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Solid-phase extraction as a tool to remove impurities and small fragments from synthetic peptides
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 344 (2005) 144–146 www.elsevier.com/locate/yabio
Notes & Tips
Solid-phase extraction as a tool to remove impurities and small fragments from synthetic peptides Mathias W. Hofmann ¤, Holger Stalz 1, Dieter Langosch Lehrstuhl für Chemie der Biopolymere, TU München, D-85354 Freising-Weihenstephan, Germany Received 9 March 2005 Available online 29 June 2005
Synthetic peptides are increasingly used in biological, pharmaceutical, and medical research where they are required in high amounts and purities. While hydrophilic peptides are routinely synthesized by solid-phase methods, faithful synthesis of highly hydrophobic sequences is still diYcult using standard Xuorenylmethyloxycarbonyl (Fmoc)2 procedures. Therefore tert-butoxycarbonyl (Boc) chemistry is frequently applied here [1,2]. Upon using p-cresol or p-thiocresol as scavengers in Boc solidphase peptide synthesis, side chain alkylation of tryptophan during cleavage of peptide by hydroXuoric acid from the synthesis resin is suppressed and the quality of the product is signiWcantly enhanced [3]. These aromatic compounds exhibit signiWcant UV absorbance and therefore interfere with peptide quantiWcation via tryptophan absorbance. Hence, a quick and reliable procedure for scavenger removal is highly desirable. Solid-phase extraction (SPE) methods are usually used to remove low-molecular-weight contaminants from peptides prior to HPLC [4]. Thereby, the peptide is transiently bound to a hydrophobic matrix, the contaminants are washed away, and the peptide is recovered by elution with an organic solvent. The main advantages of these methods are their selectivity, eYciency, and broad applicability. Here, we describe a rapid and simple alternative SPE application where p-cresol and low-molecular-weight peptide fragments bind to a matrix
*
Corresponding author. Fax: +49 8161 71 4404. E-mail address: [email protected] (M.W. Hofmann). 1 Present address: Waters Corp., EHQ, Waters SAS, 78280 Guyancourt, France. 2 Abbreviations used: AcN, acetonitrile; Boc, tert-butoxycarbonyl; Fmoc, Xuorenylmethyloxycarbonyl; SPE, solid-phase extraction; TFE, 2,2,2-triXuoroethanol; TFA, triXuoroacetic acid. 0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.06.021
derivatized with C2 chains while highly hydrophobic peptides are recovered in the Xow-through. The peptides (LV16, NH2-KKW(LV)8KKK-COOH; LV20, NH2-KKW(LV)10KKK-COOH; LV24, NH2KKW(LV)12KKK-COOH) were synthesized by Boc chemistry (PSL, Heidelberg, Germany). SepPak plus (tC2) cartridges for SPE were purchased from Waters (Milford, MA, USA). For UV spectroscopy, 2,2,2-triXuoroethanol (TFE) and analytical-grade dimethyl sulfoxide were used as solvents. Analytical-grade acetonitrile (AcN) was from Roth and triXuoroacetic acid (TFA) was from Sigma. Peptides were dissolved at a concentration of 2 mg/ml in AcN/H2O (20/80) and treated for 15 min in a bathtype sonicator. Undissolved peptide was removed by centrifugation for 10 min at 4 °C, 13,000 rpm. Peptide concentration was determined by UV spectroscopy at 282 nm using a tryptophan extinction coeYcient of 5600 M¡1 cm¡1. For SPE a tC2 SepPak plus cartridge was conditioned with 4 ml AcN and equilibrated with 3 ml AcN/H2O (5/95) prior to use; 1 ml of peptide stock solution was loaded and the Xow-through was collected and combined with a wash fraction of 0.5 ml AcN/H2O (5/ 95). These fractions containing the peptide were lyophilized overnight and dissolved in TFE. After washing the cartridge with 4 ml AcN/H2O (5/95), p-cresol was eluted using 4 ml of AcN, whereby it was regenerated. p-Cresol content was quantiWed by either HPLC with UV detection at 280 nm or UV/Vis spectrometry ( D 1665 M¡1 cm¡1) from the respective fraction using external calibration. HPLC was performed using a C4 analytical column (214TP54, 250 £ 4.6 mm, GraceVydac, USA). Gradient LC elution conditions were as follows: buVer A, AcN/H2O/TFA (20/79.9/0.1); buVer B, AcN/ H2O/TFA (90/9.9/0.1). Time program for p-cresol was
Notes & Tips / Anal. Biochem. 344 (2005) 144–146
Fig. 1. p-Cresol removal by SPE. (A) Retention time of pure p-cresol in HPLC chromatography. (B and C) Injections of peptide LV16 before (B) and after (C) SPE. Arrows denote the position of p-cresol at 20.1 min. Peaks at »43 min in B and C contain peptide LV16. Note the complete absence of a p-cresol peak after SPE.
