Hydrophobic Interaction Chromatography for Separation of Tryptophan and Methionine Oxidized Peptides from Their Native Forms

Transcription

1 Application ote 2 ydrophobic Interaction Chromatography for Separation of Tryptophan and Methionine xidized Peptides from Their ative Forms ITRDUCTI ydrophobic interaction chromatography (IC) is a technique used to separate peptides, proteins, and other biological molecules based on their degree of hydrophobicity. The IC mobile phase comprises a highconcentration salting-out agent typically ammonium sulfate that increases the hydrophobic interaction between the solute and the stationary phase. IC and reversed-phase chromatography are closely related techniques. Both are based upon interactions between solvent-accessible nonpolar groups (hydrophobic patches) on the surface of the solute and the hydrophobic ligands of the stationary phase. In practice, however, IC and reversed-phase chromatography are different, as reversed-phase stationary phases are more highly substituted with hydrophobic ligands than IC stationary phases. The techniques also use different mobile phases. Protein binding to reversed-phase stationary-phases is usually very strong, and requires polar solvents for elution, which can denature the proteins during the separation. Reversed-phase PLC has found extensive application in analytical and preparative separations of peptides and low molecular weight proteins that have stable primary structure in aqueous-organic mobile phases. IC is an alternative to exploiting the hydrophobic properties of proteins, working with nonpolar, non-denaturing mobile phases. 2 Proteins and peptides are sensitive to oxidative damage. xidizing proteins and peptides can alter their biological activity, half-life, and immunogenicity. atural biological and environmental oxidants have been suggested as causative or contributory factors in many diseases. xidation of proteins has also been reported as a natural posttranslational event mediated enzymatically by amines and oxidases. 3 Methionine, cysteine, histidine, tryptophan, and tyrosine residues are most susceptible to this oxidation. 4 Methionine is easily oxidized by atmospheric oxygen to form methionine sulfoxide, and tryptophan can be oxidized into four different oxidation products by peroxide. Peroxide is an impurity generated during protein storage, or from polysorbates commonly used for protein purification and solubilization. ther peroxide-contaminated materials include polyethylene glycol (PEG) or silicon rubber from vial stoppers. When proteins are used as pharmaceutical ingredients, methionine and tryptophan oxidation during processing or storage can affect protein activity. 5

2 In Application ote 29, the authors used a weak cation-exchange chromatography method with the ProPac WCX- to separate oxidized and native Luteinizing ormone-releasing ormone (L-R) a tryptophan-containing peptide, and oxidized and native a-melanocyte Stimulating ormone (MSA) a methionine-containing peptide. ere, the authors demonstrate separation of these oxidized and nonoxidized variants on the ProPac IC- (hydrophobic interaction) column and the ProPac WCX- (weak anion-exchange) column using ICS-3 PEEK and UltiMate 3 Titanium systems. The ProPac IC- column separates L-R isoforms not found using weak the cation-exchange column and chemistry, and the resolution of the oxidized variants is improved over the aforementioned method. Equipment ICS-3 Liquid Chromatography System ICS-3 SP (P/ 677) ICS-3 VWD (P/ 64653) ICS-3 TC (P/ 64444) AS Autosampler (P/ 56859) ProPac IC- column, 4.6 mm, (P/ 63655) ProPac WCX- column, 4.6 mm, (P/ 54993) UltiMate 3 Titanium Liquid Chromatography System SRD-36 Solvent Rack with six degasser channels (P/ ) LPG-34AB Quaternary Analytical Pump (P/ 537.5), or DPG-36AB Dual Ternary Analytical Pump (P/ 537.4) WPS-3TBPL Biocompatible Analytical Autosampler (P/ ) TCC-32B Column Compartment with two PEEK -port 2-position valves (P/ ), or TCC-3 Column Compartment (P/ 5722.) Detector VWD-34 Variable Wavelength (P/ 574.), or PDA-3 PhotoDiode Array Detector (P/ 58.2) Biocompatible Analytical Flow Cell (P/ 674.2), or Biocompatible Analytical Flow Cell for PDA (P/ 68.2) Chromeleon Data Management Software MSQ Plus Mass Spectrometer with Data System (P/ 636) AXP-MS Auxiliary Pump Kit (P/ 6684) Chromeleon MS Support (MSQ MS control software) (P/ 6726) Reagents and Samples Deionized water 8.2 (MΩ-cm) Ammonium sulfate, Molecular biology grade (Sigma-Aldrich, A448) Sodium phosphate, dibasic, anhydrous, powder (J.T. Baker, 3826) Sodium phosphate, monobasic, monohydrate (Sigma-Aldrich, S8282) Ammonium bicarbonate (Sigma-Aldrich, S9638) Ethylenediaminetetraacetic acid (EDTA), disodium dihydrate (Sigma-Aldrich, E644) ydrogen peroxide, 3% (Sigma-Aldrich, 9) Dimethyl sulfoxide (DMS), ACS reagent (Sigma-Aldrich, 4723 ) ydrochloric acid (Cl), 2, Ultrex II Ultrapure Reagent (J.T. Baker, 69-5) Acetic acid, glacial, PLC grade (J.T. Baker 955-3) Luteinizing hormone-releasing hormone (L-R) uman (Sigma-Aldrich, L734), 97% of the peptide material contains the following sequence: p-glu-is-trp-ser-tyr-gly-leu-arg-pro-gly- 2 a-melanocyte Stimulating ormone (a-ms), (Sigma- Aldrich, M-435), 78% of dry weight is peptide, 98% of the peptide material contains the following sequence -Acetyl-Ser-Tyr-Ser-Met-Glu-is-Phe-Arg-Trp-Gly- Lys-Pro-Val- 2 CDITIS Conditions for ydrophobic Interaction Chromatography Column: ProPac IC column, 4.6 mm (P/ 63655) Flow Rate:. ml/min Temperature: 3 C Inj. Volume: µl 2 ydrophobic Interaction Chromatography for Separation of Tryptophan and Methionine xidized Peptides from Their ative Forms

