Burvenich, Christian

De Spiegeleer, Bart

Pezeshki, Adel

Vergote, Valentijn

English

Ghent University Academic Bibliography

INTRODUCTION Bovine mammary gland peptibolomics:                                             Adsorption of peptides at the sample drying stepAdel Pezeshki1,2, Valentijn Vergote1, Christian Burvenich2, Bart De Spiegeleer1*1Drug Quality & Registration (DruQuaR) group, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium,                       2Department of Physiology and Biometrics, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium.*Corresponding author: bart.despiegeleer@ugent.be (O.Ref.: 2008–545c; Proteomic Europe Conference 2008, Lisbon, Portugal)EXPERIMENTALRESULTS AND DISCUSSIONCONCLUSIONSDruQuaRTable 1: HPLC method ColumnVydac Everest RP-C18250 mm × 4.6 mm, 5 µm, 300 ÅColumn temperature 30°CFlow rate 1 ml/minMobile phase constituentsA = Water with 0.1% m/V formic acidB = Acetonitrile with 0.1% m/V formic acidGradientIsocratic for 1 min: 90% A and 10%BLinear gradient to 40% B at 60 minSample temperature AmbientInjection volume 30 µLDetection UV @ 275 nm40,050,060,070,080,090,0100,0110,0120,0Speedvac LyovacRecovery (%)PlasticGlass40,050,060,070,080,090,0100,0110,0120,0Mannitol Water DMSO DMF PEG 400 n-B-D-Glucopyran.Recovery (%)PlasticGlassA405060708090100110120Mannitol Water DMSO DMF PEG 400 n-B-D-Glucopyran.Recovery (%)PlasticGlassB40,050,060,070,080,090,0100,0110,0120,0Recovery (%)PlasticGlassA40,050,060,070,080,090,0100,0110,0120,0Recovery (%)PlasticGlassBPeptide AdditiveRecovery (%)SpeedVac LyoVacPlastic Glass Plastic GlassLeucine-enkephalinMannitol 90.3 ± 3.5 98.9 ± 4.2 89.0 ± 2.9 94.6 ± 3.4 Water 97.6 ± 9.1 97.2 ± 8.3 93.2 ± 7.4 96.2 ± 9.1DMSO 96.5 ± 5.5 93.2 ± 4.7 85.1 ± 4.7 91.4 ± 3.4DMF 89.8 ± 3.5 101.9 ± 1.0 90.9 ± 1.6 93.3 ± 3.3PEG 400 89.4 ± 3.3 97.8 ± 3.6 83.4  ± 5.2 93.3 ± 3.1n-β-D-Glucopyranoside 95.9 ± 1.6 100.9 ± 2.9 91.4 ± 2.0 95.0 ± 1.4 GoserelinMannitol 92.3 ± 4.6 92.3 ± 6.7 99.2 ± 3.1 64.3 ± 3.6Water 84.4 ± 4.5 102.2 ± 8.8 100.2 ± 4.7 73.5 ± 5.3DMSO 88.8 ± 5.1 100.6 ± 5.7 96.1 ± 5.5 72.1 ± 4.5DMF 89.7 ± 6.8 90.8 ± 7.2 92.4 ± 5.8 72.8 ± 6.5PEG 400 85.7 ± 8.2 92.8 ± 7.8 71.4 ± 6.6 54.0 ± 7.7n-β-D-Glucopyranoside 93.1 ± 1.3 92.9 ± 8.3 90.4 ± 2.5 90.7 ± 2.5Bovine insulinMannitol 62.8 ± 0.8 83.6 ± 4.2 81.7 ± 4.0 94.7 ± 5.3Water 82.4 ± 5.1 83.9 ± 6.0 90.3 ± 6.1 94.8 ± 2.4DMSO 92.6 ± 7.1 92.5 ± 7.4 94.1 ± 8.4 103.6 ± 9.2DMF 74.1 ± 2.7 89.2 ± 4.5 91.8 ± 5.1 98.6 ± 3.8PEG 400 95.8 ± 5.5 81.0 ± 5.2 64.7 ± 2.1 95.7 ± 3.3n-β-D-Glucopyranoside 95.4 ± 7.1 92.1 ± 6.7 89.3 ± 5.0 88.9 ± 4.9BuserelinMannitol 91.9 ± 4.4 93.4 ± 1.4 85.0 ± 3.1 71.9 ± 0.9Water 86.4 ± 3.8 91.6 ± 8.6 89.5 ± 1.5 82.4 ± 2.7DMSO 86.4 ± 1.8 93.4 ± 1.2 103.7 ± 2.1 81.7 ± 2.1DMF 91.1  ± 2.4 90.1 ± 1.2 94.2 ± 6.7 77.7 ± 0.9PEG 400 79.0 ± 2.2 105.7 ± 3.3 94.1 ± 2.4 79.2 ± 5.0n-β-D-Glucopyranoside 96.2 ± 1.7 92.0 ± 3.8 92.0 ± 2.4 95.1 ± 1.7Mouse obestatinMannitol 95.3 ± 3.1 93.7 ± 4.4 90.8 ± 6.0 95.2 ± 4.7Water 90.8 ± 2.8 91.1 ± 11.0 91.2  ± 3.0 88.5 ± 4.0DMSO 99.8 ± 3.7 91.2 ± 6.1 95.8 ± 4.0 91.1 ± 2.7DMF 101.4 ± 7.8 90.2 ± 7.4 101.3 ± 8.0 91.8 ± 7.0 PEG 400 101.8 ± 8.3 93.2 ± 3.4 82.7 ± 4.4 87.2  ± 1.5n-β-D-Glucopyranoside 97.2 ± 2.6 102.5 ± 3.6 92.6 ± 2.8 96.1 ± 2.3Table 2: Peptide loss during sample evaporationFigure 1Effect of drying method on model peptides recovery Figure 2Effect of additives using SpeedVac (A) and LyoVac(B) Variable recovery of peptides is a well-known but often neglected phenomenon that impacts quantitative peptide analysis. Only recently, few studies have systematicallyinvestigated the factors influencing the adsorption of peptides, however, without including the drying step in the sample