IRDye

Infrared Dye Reagents

Technical Note
IRDye Peptide Labeling Application Guide
Published March 2008. The most recent version of this Technical Note is posted at http://biosupport.licor.com/support.

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TABLE

OF

CONTENTS

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 2 II. Peptide Labeling Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 2 III. Labeling Reaction Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 4 A. Solution Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 4 B. On-Bead Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 4 C. Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 4 IV. Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 4 A. HIV-1 Protease Substrate Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 4 B. Caspase-8 Substrate Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 6 V. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 7

I. INTRODUCTION
Peptides labeled with fluorescent dyes are important as probes for in vivo imaging and as substrates for enzyme activity assays. Near-infrared (NIR) fluorophores such as IRDye 800CW (excitation 778 nm; emission 794 nm) are particularly useful due to low NIR autofluorescence from tissues, cells, biological materials, or drug compounds. NIR fluorophores can also be com-

bined with an appropriate quencher dye to create fluorogenic peptide probes, such as those used for protease activity detection.1-4 IRDye QC-1 is the first nonfluorescent quencher that can efficiently quench a wide range of fluorophores across the visible and near infrared spectra (~500-800 nm) in a fluorescence resonance energy transfer (FRET) system. Together, IRDye 800CW and IRDye QC-1 provide a well-matched donor-quencher pair to generate highly efficient NIR fluorescent peptide probes (Figure 1). The IRDye infrared dye NHS esters will react with the amino groups of a peptide to form a stable, specificallylabeled compound. This reaction can be performed in solution or on solid-phase peptide synthesis beads.

II. PEPTIDE LABELING DESIGN

Design of the peptide sequence and the labeling method must take into account the features needed for the probe and the labeling chemistry constraints. Peptides will have a reactive, primary amine group at the N-terminus of the molecule that can be used for dye labeling, unless that position has been specifically protected or blocked. If the peptide contains lysine residues, the primary amine of the lysine can also be used as a dye labeling site. Each labeling reaction should be designed to label only one specific site of the peptide. When multiple sites (lysines, N-terminus) are available for labeling, a mixture of products will result, which usually is undesirable. The mixed population of labeled molecules may be difficult to use or characterize, since different labeled forms may have different properties or potency. When multiple dye molecules are coupled to a peptide, close proximity of the dyes may result in self-quenching of the fluorescence.

Figure 1. Example of a fluorogenic substrate. Schematic of NIR fluorogenic peptide probe using IRDye 800CW donor and IRDye QC-1 quencher; an unmodified C-terminus (COOH) is shown in this example.

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Table 1. Summary of Peptide Labeling Design Strategies. Peptide Sequence Contains Free N-terminal amino group and no lysines Free N-terminal amino group and one (or more) lysine(s) Free N-terminal amino group and one lysine Number of Labels Desired One

Possible Approaches Do the NHS ester reaction in aqueous solution. Organic solution labeling or labeling on the beads can also be used. Purify the product. Use on-bead labeling of the terminal amine with the NHS ester while peptide is still in the synthesis beads and lysine is protected. Cleave, deprotect and purify the product. Use on-bead labeling of the N-terminal amine with the NHS ester while peptide is in the synthesis bead and lysine is protected. Cleave, deprotect and purify the product. React the second dye with the lysine in a solution reaction. Purify the product. Synthesize the peptide in highly acid-labile synthesis beads (2-chlorotrityl chloride resin) with easily cleaved protecting group (for example, Mtt) on the specific lysine to be labeled. React the first dye NHS ester with the terminal amine while peptide is still on the synthesis beads. Cleave the peptide from the beads with mild conditions that will deprotect only the desired lysine. React the second dye with the deprotected lysine amine in a solution reaction. Complete the deprotection of the remaining lysines, and purify the product. Do the NHS ester reaction in aqueous solution. Nonaqueous solution labeling or labeling on the beads can also be used. Mixed products may result.