0 min, 0% B; 10 min, 0% B; 20 min, 40% B; 25 min, 40% B; 30 min, 0% B; 40 min, 0% B. All experiments were done at a Xow rate of 0.4 ml/min and sample volume was 20 l per run. Mass spectra of peptides were obtained with a Q-ToF Ultima ESI mass spectrometer (Waters, UK) in positive ESI mode with the following settings: capillary voltage, 2.5–2.8 kV; cone voltage, 60 V; ToF settings, MCP voltage, 2.1 kV; ToF voltage, 9.1 kV. For data processing, MassLynx 4.0 software (Waters) was used. The three peptides LV16, LV20, and LV24 mimic hydrophobic membrane-spanning domains of membrane proteins [5] (and unpublished results). Their hydrophobic core consists of 16, 20, or 24 alternating leucine and valine residues. Lysine residues are attached at both termini for better solubility and a tryptophan residue at position three allows for quantiWcation via UV absorption. Synthesis of peptides using Boc chemistry resulted in purities from 85 to 90% as judged by mass spectrometry. Although this level of purity proved suYcient for examining their functional properties [5],
145
Fig. 2. Removal of peptide fragments by SPE. (A) Mass spectrum recorded prior to SPE. LV16 gives rise to a doubly charged ion at 1273.4 m/z (asterisk) and is accompanied by some singly charged fragments (arrows) identiWed as NH2-VLVLVKKK-COOH (a), NH2KWLVLVLV-COOH (b), NH2-KWLVLVLVL-COOH (c), and NH2KWLVLVLVLV-COOH (d). (B) Upon SPE, these fragments are removed, whereas some doubly charged larger fragments remain.
they contained 0.3–1.3% p-cresol that originated from the cleavage reaction during synthesis as determined by HPLC analysis. As p-cresol is a strong UV absorber, low levels suYce to signiWcantly inXuence determination of peptide concentration via UV absorbance and therefore should be removed. Due to the strong hydrophobicity of the peptides, puriWcation by traditional reverse-phase chromatography frequently results in low yields due to irreversible binding of the peptide to the matrix. Therefore, we developed an SPE procedure for rapid removal of p-cresol. Crude peptide dissolved in AcN/H2O (20/80) was passed over a SepPak Plus tC2 column to which it did not signiWcantly bind. Hence, peptide appeared in the Xow-through, while p-cresol remained bound to the column and was eluted afterward by AcN. Using this method, p-cresol could be removed to undetectable levels from the peptide samples as revealed by the chromatograms shown in Fig. 1. Furthermore, the recovery of all peptides as determined by UV spectroscopy was excellent (LV16, 97%; LV20, 93%; LV24, 100%).
