Created: 1st December 2000, last updated: 30th December 2000, © 2000 ABRF

RESEARCH GROUP REPORTS

 

Strategies for the Synthesis of Labeled Peptides

Lisa Bibbs,a Nicholas P. Ambulos,b Steven A. Kates,c Ashok Khatri,d Katalin F. Medzihradszky,e George Ösapay,f and Susan T. Weintraubg

aThe Scripps Research Institute, La Jolla, CA; bUniversity of Maryland at Baltimore, Baltimore, MD; cPerSeptive Biosystems, Inc., Farmingham, MA; dMassachusetts General Hospital, Boston, MA; eUniversity of California, San Francisco, CA; fUniversity of California, Irvine, CA; gUniversity of Texas Health Science Center at San Antonio, San Antonio, TX

Labeled peptides synthesized by core facilities are frequently used by researchers for following trafficking of a peptide, for binding studies, to determine substrate specificity, and for receptor cross-linking studies. The membership of the Association of Biomolecular Resource Facilities was asked to participate in a study focusing on synthesis of a biotin-labeled peptide, and it was suggested that a new strategy, using Rink amide 4-methylbenzhydrylamine resin coupled with Fmoc-Lys(Dde)-OH, be used. This strategy can be used for addition of a variety of labels other than biotin and should prove useful to core facilities. Comparison of the new strategy to other strategies was performed. Biotin labeling has long been assumed to be routine and specific. Despite the assumed routine nature of synthesizing biotinylated peptides, 9 of the 34 samples submitted did not contain any of the correct product. Although synthesis using Fmoc-Lys(Dde)-OH plus biotin generally gave the highest yields, other approaches also yielded a high percentage of the correct product. Therefore, the various strategies are generally comparable. The major advantage of this new approach is that other labels such as fluorescein, dansyl groups, methyl coumarin, and potentially fluorophores and quenchers used for fluorescence resonance energy transfer (FRET) can be directly incorporated into peptides. (J Biomol Tech 2000;11:155-165)

Key Words: biotin, labeled peptides, synthesis.

Address correspondence and reprint requests to: Lisa Bibbs, The Scripps Research Institute, Core Facility, SP-3, La Jolla, CA 92037 (email: bibbs@scripps.edu).

 

Two approaches can be used to generate labeled peptides: the peptide can be synthesized using labeled amino acids, or the label can be added after peptide synthesis has been completed. When a peptide is labeled after synthesis, there occasionally are problems with the location of the label, depending on the composition of the peptide. The use of labeled amino acids during synthesis usually insures the correct positioning of the label.

The ABRF Peptide Synthesis Research Group (PSRG) of the Association of Biomolecular Resource Facilities (ABRF) conducts annual studies to help member laboratories assess their peptide synthesis capabilities.1 Concurrently, through careful design and synthesis of the test sequence, these studies also serve as an avenue to introduce new techniques to member laboratories. This study focused on the synthesis of side-chain-labeled peptides--specifically, a peptide with a C-terminal biotin-labeled lysine. Participating laboratories were asked to construct the following peptide:

H-Ala-Glu-Lys-Gly-Lys-Leu-Arg-Phe-Lys(biotin)-NH2

Although use of Fmoc-Lys(biotin)-OH was an obvious choice for direct assembly of this sequence, one goal of the study was to acquaint member laboratories with a flexible strategy that could be used for introduction of a variety of different labels other than biotin. To make this endeavor especially valuable for core laboratories, it was suggested that the new approach, based on utilization of a side-chain protected lysine residue orthogonal to Fmoc/t-Bu that employs Rink amide MBHA resin coupled with Fmoc-Lys(Dde)-OH be used.2 In this approach, first the peptide is synthesized, then the label is added for coupling to the Lys, followed by cleavage and deprotection. Participating laboratories were asked to submit the requested peptide without purification. The PSRG members used amino acid analysis, capillary electrophoresis, reverse-phase high-performance liquid chromatography (HPLC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), and electrospray ionization mass spectrometry (ESI/MS) to analyze all peptides submitted from participating laboratories in addition to a test peptide synthesized by the PSRG in preparation for the study. In this report, the results from analysis of the PSRG test peptide and submitted peptides are presented, and the method used by the PSRG for synthesis of the test peptide is described.