3 Detection: Eluents: UV, 24, 254, and 28 nm A: 2 M Ammonium sulfate in. M sodium phosphate (p 7.) B:. M Sodium phosphate, monobasic (p 7.) Gradient Program: Time (min) A% B% Comments... Precondition column before sample injection... Sample injection 2... Isocratic mobile phase during sample injection 7... Gradient 2... Wash Re-equilibration Prewash column with % B for 5 min prior to the first injection. Conditions for Weak Cation-Exchange Chromatography Column: ProPac WCX column, 4 25 mm (P/ 54993) Flow Rate:. ml/min Temperature: 3 C Inj. Volume: µl Detection: UV, 24, 254, and 28 nm Eluents: A: mm Sodium phosphate (p 6.) B: mm Sodium phosphate with 5 mm sodium chloride (p 6.) Gradient Program: Time (min) A% B% Comments Sample injection Gradient Wash Re-equilibration Mass Spectrometry Conditions An AXP-MS auxiliary pump was used to manually infuse the oxidized and non-oxidized L-R samples at a flow rate of.2 ml/min for optimization in the MSQ Plus mass spectrometer. During optimization, various cone voltages were examined, with 7V determined as optimal. As these were charged species, ESI was the preferred ionization mode, with positive ionization providing the best response. Ion Source: Positive ESI eedle voltage: 3V Cone voltage: 7V Probe temp: 35 C Scan mode: Full scan at m/z per second Scan range 5~2 m/z AXP-MS flow.2 ml/min C 3 C/ 2 (5/5, v/v) Infused Volume µl PREPARATI F SLUTIS AD REAGETS 2 M Ammonium Sulfate in. M Sodium Phosphate, Monobasic, p 7. Dissolve g ammonium sulfate and 2 g sodium phosphate monobasic in 65 ml DI water (8.2 MΩ cm) in a L volumetric flask. Adjust the p to 7. with approximately 2.5 ml 5% sodium hydroxide and bring to volume with DI water. Filter through a.22 µm filter.. M Sodium Phosphate, Monobasic, p 7. Dissolve 2 g sodium phosphate monobasic in 9 ml DI water in a L volumetric flask. Adjust the p to 7. with approximately.5 ml of 5% sodium hydroxide and bring to volume with DI water. Filter through a.22 µm filter.. M Ammonium Bicarbonate, p 8.8 Combine 7.9 g ammonium bicarbonate with 9 ml DI water in a ml volumetric flask. Adjust the p to 8.8 with -2 Cl solution. Bring to volume with DI water. Filter through a.22 µm filter. 75 mm EDTA, p 8. Combine 78.8 g EDTA disodium dihydrate with 9 ml DI water in a L volumetric flask. Adjust the p to 8. using a.5% a (w/w) solution. Bring to volume with DI water. Filter through a.22 µm filter. 4 mm ydrogen Peroxide Combine.455 ml hydrogen peroxide (3%, 8.79 M) with 9.55 ml water in a ml volumetric flask. Make this solution fresh daily. M Sodium Chloride Dissolve g sodium chloride in deionized water in a L volumetric flask and bring to volume. Filter through a.22 µm filter. Application ote 2 3