146
Notes & Tips / Anal. Biochem. 344 (2005) 144–146
Mass spectra of the peptides acquired by electrospray mass spectrometry are compared before and after SPE in Fig. 2 which shows exemplary spectra of LV16. It is evident that samples that had been stored for a prolonged period of time contain small singly charged hydrophobic peptide fragments that possibly originate from peptide breakdown (Fig. 2A). Interestingly, these fragments were also eliminated by SPE, as shown in a spectrum (Fig. 2B) recorded under identical conditions. Larger hydrophobic fragments likely to originate from prematurely terminated synthesis could not be removed by the SPE procedure. Similar results were obtained with peptides LV20 and LV24 (data not shown). Recapitulating, peptide puriWcation by SPE eVectively removes small-molecule contaminants and small peptide fragments while loss of peptide is almost negligible. Hence, this procedure is a fast and cost-eYcient alternative to complex HPLC-based techniques. In addition to its ease of use, its applicability to highly hydrophobic sequences is its main advantage. It facilitates the usage of peptides mimicking transmembrane protein domains in biological, medical, and pharmaceutical research.
Acknowledgment This research was supported by a grant from the Volkswagen Foundation as part of the project “Conformational Control of Biomolecular Function.” References [1] L.P. Miranda, P.F. Alewood, Accelerated chemical synthesis of peptides and small proteins, Proc. Natl. Acad. Sci. USA 96 (1999) 1181–1186. [2] A. Karlstrom, K. Rosenthal, A. Unden, Study of the alkylation propensity of cations generated by acidolytic cleavage of protecting groups in Boc chemistry, J. Pept. Res. 55 (2000) 36–40. [3] L.P. Miranda, A. Jones, W.D.F. Meutermans, P.F. Alewood, pCresol as a reversible acylium ion scavenger in solid-phase peptide synthesis, J. Am. Chem. Soc. 120 (1998) 1410–1420. [4] T. Herraiz, V. Casal, Evaluation of solid-phase extraction procedures in peptide analysis, J. Chromatogr. A 708 (1995) 209–221. [5] M.W. Hofmann, K. Weise, J. Ollesch, P. Agrawal, H. Stalz, W. Stelzer, F. Hulsbergen, H. de Groot, K. Gerwert, J. Reed, D. Langosch, De novo design of conformationally Xexible transmembrane peptides driving membrane fusion, Proc. Natl. Acad. Sci. USA 101 (2004) 14776–14781.
Notes & Tips
Solid-phase extraction as a tool to remove impurities and small fragments from synthetic peptides Mathias W. Hofmann ¤, Holger Stalz 1, Dieter Langosch Lehrstuhl für Chemie der Biopolymere, TU München, D-85354 Freising-Weihenstephan, Germany Received 9 March 2005 Available online 29 June 2005
Synthetic peptides are increasingly used in biological, pharmaceutical, and medical research where they are required in high amounts and purities. While hydrophilic peptides are routinely synthesized by solid-phase methods, faithful synthesis of highly hydrophobic sequences is still diYcult using standard Xuorenylmethyloxycarbonyl (Fmoc)2 procedures. Therefore tert-butoxycarbonyl (Boc) chemistry is frequently applied here [1,2]. Upon using p-cresol or p-thiocresol as scavengers in Boc solidphase peptide synthesis, side chain alkylation of tryptophan during cleavage of peptide by hydroXuoric acid from the synthesis resin is suppressed and the quality of the product is signiWcantly enhanced [3]. These aromatic compounds exhibit signiWcant UV absorbance and therefore interfere with peptide quantiWcation via tryptophan absorbance. Hence, a quick and reliable procedure for scavenger removal is highly desirable. Solid-phase extraction (SPE) methods are usually used to remove low-molecular-weight contaminants from peptides prior to HPLC [4]. Thereby, the peptide is transiently bound to a hydrophobic matrix, the contaminants are washed away, and the peptide is recovered by elution with an organic solvent. The main advantages of these methods are their selectivity, eYciency, and broad applicability. Here, we describe a rapid and simple alternative SPE application where p-cresol and low-molecular-weight peptide fragments bind to a matrix