 

METHODS

Synthesis of the Reference Peptide

For testing by the PSRG, H-Ala-Glu-Gly-Lys-Leu-Arg-Phe-Lys(biotin)-NH2 was synthesized on an AB 431A synthesizer (Applied Biosystems, Foster City, CA). Rink amide MBHA resin was coupled with Fmoc-Lys(Dde)-OH using HBTU/HOBt/DIEA activation.3 All remaining residues except the N-terminal Ala were incorporated as Fmoc-amino acids with the same activation chemistry, using Pbf for Arg, Boc for Lys, and OtBu for Glu as the side-chain protecting groups. The N-terminal Ala was incorporated as t-Boc-Ala. After completion of the synthesis, the resin was washed twice with N,N-dimethylformamide (DMF). The Dde side-chain protection was removed by treatment with 2% hydrazine (Sigma, St. Louis, MO) in DMF (7 mL/0.1 mmole of the protected peptide resin; 2 times, 5 minutes each). The resin was then washed 3 times with DMF followed by 2 washes with DMF:dimethyl sulfoxide (DMSO; 1:1, v/v). A 10-fold molar excess of biotin (Aldrich, Milwaukee, WI) was dissolved in 5 mL of DMF:DMSO (1:1). It was necessary to warm the mixture and vortex it for several minutes to dissolve the biotin completely. The biotin solution was then treated with 2.1 mL of 0.45-M HBTU/HOBt in DMF followed by 0.3 mL of diisopropylethylamine (DIEA). The activated biotin solution was added to the resin, and the mixture was stirred overnight. The resin was washed with DMF:DMSO (1:1; 3 times) followed by dichloromethane:methanol (1:1; 2 times). After the resin was thoroughly dried, the peptide was cleaved and deprotected with Reagent-K.

Amino Acid Analysis

Crude peptide samples were hydrolyzed for 24 hours at 110°C using vapor-phase hydrolysis in 6-N HCl containing 2% saturated phenol/water. The samples were then analyzed by Waters AccQ*Tag chemistry using a Waters Alliance HPLC system (Milford, MA) equipped with a fluorescence detector.

High-Performance Liquid Chromatography

Analyses were conducted on a Waters HPLC system consisting of two model 600 solvent delivery systems, a Wisp model 712 automatic injector, a model 490 programmable wavelength ultraviolet detector, and a DEC model 860 Networking Computer to control system operation and collect data. HPLC conditions were as follows: column, Delta Pak C18 (Waters), 3.9 X 150 mm, 5 µm, 100 Å; buffer A, 0.1% trifluoroacetic acid (TFA) in water; buffer B, 0.1% TFA in acetonitrile; linear gradient, 5% B to 60% B in 45 minutes; flow rate, 1.0 mL/minute; detector wavelengths, 220 and 279 nm.

Capillary Electrophoresis

Peptides were analyzed on a PACE-MDQ CE system (Beckman Instruments, Palo Alto, CA) using an amine-coated capillary and acetate buffer, pH 4.5. The capillary was preconditioned with an amine regenerator and a buffer rinse. Samples were applied by a 5-second pressure injection (2 psi) and separated at 30 kV for 10 minutes using reversed polarity.

Electrospray Ionization Mass Spectrometry

ESI/MS analyses were conducted on a ThermoFinnigan LCQ ion trap mass spectrometer (San Jose, CA). Samples were dissolved in 1 mL of water and diluted at least 100-fold in 70% aqueous acetonitrile/0.5% acetic acid. Peptide samples were introduced into the electrospray interface by flow injection at a rate of 5 µL/minute. MS conditions were as follows: capillary temperature, 200°C; spray voltage, 5 kV; capillary voltage, 30 V; tube lens offset, 18 V; sheath gas, 30 L/min. For MSn, the relative collision energy was 35%. Spectral averaging for 10 scans was used prior to data acquisition.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