4 2 mm Sodium Phosphate, Dibasic Dissolve g anhydrous dibasic sodium phosphate in L DI water in a volumetric flask. Filter through a.22 µm filter. 2 mm Sodium Phosphate, Monobasic Dissolve 27.6 g of monohydrate monobasic sodium phosphate in L DI water in a volumetric flask. Filter through a.22 µm filter. mm Sodium Phosphate, p 6. Combine 4 ml 2 mm dibasic sodium phosphate, 86 ml 2 mm monobasic sodium phosphate, and 9 ml water in a 2 L volumetric flask. Adjust the relative proportions of 2 mm dibasic and monobasic sodium phosphate used to achieve a p of 6. while maintaining a total volume of ml. mm Sodium Phosphate with 5 mm Sodium Chloride, p 6. Combine 35 ml 2 mm dibasic sodium phosphate, 65 ml 2 mm monobasic sodium phosphate, ml M sodium chloride, and 9 ml DI water in a 2 L volumetric flask. Adjust the relative proportions of 2 mm dibasic and monobasic sodium phosphate to achieve a p of 6. while maintaining a total volume of ml. SAMPLE PREPARATI xidation of Tryptophan in L-R A vial containing mg L-R was reconstituted with a solution consisting of 48.6 µl glacial acetic acid, 6.5 µl 2 Cl, and.3 µl DI 2, and labeled on-xidized L-R. Another vial containing mg L-R was reconstituted with a solution consisting of 48.6 µl glacial acetic acid, 6.5 µl 2 Cl, and.3 µl DMS and labeled xidized L-R. A nonoxidized buffer control was prepared by combining 4.7 µl glacial acetic acid, 5.6 µl -2 Cl, and. µl water. An oxidized buffer control was prepared by combining 4.7 µl glacial acetic acid, 5.6 µl 2 Cl, and. µl DMS. All vials were incubated at room temperature for 5 min. After incubation, 94.7 µl DI 2 was added to each vial. The samples were diluted -fold by adding µl of sample to 495 µl of eluent B, then adding 495 µl of eluent A. The samples were maintained at room temperature and analyzed within 48 h. When stored at 4 ºC, the injection solution can be used for up to one week. Samples can be stored in a -4 C freezer for up to one month, with one thaw cycle. Samples stored frozen should be stored in single-use size aliquots. xidation of Methionine in a-ms A vial containing mg MS was reconstituted with 533 µl water to make a.5 mg/ml MS solution. A mg/ml non-oxidized MS control was prepared by combining 5 µl.5 mg/ml MS solution with 5 µl M ammonium bicarbonate, µl 75 mm EDTA, and 2 µl water. A mg/ml Met-xidized MS sample was prepared by combining 5 µl.5 mg/ml MS solution with 5 µl of M ammonium bicarbonate, µl 75 mm EDTA, and 2 µl 4 mm hydrogen peroxide. A buffer control was prepared by combining 5 µl water with 5 µl M ammonium bicarbonate, µl 75 mm EDTA, and 2 µl 4 mm hydrogen peroxide. All the oxidized samples had a final concentration of. M ammonium bicarbonate, 5 mm EDTA, and 53 mm hydrogen peroxide. All samples were incubated for 3 min in an ice water bath. After incubation, each sample was diluted -fold by adding 3 µl to 35 µl eluent B, then adding 35 µl eluent A. The samples are stable for 48 h at room temperature, one week at 4 C, and one month at -4 C (with one thaw cycle). Results and Discussion Figure A shows the elution of two non-oxidized L-R peaks at and 8 min (peaks 5 and 6) on the IC- column at 24 nm. (ote, data was collected at 254 and 28 nm as well, but 24 nm provided optimum response for this analysis.) The peak eluting within the first 3 min was also seen in the buffer control (data not shown). Mass spectrometric analysis of the nonoxidized L-R (Figure 2B) revealed only a single major component with a mass-to-charge ratio (m/z) equal to that expected for L-R. Figure 2B shows the presence of two MS peaks. The first MS peak was identified as L-R [M+2] 2+, and the second peak was identified as an acetonitrile adduct of L-R. Fractions of peaks 5 and 6 in Figure A were collected, dialysed against water, vacuum dried to concentrate, and infused for MS analysis. The m/z of these 4 ydrophobic Interaction Chromatography for Separation of Tryptophan and Methionine xidized Peptides from Their ative Forms