*
Corresponding author. Fax: +49 8161 71 4404. E-mail address: [email protected] (M.W. Hofmann). 1 Present address: Waters Corp., EHQ, Waters SAS, 78280 Guyancourt, France. 2 Abbreviations used: AcN, acetonitrile; Boc, tert-butoxycarbonyl; Fmoc, Xuorenylmethyloxycarbonyl; SPE, solid-phase extraction; TFE, 2,2,2-triXuoroethanol; TFA, triXuoroacetic acid. 0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.06.021
derivatized with C2 chains while highly hydrophobic peptides are recovered in the Xow-through. The peptides (LV16, NH2-KKW(LV)8KKK-COOH; LV20, NH2-KKW(LV)10KKK-COOH; LV24, NH2KKW(LV)12KKK-COOH) were synthesized by Boc chemistry (PSL, Heidelberg, Germany). SepPak plus (tC2) cartridges for SPE were purchased from Waters (Milford, MA, USA). For UV spectroscopy, 2,2,2-triXuoroethanol (TFE) and analytical-grade dimethyl sulfoxide were used as solvents. Analytical-grade acetonitrile (AcN) was from Roth and triXuoroacetic acid (TFA) was from Sigma. Peptides were dissolved at a concentration of 2 mg/ml in AcN/H2O (20/80) and treated for 15 min in a bathtype sonicator. Undissolved peptide was removed by centrifugation for 10 min at 4 °C, 13,000 rpm. Peptide concentration was determined by UV spectroscopy at 282 nm using a tryptophan extinction coeYcient of 5600 M¡1 cm¡1. For SPE a tC2 SepPak plus cartridge was conditioned with 4 ml AcN and equilibrated with 3 ml AcN/H2O (5/95) prior to use; 1 ml of peptide stock solution was loaded and the Xow-through was collected and combined with a wash fraction of 0.5 ml AcN/H2O (5/ 95). These fractions containing the peptide were lyophilized overnight and dissolved in TFE. After washing the cartridge with 4 ml AcN/H2O (5/95), p-cresol was eluted using 4 ml of AcN, whereby it was regenerated. p-Cresol content was quantiWed by either HPLC with UV detection at 280 nm or UV/Vis spectrometry ( D 1665 M¡1 cm¡1) from the respective fraction using external calibration. HPLC was performed using a C4 analytical column (214TP54, 250 £ 4.6 mm, GraceVydac, USA). Gradient LC elution conditions were as follows: buVer A, AcN/H2O/TFA (20/79.9/0.1); buVer B, AcN/ H2O/TFA (90/9.9/0.1). Time program for p-cresol was
Notes & Tips / Anal. Biochem. 344 (2005) 144–146
Fig. 1. p-Cresol removal by SPE. (A) Retention time of pure p-cresol in HPLC chromatography. (B and C) Injections of peptide LV16 before (B) and after (C) SPE. Arrows denote the position of p-cresol at 20.1 min. Peaks at »43 min in B and C contain peptide LV16. Note the complete absence of a p-cresol peak after SPE.
0 min, 0% B; 10 min, 0% B; 20 min, 40% B; 25 min, 40% B; 30 min, 0% B; 40 min, 0% B. All experiments were done at a Xow rate of 0.4 ml/min and sample volume was 20 l per run. Mass spectra of peptides were obtained with a Q-ToF Ultima ESI mass spectrometer (Waters, UK) in positive ESI mode with the following settings: capillary voltage, 2.5–2.8 kV; cone voltage, 60 V; ToF settings, MCP voltage, 2.1 kV; ToF voltage, 9.1 kV. For data processing, MassLynx 4.0 software (Waters) was used. The three peptides LV16, LV20, and LV24 mimic hydrophobic membrane-spanning domains of membrane proteins [5] (and unpublished results). Their hydrophobic core consists of 16, 20, or 24 alternating leucine and valine residues. Lysine residues are attached at both termini for better solubility and a tryptophan residue at position three allows for quantiWcation via UV absorption. Synthesis of peptides using Boc chemistry resulted in purities from 85 to 90% as judged by mass spectrometry. Although this level of purity proved suYcient for examining their functional properties [5],
145
Fig. 2. Removal of peptide fragments by SPE. (A) Mass spectrum recorded prior to SPE. LV16 gives rise to a doubly charged ion at 1273.4 m/z (asterisk) and is accompanied by some singly charged fragments (arrows) identiWed as NH2-VLVLVKKK-COOH (a), NH2KWLVLVLV-COOH (b), NH2-KWLVLVLVL-COOH (c), and NH2KWLVLVLVLV-COOH (d). (B) Upon SPE, these fragments are removed, whereas some doubly charged larger fragments remain.