One microliter of a sample solution of approximately 10 to 50 pmole/µL was combined with 1 µL of 4-hydroxy-alpha-cyano-cinnamic acid matrix solution (Hewlett-Packard, Palo Alto, CA), and the mixture was deposited on a stainless steel target (2 spots/sample). MALDI-TOF analyses were performed on either a Voyager Elite or a Voyager DE-STR mass spectrometer (Applied Biosystems) in reflectron mode with delayed extraction. A two-point external calibration based on angiotensin II (m/z 1046.54, MH+) and adrenocorticotropic hormone (ACTH) 18 to 39 Clip peptide (m/z 2465.2, MH+) was used for the molecular mass determinations. Post-source decay (PSD) experiments were performed on the Voyager DE-STR. Data were acquired in 10 consecutive frames, lowering the reflectron voltage by 25% at each step. Air was used as collision gas while focusing on the low mass region (m/z < 200). The data were smoothed to yield mostly average masses. ACTH-Clip peptide was used for PSD calibration.

Sequence Analysis

Selected peptides were sequenced by Edman degradation using an Applied Biosystems model 494 protein sequencer and an Applied Biosystems model 120A HPLC analyzer fitted with a reverse-phase (RP) HPLC column.

 

RESULTS AND DISCUSSION

Analysis of the Test Peptide

The integrity of the reference peptide was confirmed by amino acid analysis, capillary electrophoresis, Edman degradation, RP-HPLC, MALDI-TOF/MS, and ESI/MS. All analyses gave the anticipated results. As shown in Figure 1, PTH-Lys(biotin) was well resolved from the other amino acids during Edman degradation analysis.

FIGURE 1. Elution position of PTH-Lys(biotin) by Edman sequence analysis. The Lys(biotin) is well resolved from other amino acids and can easily be identified.

 

The ESI mass spectrum of the test peptide is shown in Figure 2. Three charge states of the peptide were readily detected: m/z 1173.7, [M+H]+, m/z 587.5, [M+2H]2+, and m/z 392.1, [M+3H]3+. The tandem mass spectrometry (MS/MS) spectrum obtained by collision-induced dissociation of the 2+ ion confirmed the expected sequence (Fig. 3).

FIGURE 2. Electrospray ionization mass spectrum of the test peptide, AEGKLRFK(biotin). Three charge states of the peptide are readily detected, verifying a molecular mass of 1173 daltons.

 

FIGURE 3. Tandem mass spectrum obtained by collision-induced dissociation of [M+2H]2+(m/z 587.5) for the test peptide. The fragmentation pattern confirms the expected sequence.

 

Analysis of Submitted Peptides

Thirty-four peptides were submitted in crude form by participating laboratories. Eight different methods were used for the synthesis. The best crude material was found to be 96% pure by RP-HPLC. A summary of the analytical data for the submitted peptides is presented in Table 1. The following results were obtained:

 