One

Two different dyes: one on the N-terminus, one on lysine Two different dyes: one on the N-terminus, and one on a specific lysine

Free N-terminal amine and two or more lysines

Several amines (N-terminal and /or lysines)

Multiple labels of the same dye

If the desired peptide sequence contains no lysines and only a single label is needed, reaction of the N-terminal amino group with the NHS ester dye is the simplest approach. This can be done either in homogenous solution or in the solid-phase synthesis beads; however, the solution method is preferred. In solution, the peptide should be suspended in a phosphate buffer or other amine-free buffer at pH 8-8.5. At this pH, the N-terminus will react with the NHS ester to form a stable, covalent amide bond. For on-bead labeling, a peptide is synthesized using standard peptide chemistry with the C-terminus attached to the polymer and the N-terminal amine deprotected.5 The free N-terminal amino group can react with the NHS ester dye to produce the desired dye-peptide conjugate. This reaction is performed in dimethylformamide (DMF). After conjugation, the peptide is cleaved from the resin and the natural C-terminus is restored. The solid-phase synthesis bead labeling approach will generally require more dye because the peptide is synthesized in the interior of the bead, causing access and, ultimately, reactivity to be limited.

If the desired peptide sequence contains one or more lysines and just a single dye label is needed, solidphase synthesis bead labeling is the method of choice. The peptide is synthesized with the N-terminus unblocked and all lysine groups protected. During the conjugation, only the free amine on the N-terminus is labeled. After treatment with peptide cleavage solution, the desired mono-labeled peptide conjugate can be isolated. The cleavage solution removes any protecting groups from amino acids such as lysines. To label a peptide with two different dyes, place one label on the N-terminus and the second on a lysine. The on-bead labeling approach is used to add the first dye to the N-terminus. The peptide is then cleaved from the resin, and lysine groups are deprotected in the process. After purification of the mono-labeled peptide, the second dye is conjugated via the lysine in a solution reaction at pH 8-8.5. Provided the peptide has one lysine, one dual-labeled product will be produced. If possible, avoid peptides with more than one lysine. If the peptide has multiple lysines, a family of products will be generated with different numbers and positions of labels.

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A general approach for dealing with multiple lysines has been described by others, using specialized peptide synthesis chemistry.6 The peptide is prepared in highly acid-labile synthesis beads (2-chlorotrityl chloride resin) with an easily cleaved protecting group (for example, Mtt) on the specific lysine to be labeled. The process involves reacting the first NHS ester dye with the terminal amine while the peptide is still in the synthesis beads and all lysines are protected. Cleavage of the peptide is then done under mild conditions to deprotect the desired lysine, while leaving the other lysines protected. The deprotected lysine is labeled in solution with an NHS ester dye in the normal manner, followed by deprotection of the remaining lysines.

minimize cost, unless the peptide is the more expensive reagent. The use of fresh, high-quality dimethylformamide (DMF) is necessary to achieve efficient on-bead peptide labeling results. Care must be taken to ensure that the DMF is amine free and anhydrous. Pure DMF will decompose upon standing and generate traces of dimethylamine, which will react with NHS esters. The recommended deprotection solution is a fresh mixture of trifluoroacetic acid (TFA, 95%), water (2.5%) and triisopropylsilane (2.5%). Deprotection mixtures containing thiols or other nucleophilic groups should not be used. Avoid leaving the labeled peptide in the deprotection solution longer than 5 hours. The length and sequence of the peptide may also affect the efficiency of the labeling reaction. See Section IV for more information about labeling conditions.

III. LABELING REACTION CONDITIONS

A. Solution Labeling
A labeling reaction in aqueous buffer should be used when the peptide design requirements for the product allow (see previous section). The following conditions provide good labeling efficiency: Buffer: Temperature: Time: Dye equivalents per labeling site: Phosphate, pH 8-8.5 Ambient 2-3 hr 1-2

C. Purification
HPLC purification of the peptides is required after cleavage from the beads and after each solution labeling step. Zorbax C18 HPLC columns from Agilent work well, and are available in different sizes for analytical and preparatory scale. The recommended mobile phase is a gradient mixture of acetonitrile and water with triethylammonium acetate (TEAA) buffer (pH 6). The triethylamonium acetate buffer serves to provide ion pairing for better retention behavior of the hydrophilic compounds on the column. For flexibility in development and/or troubleshooting, the HPLC system should be equipped with a diode array detector and be able to monitor at the absorption maxima of all dyes used.

It is critical in all NHS ester labeling reactions to avoid nucleophilic molecules and ions which can also react with NHS ester groups. Peptides need to be in a solution free of primary amines (e.g. Tris), azide and thiols. Phosphate buffer is readily available and contains no free amines. The rate of the desired reaction between the free amines on the peptide and the NHS ester of the dye is fast compared to the rate of the NHS ester hydrolysis (half life about 2 hours). The reaction should be run at the highest practical peptide concentration. See Section IV for more details.

