-Golay method and baseline subtracted. Complexes masses were estimated using an in-built ruler (BRUKER DataAnalysis v3.2 software). For denaturing analysis, desalted samples were diluted in a water/acetonitrile/formic acid mix (H2O/ACN/FA 50 :50 :1 volume) at a final concentration of approximately 4 µM (for 75 kDa subunits). The nebulization gas (N2) pressure was 0.3 bar, drying gas flow was 3 l/min, and the capillary exit voltage was set to 160 V. Calibration was performed using myoglobin (SIGMA-ALDRICH). Mass spectra were analysed using maximum entropy deconvolution (BRUKER DataAnalysis v3.2 software). In the following, denaturing analysis refers to ESI-MS analysis in H2O/ACN/FA with classical instrumental parameters. In these conditions, all non-covalent interactions are broken. Non-covalent analysis refers to ESI-MS analysis in AcNH4 with "gentle" instrumental parameters. In this case, non-
3.2. MANUSCRIT : STRUCTURAL STUDY OF C. MAENAS HC BY ESI-MS 115 covalent interactions are preserved during the analysis, even if biochemical treatments can partially dissociate the complexes before analysis. Dissociation in TEA For alkaline denaturation of hemocyanin, desalted samples were diluted in 10 mM AcNH4 with various TEA concentrations (from 0.005 % to 0.05 % TEA in volume, pH 7.52 to 9.02 respectively) and incubated for 15 min at ambient temperature before non-covalent ESI-MS analysis was performed. Reassociation with formic acid For acidic reassociation, desalted hemocyanin was first dissociated in 10 mM AcNH4, 0.03 % TEA (pH 8.6) for 15 min at ambient temperature and then formic acid was added to a final concentration of 0.001 % to 0.04 % (pH 8.48 to 4.22, respectively) ; the preparation was incubated for another 15 min before performing non-covalent ESI-MS analysis to allow for reassociation. Effect of L-lactic acid For investigation of L-lactic acid effect, desalted hemocyanin was first dissociated in 10 mM AcNH4, 0.03 % TEA (pH 8.6) for 15 min at ambient temperature and then lactic acid was added to a final concentration of 2 mM (L(+)-lactic acid, SIGMA-ALDRICH), along with TEA to counterbalance the acidifying effect of lactic acid. After addition, the final TEA concentration ranged from the original 0.03 % to 0.07 %, and pH values ranged from 5.89 to 8.58. The preparation was left to incubate for 15 min at ambient temperature before non-covalent ESI-MS analysis. Effect of chelation of divalent cations by EDTA Divalent cations were removed using EDTA in order to investigate their structural role. Two 10 mM Na+-EDTA, 10 mM AcNH4 mixes were prepared with different pH, one with 0.11 % TEA (approximative pH 7.5) and the other with 0.15 % TEA (approximative pH 9). A desalted total hemolymph sample was diluted 20-fold in each of these mixes, concentrated on a 10 kDa Microcon filter, diluted in the same EDTA mix again and then washed by 6 concentration-dilution steps in 10 mM AcNH4, 0.005 % TEA and 0.05 % TEA respectively in order to remove the Na+ and EDTA salts along with the chelated cations. Non-covalent analysis was performed at this step. Then, L-lactic acid was added up to 2 mM final concentration with TEA to adjust pH either at 7.5 or 9 approximately and the preparation was left to incubate for 15 min at ambient temperature before analysis.
Page 1 and 2:
THESE DE DOCTORAT DE L’UNIVERSITE
Page 3:
i Résumé / Abstract Réponse adap
Page 6 and 7:
iv SOMMAIRE 4.2 Objectifs de l’é
Page 8 and 9:
2 CHAPITRE 1. INTRODUCTION Les éco
Page 10 and 11:
4 CHAPITRE 1. INTRODUCTION 1.1 Mili
Page 12 and 13:
6 CHAPITRE 1. INTRODUCTION Les prin
Page 14 and 15:
8 CHAPITRE 1. INTRODUCTION TAB. 1.1
Page 16 and 17:
10 CHAPITRE 1. INTRODUCTION et cycl
Page 18 and 19:
FIG. 1.5 - Localisation des princip
Page 20 and 21:
(a) FIG. 1.6 - Fonctionnement d’u
Page 22 and 23:
16 CHAPITRE 1. INTRODUCTION TAB. 1.
Page 24 and 25:
18 CHAPITRE 1. INTRODUCTION FIG. 1.
Page 26 and 27:
20 CHAPITRE 1. INTRODUCTION Différ
Page 28 and 29:
Page 30 and 31:
24 CHAPITRE 1. INTRODUCTION le mili
Page 32 and 33:
26 CHAPITRE 1. INTRODUCTION Capacit
Page 34 and 35:
28 CHAPITRE 1. INTRODUCTION TAB. 1.
Page 36 and 37:
30 CHAPITRE 1. INTRODUCTION 700 600
Page 38 and 39:
32 CHAPITRE 1. INTRODUCTION férent
Page 40 and 41:
34 CHAPITRE 1. INTRODUCTION (a) (b)
Page 42 and 43:
Page 44 and 45:
Page 46 and 47:
40 CHAPITRE 1. INTRODUCTION 1 P 50
Page 48 and 49:
42 CHAPITRE 1. INTRODUCTION presque
Page 50 and 51:
44 CHAPITRE 1. INTRODUCTION affecte
Page 52 and 53:
Page 54 and 55:
Page 56 and 57:
50 CHAPITRE 1. INTRODUCTION fure co
Page 58 and 59:
52 CHAPITRE 1. INTRODUCTION amélio
Page 60 and 61:
54 CHAPITRE 1. INTRODUCTION 1.5 Que
Page 62 and 63:
56 CHAPITRE 2. ESI-MS ET MALLS APPL
Page 64 and 65:
Page 66 and 67:
Page 68 and 69:
Page 70 and 71: 64 CHAPITRE 2. ESI-MS ET MALLS APPL
Page 78 and 79: The Structural Analysis of Large No
Page 108 and 109: 102 CHAPITRE 2. ESI-MS ET MALLS APP
Page 110 and 111: 104 CHAPITRE 3. STRUCTURE DE L’HC
Page 140 and 141: 134 CHAPITRE 4. PLASTICITÉ PHÉNOT
Page 170 and 171:
164 CHAPITRE 4. PLASTICITÉ PHÉNOT
Page 172 and 173:
Dénaturant Natif
Page 174 and 175:
Page 176 and 177:
Page 178 and 179:
172 CHAPITRE 5. ADAPTATIONS RESPIRA
Page 180 and 181:
Page 182 and 183:
Page 184 and 185:
Page 186 and 187:
Page 188 and 189:
Page 190 and 191:
Page 192 and 193:
Page 194 and 195:
Page 196 and 197:
Page 198 and 199:
Page 200 and 201:
Page 202 and 203:
Page 204 and 205:
Page 206 and 207:
Page 208 and 209:
Page 210 and 211:
Page 212 and 213:
Page 214 and 215:
Page 216 and 217:
Page 218 and 219:
Page 220 and 221:
Page 222 and 223:
216 CHAPITRE 6. CONCLUSION ET PERSP
Page 224 and 225:
Page 226 and 227:
Page 228 and 229:
Page 230 and 231:
Page 233 and 234:
Annexe A Détermination des masses
Page 235 and 236:
229 10000 h280 .M h) h280−h337 80
Page 237 and 238:
Annexe B Détermination des distrib
Page 239 and 240:
233 Modèle 2 : 2 sous-unités lég
Page 241 and 242:
235 1 0.9 0.8 6 s-u aléatoires 2 s
Page 243 and 244:
Bibliographie Adair, G. S. The hemo
Page 245 and 246:
BIBLIOGRAPHIE 239 Catalina, M. I.,
Page 247 and 248:
BIBLIOGRAPHIE 241 Farmer, C. S., J.
Page 249 and 250:
BIBLIOGRAPHIE 243 Hourdez, S. Adapt
Page 251 and 252:
BIBLIOGRAPHIE 245 Mangum, C. P. Sub
Page 253 and 254:
BIBLIOGRAPHIE 247 Rainer, J., C. P.
Page 255 and 256:
BIBLIOGRAPHIE 249 Svedberg, T. et I
Page 257 and 258:
BIBLIOGRAPHIE 251 Vanin, S., E. Neg
Page 259 and 260:
Liste des figures 1.1 Cycle des mar
Page 261:
LISTE DES FIGURES 255 4.17 Profils
Page 265 and 266:
Table des matières Résumé Sommai
Page 267 and 268:
TABLE DES MATIÈRES 261 4.3.5 Spect
Page 269 and 270:
TABLE DES MATIÈRES 263 Table des m
show all

maenas (intertidal zone) and Segonzacia mesatlantica - Station ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?