5 Column: ProPac IC- (4.6 mm ) Eluent: (A) 2 M Ammonium Sulfate in. M Sodium Phosphate (p 7.) (B). M Sodium Phosphate Monobasic (p 7.) Temperature: 3 C Flow Rate:. ml/min Inj. Volume: µl Detection: Absorbance, UV 24 nm Peaks:. Unknown 2. Unknown 3. xy L-R 4. xy L-R 5. L-R peak 6. L-R peak 2 7. xy L-R 8. xy L-R AXP-MS Flow:.2 ml/min C 3 C/ 2 (5/5, v/v) Injection: µl eedle Voltage: 3 V Cone Voltage: 7V Probe Temp: 35 C Scan Mode: Full scan at m/z per second Scan Range: 5 2 m/z (55 7 m/z is shown) Peaks: See below 5 mau 5 mau 2 A. on xidized L-R % B B. xidized L-R % B 3 Minutes A. xidized L-R.3E m/z B. on xidized L-R E m/z Relative Abundance Relative Abundance Figure : The separation of (A) non-oxidized L-R and (B) oxidized L-R using the ProPac IC- column. Figure 2. Mass spectra of (A) oxidized L-R and (B) non-oxidized L-R. two peaks were identical (data not shown). The source of the two impurity peaks observed on the IC- column is uncertain, as the supplier reports the product is 97% pure by reversed-phase chromatography. The Trp in L-R was forcibly oxidized with DMS and Cl. Figure B shows four additional peaks, or two oxidation products for each retained peak in the nonoxidized L-R chromatogram. The presence of peaks 5 and 6, with reduced peak areas in the oxidized L-R indicates incomplete oxidation. All six peaks were well resolved using the IC- column. Figure 3 shows a variety of Trp oxidation products described by E.L. Finley et al. 7 The expected products were: hydroxy-tryptophan (TRP), -formylkyurenine (FK), kynurenine (KY), and 3-hydroxykyurenine (3-KY). The authors were able to identify two of the products using MS detection. Figure 2B shows the presence of four additional major ions, three of which were identified. The following assignments were made based on mass/charge ratios. Peak L-R, [M+2] 2+ Peak 2 ydroxy-tryptophan (TRP), oxidized product of L-R, [(M+6)+2] 2+ Peak 3 -formylkyurenine (FK), oxidized product of L-R, [(M+32)+2] 2+ Peak 4 Acetonitrile adduct of L-R, [L-R+C 3 C+2] 2+ Peak 5 Acetonitrile adduct of ydroxy-tryptophan (TRP), oxidized product of L-R, [TRP+C 3 C+2] 2+ Peak 6 Acetonitrile adduct of -formylkuyrenine, oxidized product of L-R, [FK+C 3 C+2] 2+ Application ote 2 5