TABLE 1
Summary of Results from ABRF Peptide Synthesis Research Group Study
Mass Spectrometry Data
Percentage
Correct Product Major Product
Other Lys
RP- Correct Compo- Protec- Cleavage
Samplea     HPLC     CE     Mass     Identity     nents     tion     Biotin     Chemistry     Instrument     Reagent
1340A 88.9 85.9 Yes Aloc D-Biotin Fmoc/HBTU Rainin Modified K
Symphony
4761 12.5 31 Yes Multiple Aloc EZ-link Fmoc Milligen R
NHS 9050
Biotin
0715 86.4 88 Yes - Lys Biotin Fmoc/PyBOP/ Shimadzu R
(biotin) HOBt Pssm-8
1231 82.2 81.7 Yes - Glu, Biotin Fmoc/HBTU/ Rainin Modified B
- Ala, HOBt Symphony
- Lys
(biotin)
1354 54.9 76.8 Yes - Lys Biotin Fmoc/HBTU/ ABI 431 Modified B
(biotin) HOBt
4612 28.5 58.9 Yes peptide Biotin Fmoc/HBTU/ ABI 431A B
+16, HOBt
- Lys
(biotin)
8105 0 0 No - Lys Glu-to- Biotin Fmoc/HBTU/ ABI 433A Modified K
(biotin) Gln HOBt
and Glu-
to-Gln
8398A 73.8 80.1 Yes Biotin Fmoc/HATU Rainin B
Symphony
8583 11.7 12.7 No - Lys Biotin Fmoc/HBTU/ ABI 433A K
(biotin) HOBt
MPCF 76.6 83.8 Yes + Pmc Biotin Fmoc/HBTU Rainin Modified B
Symphony
8398B 59.3 81.7 Yes (Biotin + Extra Biotin Fmoc/HATU Rainin B
caproate) Lys(biotin caproate Symphony
caproate) resin
2958 50.4 47 Yes + 91 Amu, Boc-Lys D-Biotin Boc/HBTU/ AB 430 5 mL HF/
possible (2ClZ) HOBt 500 µL
bzl on Glu Anisole
0690 61.2 84 Yes (Biotin + Pmc Dde (±)Biotin Fmoc/HBTU/ AB 433A Reagent-K
caproate) HOBt
2311 0 0 No - Arg Dde Biotin Fmoc/HBTU/ AB 430 Reagent-K
HOBt
2913 52.9 71.1 Yes + Pbf Dde D-Biotin Fmoc/HBTU/ AB 431A Modified
HOBt Reagent-B
4005 0 0 No 2 Biotin 1902 Dde Biotin-- Fmoc/PyBOP AMS 422 Modified
caproatesb daltonsc amido- Reagent-B
caproate
NHS ester
4209D 61.7 65.6 Yes Dde (±)Biotin Fmoc/HBTU ACT Modified
396MBS Reagent-B
IRBJ 0 0 No 2 Biotinsb Dde Biotin-N- Fmoc/HBTU ACT Modified
hydroxy- MPS 396 Reagent-B
succinimide
1068 84.3 87.2 Yes + Pbf Dde NHS-LC- Fmoc/PyBOP Perseptive Reagent-K
Biotin Pioneer
1374 82.8 83.2 Yes - Ala Dde Succin- Fmoc/HBTU/ AB 431 Reagent-R
imidyl HOBt
D-Biotin
5130 72.9 82.9 Yes Dde D-Biotin Fmoc/HOBt/ AB 431A Reagent-K
DCC
5557 96.1 90 Yes Dde D-Biotin Fmoc/HBTU/ AB 433 Reagent-K
HOBt
9818 92.1 92.5 Yes Dde D-Biotin Fmoc/HBTU/ AB 432 Modified
HOBt Reagent-B
9453 81.6 81.5 Yes Peptide Fmoc (±)Biotin Boc AB 430 90:10
+ 90 HF:Anisole
1340T 96 90 Yes MeOtrt D-Biotin Fmoc/HBTU Rainin Modified
Symphony Reagent-K
0775 85.1 84 Yes + Biotinb Mtt (±)Biotin Fmoc/TBTU Rainin Modified
Symphony Reagent-B
1524 33.7 63.4 Yes (Biotin - Biotin Mtt Sulfo-NHS- Fmoc/TBTU/ Milligen Reagent-K
caproate) caproate LC-Biotin HOBt 9050
1609 74.1 66.1 Yes + Biotinb Mtt D-Biotin Fmoc/HBTU/ AB 432 Reagent-R
HOBt
1633 60 55.8 Yes - Biotin Mtt EZ-link Fmoc/HOBt/ AB 431A Reagent-K
NHS DCC
Biotin
3845 0 0 No - Biotin Mtt NHS-LC- Fmoc Opfp/ Milligen Modified
Biotin HOBt 9050 Plus Reagent-B
4209M 7 6.8 No - Biotin Mtt (±)Biotin Fmoc/HBTU ACT Modified
396MBS Reagent-B
7298 81.2 70.8 Yes + Biotinb Mtt D-Biotin Fmoc/HBTU/ AB 433 Reagent-K
HOBt
7299 89.9 80.8 Yes Mtt D-Biotin Fmoc/HBTU/ AB 433 Reagent-K
HOBt
7347 78 83.5 Yes + Biotinb Mtt (±)Biotin Fmoc/HBTU Perseptive Reagent-B
Pioneer

aThe samples are grouped according to Lys protection and then listed in numerical order.
bDid not use Boc-Ala-OH.
cThe extra mass is on the Lys(biotin) by tandem mass spectrometry.
±, with and without; CE, capillary electrophoresis; RP-HPLC, reverse-phase high-performance liquid chromatography.