preparation.1,2The sample concentrating and drying step can be a significant source of peptide loss, mainly due to adsorption.3 Efficient and careful removal of solvent from samples bycentrifugal evaporation or freeze-drying methods is an important step in peptidomics. The recovery of peptides has not yet been fully investigated with these sampledrying methods. Moreover, the surface adsorption of the peptides by the container and efforts to reduce this adsorption by organic additives is only scarcely elaborateduntil now. In this experiment, the recovery of five model peptides was investigated applying different organic additives in function of the two applied solvent evaporationprocesses and vials (polypropylene and glass).EXPERIMENT 1: EFFECT OF ADDITIVES ON MODEL PEPTIDES RECOVERY:A mixture of model peptides (0.2 µg/µl each), i.e. bovine insulin, buserelin, goserelin, leucine-enkephalin, and mouseobestatin prepared by water/acetonitrile mixture (95:5 V/V) was pipetted into total recovery glass vials containing thefive separate additives, i.e. DMSO, DMF, PEG 400, mannitol (0.5% W/V aqueous solution) and n-nonyl-β-D-glucopyranoside (0.05% W/V aqueous solution). 95 µl of model peptides mixture was added to vials containing the fiveµl of selected additives. Without drying involved, the samples were analyzed by the HPLC method (Table 1).EXPERIMENT 2: EFFECT OF ADDITIVES ON MODEL PEPTIDE RECOVERY INCLUDING DRYING AND VIAL MATERIAL EFFECTS:A 0.2 µg/µl mixture of model peptides was prepared in water. Five µl of additives were pipetted into vials made ofpolypropylene and glass containing 95 µl of the model peptides mixture. After drying the samples by centrifugalevaporation or freeze-drying, 95 µl of water/acetonitrile mixture (95:5 V/V) was added to the samples (recovery valueswere corrected for water-drying loss in case of mannitol and n-nonyl-β-D-glucopyranoside aqueous solutions, water,DMSO, and DMF). Samples were analyzed in triplicate (n=3) by the HPLC (Table 1). The drying action resulted in a decreased recovery of model peptides (Table 2). Maximum amounts of model peptides were recovered for the following vial type-evaporation techniquecombinations: polypropylene-lyophilization and glass-centrifugal evaporation (Figure 1). n-nonyl-β-D-Glucopyranoside is the best additive for recovery of peptides during centrifugal evaporation aswell as during freeze-drying (Figure 2). Recovery of goserelin and buserelin was significantly lower on glass vials when LyoVac was used, while thiswas not consistent using SpeedVac (Figure 3).Drying step (e.g. as part of the sample preparation) can cause significant peptide loss. Applying n-nonyl-β-D-glucopyranoside as an additive along with choosingappropriate vial material (i.e. polypropylene for LyoVac and glass for SpeedVac methods) can prevent or decrease peptide loss during the selected solvent removalevaporation procedure.REFERENCES1P. Hyenstrand, J.S. Metcalf, K.A. Beattie, G.A. Codd, Toxicon, 39, 2001, 589-594.2H. John, M. Walden, S. Schäfer, S. Genz, W.G. Anal. Bioanal. Chem. 378, 2004, 883-897.3A. Pezeshki, V. Vergote, S. Van Dorpe, B. Baert, C. Burvenich and B. De Spiegeleer, JPBA, submitted, 2008.ACKNOWLEDGEMENTS: We would like to thank Koen Depoorter for his operational help in the experiments.Figure 3Effect of peptides using SpeedVac (A) and LyoVac(B) Drug Quality & Registration (DruQuaR) format Man06.001.006-v01

Bovine mammary gland peptibolomics: Adsorption of peptides at the sample drying step

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Bovine mammary gland peptibolomics: Adsorption of peptides at the sample drying step

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