they contained 0.3–1.3% p-cresol that originated from the cleavage reaction during synthesis as determined by HPLC analysis. As p-cresol is a strong UV absorber, low levels suYce to signiWcantly inXuence determination of peptide concentration via UV absorbance and therefore should be removed. Due to the strong hydrophobicity of the peptides, puriWcation by traditional reverse-phase chromatography frequently results in low yields due to irreversible binding of the peptide to the matrix. Therefore, we developed an SPE procedure for rapid removal of p-cresol. Crude peptide dissolved in AcN/H2O (20/80) was passed over a SepPak Plus tC2 column to which it did not signiWcantly bind. Hence, peptide appeared in the Xow-through, while p-cresol remained bound to the column and was eluted afterward by AcN. Using this method, p-cresol could be removed to undetectable levels from the peptide samples as revealed by the chromatograms shown in Fig. 1. Furthermore, the recovery of all peptides as determined by UV spectroscopy was excellent (LV16, 97%; LV20, 93%; LV24, 100%).
146
Notes & Tips / Anal. Biochem. 344 (2005) 144–146
Mass spectra of the peptides acquired by electrospray mass spectrometry are compared before and after SPE in Fig. 2 which shows exemplary spectra of LV16. It is evident that samples that had been stored for a prolonged period of time contain small singly charged hydrophobic peptide fragments that possibly originate from peptide breakdown (Fig. 2A). Interestingly, these fragments were also eliminated by SPE, as shown in a spectrum (Fig. 2B) recorded under identical conditions. Larger hydrophobic fragments likely to originate from prematurely terminated synthesis could not be removed by the SPE procedure. Similar results were obtained with peptides LV20 and LV24 (data not shown). Recapitulating, peptide puriWcation by SPE eVectively removes small-molecule contaminants and small peptide fragments while loss of peptide is almost negligible. Hence, this procedure is a fast and cost-eYcient alternative to complex HPLC-based techniques. In addition to its ease of use, its applicability to highly hydrophobic sequences is its main advantage. It facilitates the usage of peptides mimicking transmembrane protein domains in biological, medical, and pharmaceutical research.
Acknowledgment This research was supported by a grant from the Volkswagen Foundation as part of the project “Conformational Control of Biomolecular Function.” References [1] L.P. Miranda, P.F. Alewood, Accelerated chemical synthesis of peptides and small proteins, Proc. Natl. Acad. Sci. USA 96 (1999) 1181–1186. [2] A. Karlstrom, K. Rosenthal, A. Unden, Study of the alkylation propensity of cations generated by acidolytic cleavage of protecting groups in Boc chemistry, J. Pept. Res. 55 (2000) 36–40. [3] L.P. Miranda, A. Jones, W.D.F. Meutermans, P.F. Alewood, pCresol as a reversible acylium ion scavenger in solid-phase peptide synthesis, J. Am. Chem. Soc. 120 (1998) 1410–1420. [4] T. Herraiz, V. Casal, Evaluation of solid-phase extraction procedures in peptide analysis, J. Chromatogr. A 708 (1995) 209–221. [5] M.W. Hofmann, K. Weise, J. Ollesch, P. Agrawal, H. Stalz, W. Stelzer, F. Hulsbergen, H. de Groot, K. Gerwert, J. Reed, D. Langosch, De novo design of conformationally Xexible transmembrane peptides driving membrane fusion, Proc. Natl. Acad. Sci. USA 101 (2004) 14776–14781.