 

1. Nine of the peptides were constructed using a prelabeled Lys(biotin), yielding from 0% to 88% correct product by HPLC. In three of these peptides, the Lys(biotin) was not efficiently coupled.

2. Eleven laboratories used Fmoc-Lys(Dde)-OH (as did the PSRG). Three of these submitted samples contained no correct product. One contained the Lys(biotin) but was missing the Arg. The other two samples contained two biotins. The attachment of the extra biotin was the result of the fact that Boc-Ala-OH was not employed as the N-terminal residue. These last two laboratories also used modified biotins.

3. Nine laboratories used Fmoc-Lys(Mtt)-OH. Of these samples, one had no correct product and was missing the biotin, and another contained only low levels of the requested peptide. For this synthesis, the Mtt group must be selectively removed using dilute TFA or acetic acid/trifluoroethanol/methylene chloride (AcOH/TFE/DCM).4

4. Two laboratories used Fmoc-Lys(Aloc)-OH. One of these samples contained multiple components, with the product of interest at only 12.5%. For this synthesis, the Aloc group must be selectively removed using Pd(0),Bu3SnH,5 or Pd(0), HOAc, N-methylmorpholine in chloroform.6

5. Two laboratories constructed their peptides using t-Boc chemistry. In both of these samples, the product of interest was the major component by HPLC.

Overall, nine of the samples did not have the product of interest as the major component by HPLC.

Two types of biotin were used by the participating laboratories: biotin and biotin caproate (b-c). The caproate arm increased the mass of the correct peptide to 1286 daltons (Fig. 4) and caused an extra peak in the amino acid analysis corresponding to the derivatized caproate. (Fig. 5) The retention time of PTH-Lys(b-c) during Edman degradation analysis was within 0.1 minute of that of PTH-Tyr and therefore was not well resolved (Fig. 6).

FIGURE 4. Electrospray ionization mass spectrum of a submitted sample of AEGKLRFK(biotin) in which there was a caproyl linker between biotin and the C-terminal Lys. The three charge states detected for this peptide indicated a molecular mass of 1286 daltons.

 

FIGURE 5. Amino acid analysis of a peptide constructed with a caproyl linker to biotin. An extra peak was observed at 28.6 minutes between Met and Lys.

 

FIGURE 6. Elution position of PTH-Lys biotin caproate(Lys(BC)) by Edman sequence analysis. Lys(BC) was not readily resolved from PTH-Tyr using the gradient conditions described in Methods.

 

ESI/MS was a valuable tool in the analysis of these crude products. In peptide 8105, ESI/MS and subsequent ESI/MS/MS analysis rapidly identified a Glu-to-Gln substitution in the peptide in addition to a significant quantity of a truncated product (Figs. 7 and 8). Analysis of this peptide illustrates the fact that tandem mass spectrometry can readily identify an amino acid substitution that would be undetected by amino acid analysis.

FIGURE 7. Electrospray ionization mass spectrum of sample 8105 containing a Glu-to-Gln substitution in the requested biotin-labeled peptide (A) in addition to a substantial quantity of a truncated product (B).

 

FIGURE 8. Tandem mass spectrum obtained by collision-induced dissociation of [M+2H]2+ (m/z 587.0) for the biotin-containing peptide in sample 8105 indicating the Glu-to-Gln substitution.

 

Peptide 8398B was synthesized using a prelabeled resin. ESI/MS analysis indicated that, in addition to the expected biotin caproate peptide, there was an additional product which presumably corresponds to the peptide plus an extra Lys(b-c) (Fig. 9). This was verified by subsequent MS/MS analysis (Fig. 10). The mono-Lys(b-c) peptide constituted 17.5% of the sample by RP-HPLC.

FIGURE 9. Electrospray ionization mass spectrum of sample 8398B indicating the presence of peptides containing one (A) and two (B) residues of biotin caproate.

 

FIGURE 10. Tandem mass spectrum obtained by collision-induced dissociation of [M+2H]2+ (m/z 877.5) for the (biotin caproate)2-containing peptide in sample 8398B. The fragmentation pattern verifies the presence of two adjacent biotin caproates.