IV. EXAMPLES
The following examples highlight on-bead labeling of the deprotected N-terminal amine followed by solution labeling of lysines, which are deprotected during cleavage of the peptide from the bead. In the HIV-1 Protease Substrate example, the peptide is first labeled with IRDye 800CW, followed by IRDye QC-1. Alternatively, the labeling process may be performed in reverse order, as seen in the Caspase-8 Substrate example. Calculations of labeling efficiency and yield of final product must use the correct dye molecular weights and extinction coefficients.

B. On-Bead Labeling
Solid-phase synthesis methods for peptides use a variety of polymer supports, protecting groups, linkages, and deprotection/cleavage reagents. Peptides synthesized on NovaSyn TGA resin or Rink amide resin using Fmoc solid-phase chemistry with TFA labile side chain protecting groups have been successfully used for onbead labeling. Peptides can be custom synthesized from a number of commercial sources. Other systems have not been specifically tested. For on-bead labeling, use the dye NHS ester as the limiting reagent in the labeling reaction in order to

A. HIV-1 Protease Substrate Peptide

The peptide H2N-VSQNYPIVQNK-COOH can be specifically cleaved by HIV-1 protease. Attaching a fluorophore and a quencher to the opposite ends of the peptide produces a fluorogenic substrate for measuring HIV-1 protease activity.

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1. On-bead Labeling of the Peptide with IRDye 800CW NHS Ester

m. Spin the filters in a benchtop centrifuge; collect the filtrate, which contains the labeled peptide. n. Use an additional 1000 L of trifluoroacetic acid to rinse any remaining material from the cleavage vial into the spin filters. o. Spin the filters in the centrifuge again to collect the second filtrate. p. Combine the two filtrates and mix. q. Add the combined filtrate drop-wise into a centrifuge tube containing 40 mL of diethyl ether. r. Centrifuge the suspension formed in the centrifuge tube at ~1500 g for 10 minutes; decant and discard the ether solution from the green precipitate. s. Wash the precipitate twice with 40 mL of diethyl ether. Centrifuge and decant; save the solid green precipitate. t. Dry the crude, green solid product, which is dyelabeled peptide (IRDye 800CW-VSQNYPIVQNKCOOH), under vacuum at room temperature for 2 hours, protected from light. 2. HPLC Purification of Dye-Labeled Peptide a. Dissolve the crude dye-labeled peptide (IRDye 800CW-VSQNYPIVQNK-COOH) in 3 mL of 1x Phosphate Buffered Saline (PBS). b. Aliquot the dye-labeled peptide equally onto several spin filters; centrifuge. c. Purify the dye-labeled peptide by preparative HPLC using the following parameters as a guideline. Column: Gradient: Agilent Zorbax 300SB-C18 semi-prep column (9.4 x 250 mm) 20% to 30% Acetonitrile in aqueous 50 mM Triethylammonium Acetate (TEAA) buffer (pH 6) over 30 minutes 2.8 mL/min near 778 nm (to track the IRDye 800CW absorbance)

Note: The procedure yields approximately 4 mg of dye-labeled peptide. It uses 10 mg of IRDye 800CW NHS ester dye and 150 mg of resin-bound peptide.
a. Synthesize the peptide in NovaSyn TGA resin with an unmasked N-terminus and appropriate protecting groups for the amino acids: H2N-VS(tBu)Q(Trt)N(trt)Y(tBu)PIVQ(Trt)N(Trt)K (Boc)-NovaSyn TGA resin. b. Swell 150 mg of peptide resin in 1150 L of DMF and 50 L of anhydrous diisopropylethylamine (DIPEA) by incubating overnight at ambient temperature. c. Use the supernatant from the bead swelling step to dissolve 10 mg IRDye 800CW NHS ester dye (2x LI-COR part # 929-70021); vortex thoroughly. d. Add the dye solution to the vial containing the peptide resin. Note: The peptide-to-dye molar ratio is about 3 to 1 (~27 mole peptide in 150 mg of peptide beads and 8.5 mole IRDye 800CW). The DIPEA is ~10 fold excess over the peptide (~290 mole DIPEA to ~27 mole peptide). e. Cap the vial tightly and rotate the vial on a tube rotator at room temperature overnight, protected from light. f. Collect the resin beads using two spin filters and a benchtop centrifuge; discard the filtrates. g. Wash the dark green beads on the spin filters repeatedly with DMF (5 x 0.5 mL), then with methanol (5 x 0.5 mL), discarding the wash solutions each time. h. Dry the resin under vacuum at room temperature for 1 hour, protected from light. i. Transfer the dry resin from the frits of the spin filters into a 2 mL tube. j. Add freshly-prepared peptide cleavage solution, which contains 1330 L of trifluoroacetic acid, 35 L of water and 35 L of triisopropylsilane (95:2.5:2.5). Caution: Trifluoroacetic acid is a strong acid that can cause severe eye and skin burns. k. Cap the vial tightly, and place it on a tube rotator. Rotate the vial at room temperature for 3 hours, protected from light. l. Transfer the cleavage solution and resin to the frits of two spin filters.