6 C Column: ProPac WCX- (4 25 mm ) Eluent: mm 8 mm sodium chloride in mm sodium phosphate (p6.) over 3 min Temperature: 3 C Flow Rate:. ml/min Inj. Volume: µl Detection: Absorbance, UV 254 nm Samples: L-R and xy-l-r,.5 mg/ml 4 A. on xidized L-R Peaks. Unknown 2. Unknown 3. xy L-R 4. L-R 5. Unknown -formylkynurenine (FK) MW 28 ydroxytryptophan (TRP) MW 22 µs Tryptophan MW 86 4 B. xidized L-R µs 3-hydroxykynurenine (3-KY) MW 26 Kynurenine (KY) MW Minutes Figure 3. The chemical structure of tryptophan and its major oxidation products. 7 Figure 4. Separation of (A) non-oxidized L-R and (B) oxidized L-R using the ProPac WCX- column. The mass spectroscopy results support our interpretation of the chromatography results for the oxidation of L-R. Figure 4A shows the elution of non-oxidized L-R at 22 min on the ProPac WCX- column at 254 nm. These results show a single non-oxidized L-R at all wavelengths (24, 254, and 28 nm) used in this study, confirming the data previously published in A29. The ProPac WCX- did not resolve the two forms of this peptide observed on the IC column. After tryptophan oxidation, oxidized L-R isoforms eluted earlier at 6 7 min, as a single peak (Figure 4B). As observed with the IC column, the presence of a small nonoxidized L-R component (peak 4, with a retention time of 22 min) remaining in the oxidized L-R sample suggested that L-R was not completely oxidized. Figure 5A shows non-oxidized a-ms (peak ) at 26 min on the IC- column. After forced oxidation of the methionine in a-ms, a new peak for the oxidized a-ms (peak 2) was observed at 2 min (Figure 4B). The presence of a remaining non-oxidized MS peak at 26 min indicated that a-ms was not completely oxidized. Although a-ms also contains tryptophan residue, forcible oxidation was not attempted. These results show an orthogonal method to A 29, which uses the ProPac WCX- column for separation of methionine oxidation variants. 6 ydrophobic Interaction Chromatography for Separation of Tryptophan and Methionine xidized Peptides from Their ative Forms

7 Column: ProPac IC- (4.6 mm ) Eluent: A: 2 M ammonium sulfate in. M sodium phosphate (p 7.) B:. M sodium phosphate monobasic (p 7.) Temperature: 3 C Flow Rate:. ml/min Inj. Volume: µl Detection: Absorbance, UV 24 nm 5 A. on xydized α-ms mau 5 mau B. xydized α-ms 2 % B % B Minutes Peaks: Figure 5. Separation of (A) non-oxidized a-ms and (B) oxidized a-ms using the ProPac IC- column. Precautions L-R and a-ms are bioactive peptides. bserve all safety precautions when handling these materials. Review the material safety data sheets (MSDS) for these materials prior to handling, use, and disposal. Summary The method described herein demonstrates separation of peptides with differences as small as the oxidation of a single amino acid residue using the ProPac IC- column. The separations occur in a non-denaturing environment, and further demonstrate that IC is a viable alternative to reversed-phase PLC for separation of peptide variants. This technique is also an alternative to weak cation-exchange chromatography for separation of peptide variants as shown in A 29, and may provide improved sensitivity and selectivity over that method.. α-ms 2. xy α-ms Suppliers Sigma-Aldrich, 35 Spruce Street, St. Louis, M 633, Tel: , Mallinckrodt Baker, Inc., 222 Red School Lane, Phillipsburg, J 8865, Tel: , References. Rao, S., Bordunov, A., and Pohl, C., igh-resolution silica hydrophobic interaction chromatography (IC) column for protein/peptide separations with improved hydrolytic stability, LP 864-, Dionex Corporation, Sunnyvale, C.A. 2. Jennissen,. P., ydrophobic interaction chromatography ature Encyclopedia of Life Sciences 22, Vol. 9, Walsh, C.T., Post-translational modification of proteins. Roberts & Co., Englewood, C, USA 26 pps Kim, Y.., Berry, A.., Spencer, D. S., and Sites, W. E., Comparing the effect of protein stability of methionine oxidation versus mutagenesis: steps toward engineering oxidative resistance proteins, Protein Engineering 2, Vol.4 no.5, Zang, J., Kalonia, D.S., "The effect of neighboring amino acid residues and solution environment on the oxidative stability of tyrosine in small peptides." AAPS PharmSci Tech, 27: 8(4), Article Dionex Corporation, Separation of Tryptophan and Methionine xidized Peptides From Their Unoxidized Forms, Application ote 29, LP 53-2, Sunnyvale, CA, Finley, E. L., Dillon, J., Crouch, R. K., and Schey, K. L., Identification of tryptophan oxidation products in bovine a-crystalline, Protein Science, 998, 7, Application ote 2 7

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