 

It is interesting to note that a variety of different cleavage cocktails were developed in the 1990s. These reagents contain different scavengers in TFA to reduce side reactions of the reactive cations generated from cleaved protecting groups. Reagent-K (TFA-phenol-H2O-thioanisole-1,2-ethandithiol 82.5:5:5:5:2.5),7 Reagent-B (TFA-phenol-H2O-triisopropylsilane 88:5:5:2),8 and Reagent-R (TFA thioanisole-1,2-ethanedithiol-anisole (90:5:3:2)9 are the most commonly used. Reagent-B is becoming more popular because it provides clean, highly efficient cleavage and does not contain any malodorous thiol components. Forty-one percent of the respondents used cleavage Reagent-B for this study.

The results of this study showed that use of either a prelabeled amino acid or resin did not guarantee a good product; 37% of the peptides constructed using prelabeled starting materials were failures. It is also clear that when an Fmoc-Lys(Dde)-OH derivative is employed to introduce a label into a peptide, a Boc amino acid must be utilized for the N-terminus because the 2% hydrazine3 employed for selective deprotection of the Dde group will also remove all Fmoc groups on the peptide. Finally, for characterization of synthetic peptides, MALDI-TOF/MS and ESI/MS were extremely valuable for qualitative, but not quantitative, analysis.

Considerable potential exists for the use of the Rink amide MBHA resin coupled with the Fmoc-Lys(Dde)-OH approach for the incorporation of various labels into peptides. One new and exciting application would be in the synthesis of peptides for FRET. FRET is based on the ability of molecules called quenchers to block fluorescence when in close proximity to fluorescent dyes. FRET has been used for the identification of novel proteases.10 Incorporating a fluorophore and a quencher during peptide synthesis would avoid labeling and purification problems in peptides used for future FRET studies. It is hoped that core facilities will be able to make use of this strategy.

 

ACKNOWLEDGMENTS

We thank all the ABRF core facilities that participated in this study and Lynda Bonewald, our ABRF Executive Board liaison, for her valuable advice.

 

REFERENCES

1. Angeletti RH, Bonewald LF, Fields G. Six-year study of peptide synthesis. Methods Enzymol 1997;289:697-717.

2. Blackburn C, Kates SA. Solid-phase synthesis of cyclic homodetic peptides. Methods Enzymol 1997;289:175-197.

3. Bycroft BW, Chan WC, Chhabra SR, Hone ND. A novel lysine protecting procedure for continuous flow solid phase synthesis of branched peptides. J Chem Soc Chem Commun 1993;778-779.

4. Aletras A, Barlos K, Gatos D, Koutsogianni S, Mamos P. Preparation of the very acid-sensitive Fmoc-Lys(Mtt)-OH. Application in the synthesis of side-chain to side-chain cyclic peptides and oligolysine cores suitable for the solid-phase assembly of MAPs and TASPs Int J Pept Protein Res 1995;45:488-496.

5. Loffet A, Zhang HX. Allyl-based groups for side-chain protection of amino-acids. Int J Pept Protein Res 1993;42:346-351.

6. Kates SA, Daniels SB, Albericio F. Automated allyl cleavage for continuous-flow synthesis of cyclic and branched peptides. Anal Biochem 1993;212:303-310.

7. King DS, Fields CG, Fields GB. A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis. Int J Pept Protein Res 1990;36:255-266.

8. Sole NA, Barany G. Optimization of solid-phase synthesis of [Ala8]-dynorphin A. J Org Chem 1992;57:5399-5403.

9. Albericio F, Kneib-Cordonier N, Biancalana S, Gera L, Masada RI, Hudson D, Barany G. Preparation and application of the 5-(4-(9-fluorenylmethyloxycarbonyl)aminomethyl-3,5-dimethoxyphenoxy)valeric acid (PAL) handle for the solid phase synthesis of C-terminal peptide amides under mild conditions. J Org Chem 1990;55:3730-3743.

10. Olsen MJ, Stephens D, Griffiths D, Daugherty P, Georgiou G, Iverson BL. Function-based isolation of novel enzymes from a large library. Nat Biotechnol 2000;18:1071-1074.

 

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