Flow rate: Monitor:

d. Collect and combine the fractions containing the dye-labeled peptide. Note: Do not allow the product to stand in the TEAA buffer longer than necessary as it may be harmful to the dye. e. Calculate the dye-labeled peptide concentration by measuring the net absorbance of a sample diluted in 1x PBS at 778 nm (baseline subtraction 950-1000 nm)

Page 6 IRDye Peptide Labeling Application Guide

In which: A778 is the measured absorbance of the solution at 778 nm 800CW is the extinction coefficient for the dye at 778 nm in 1x PBS solution, which is 240,000 M-1cm-1 Dilution factor is the fold dilution of the IRDye 800CW labeled peptide solution MW800CW-peptide is the molecular weight of dye-labeled peptide (2340.6 mg/mmole in this case) f. Aliquot the purified product (typical yield 4 mg) as appropriate. g. Lyophilize or SpeedVac to dryness without heating. Store at -20 C. 3. Conjugation of IRDye QC-1 NHS Ester with Dye-Labeled Peptide Note: The procedure uses 3.5 mg of IRDye 800CWHIV peptide and 5 mg of IRDye QC-1 NHS ester quencher dye. a. Dissolve 5 mg of IRDye QC-1 NHS ester (LI-COR part # 929-70031) in 3.0 mL of 0.4 M potassium phosphate buffer (pH 8.0); vortex the solution thoroughly. b. Immediately add the dye solution to the vial(s) containing dye-labeled peptide and vortex briefly to dissolve. Pool solutions in a 15 mL conical tube, as necessary. c. Cap the reaction tube and rotate it on a tube rotator at room temperature for 3 hours, protected from light. d. Filter the reaction solution through a spin filter. e. Purify the dual-labeled peptide by HPLC as in the preceding section. Proceed immediately to the next steps. Note: Measurement of the amount of the duallabeled peptide in a given fraction is complicated by the spectral overlap of the two dyes. The following calculation is based on the IRDye 800CW peak with a correction for the IRDye QC-1 dye peak. f. Measure the absorbance at 778 nm and 850 nm (baseline subtraction from 950 to 1000 nm) of the product solution diluted in methanol.

In which: A778 is the measured absorbance of the solution at 778 nm wavelength (IRDye 800CW and IRDye QC-1) A850 is the measured absorbance of the solution at 850 nm wavelength (IRDye QC-1 only) 800CW is the extinction coefficient for IRDye 800CW at 778 nm in methanol, which is 300,000 M-1cm-1 1.265 is the ratio of A778/A850 of IRDye QC-1 in methanol Dilution factor is the fold dilution of the product sample when preparing the UV-Vis measurement solution g. Aliquot the dual-labeled peptide (typical yield 3 mg) as appropriate. h. Lyophilize or SpeedVac to dryness without heating. i. Store the aliquots at -20 C for up to 1 year.

B. Caspase-8 Substrate Peptide

Attaching a fluorophore and a quencher to the opposite ends of the peptide H2N-GIETDGAK-OH produces a fluorogenic substrate for measuring Caspase-8 activity. The N-terminus of the peptide is labeled with IRDye QC-1 NHS ester on-bead; the unblocked lysine is then labeled in solution with IRDye 800CW. 1. On-Bead Labeling of the Peptide with IRDye QC-1 NHS Ester The labeling is performed as detailed above for the on-bead labeling of HIV-1 Protease Substrate Peptide with IRDye 800CW NHS ester. Substitute 10 mg of IRDye QC-1 NHS ester for IRDye 800CW; use 200 mg of resin-bound Caspase-8 Substrate Peptide. This procedure produces approximately 3 mg of dyelabeled product after preparative HPLC. 2. HPLC Purification of Dye-labeled Peptide with IRDye QC-1 Note: The HPLC purification of dye-labeled peptide is slightly different than for the HIV-1 dye-labeled peptide. a. Dissolve the crude dye-labeled peptide in 3 mL of 1x PBS. b. Aliquot the dye-labeled peptide equally onto several spin filters; centrifuge. c. Purify the dye-labeled peptide by preparative HPLC using the following parameters as a guideline.

IRDye Peptide Labeling Application Guide Page 7

Column: Gradient:

Flow rate: Monitor:

Agilent Zorbax 300SB-C18 semi-prep column (9.4 x 250 mm) 15% to 30% Acetonitrile in aqueous 50 mM Triethylammonium Acetate (TEAA) buffer (pH 6) over 30 minutes 2.8 mL/min Near 778 nm (to track the IRDye QC-1 absorbance)

3. Conjugation of IRDye 800CW NHS Ester with Dye-Labeled Caspase-8 Peptide The conjugation of IRDye QC-1 to the dye-labeled peptide is performed in solution as previously detailed for dual-labeled HIV-1 Protease Peptide Substrate. The procedure uses 3.0 mg of dye-labeled peptide and 5 mg of IRDye 800CW NHS ester. This procedure produces approximately 2.5 mg of dual-labeled peptide after preparative HPLC purification. The HPLC purification should be modified as outlined below: Column: Gradient: Agilent Zorbax 300SB-C18 semi-prep column (9.4 x 250 mm) 15% to 30% Acetonitrile in aqueous 50 mM Triethylammonium Acetate (TEAA) buffer (pH 6) over 30 minutes 2.8 mL/min near 778 nm (to track the IRDye 800CW absorbance)

d. Calculate the dye-labeled peptide concentration by measuring the absorbance at 788 nm (baseline subtraction from 950 to 1000 nm) of the product solution diluted in methanol.

In which: A788 is the measured absorbance value of the solution at 788 nm QC-1 is the extinction coefficient for IRDye QC-1 at 788 nm in methanol solution, which is 98,000 M-1cm-1 Dilution factor is the fold dilution of the dyelabeled peptide when preparing the UV-Vis measurement solution MWQC-1 peptide is the molecular weight of the dye-labeled peptide, which is 1918.5 mg/mmole in this case

Flow rate: Monitor:

The UV-Vis analysis of the product is the same as for the dual-labeled HIV-1 Protease Substrate Peptide. For questions, please send a detailed inquiry to biohelp@licor.com.

V. REFERENCES
1. E. D. Matayoshi, G. T. Wang, G. A. Krafft, J. Erickson. Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer, Science 247(1990), 954-958. 2. K. E. Bullok, D. Maxwell, A. H. Kesarwala, S. Gammon, J. L. Prior, M. Snow, S. Stanley, D. PiwnicaWorms. Biochemical and in vivo characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis, Biochem. 46 (2007), 4055-4065. 3. G. Zheng, J. Chen, K. Stefflova, M. Jarvi, H. Li, B. C. Wilson. Photodynamic molecular beacon as an activatable photosensitizer based on proteasecontrolled singlet oxygen quenching and activation, Proc. Natl. Acad. Sci. 104 (2007), 8989-8994. 4. G. Blum, S. R. Mullins, K. Keren, M. Fonovic, C. Jedeszko, M. J. Rice, B. F . Sloane, M. Bogyo. Dynamic imaging of protease activity with fluorescently quenched activity-based probes, Nature Chem. Biology 1 (2005) 203-209. 5. M. Amblard, J.-A. Fehrentz, J. Martinez, G. Subra. Fundamentals of Modern Peptide Synthesis, In: J. Howl (Eds.) Methods in Molecular Biology Vol. 298: Peptide Synthesis and Applications, Humana Press Inc., Totowa, NJ., 2005, pp. 3-24. 6. R. T. Cummings, S. P . Salowe, B. R. Cunningham, J. Wiltsie, Y. W. Park, L. M. Sonatore, D. Wisniewski, C. M. Douglas, J. D. Hermes, E. M. Scolnick. A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease, Proc. Natl. Acad. Sci. 99 (2002) 6603-6606.

LI-COR is an ISO 9001 registered company. 2008 LI-COR, Inc. LI-COR and IRDye are trademarks or registered trademarks of LI-COR, Inc. IRDye 800CW, IRDye QC-1 and IRDye infrared dye-labeled biomolecules are covered by U.S. and foreign patents and patents pending. All other trademarks belong to their respective owners. LI-COR reagent products are intended for research use only and are not intended for therapeutic or diagnostic use.

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