Labelling of peptides with CyDye fluors for fluorescent applications on the LEADseeker Homogeneous Imaging System

Transcription

1 Issue No. 7 abelling of peptides with CyDye fluors for fluorescent applications on the EADseeker Homogeneous Imaging System EADseeker TM homogeneous imaging system is a digital imager comprising a chargecoupled device (CCD)-camera with associated software and assay systems. The EADseeker multimodality imager has been developed as part of a collaboration between Amersham Biosciences and Imaging Research Inc. (IRI) of St. Catharines, Canada, as a completely automated system to include fluorescent assay screening. Fluorescent reagents have also been designed to complement the technology relating to EADseeker. These consist of labelling systems based around proprietary CyDye TM fluors and associated biological applications. This article describes typical methodologies for labelling peptide molecules using these fluorophores. abelling peptides with a single fluorophore abelled peptides are frequently employed in binding event assays. In the past, these binding event assays involved peptide ligands which have been labelled with radioisotopes. In many cases, radioisotopic labelling has a relatively small effect on the biological activity of the peptide. However in the case of fluorescent probes, the fluorescent moiety is often quite a large molecule in its own right. This may have a significant effect on the biological activity of the peptide being labelled and therefore careful consideration should be given to the selection of the residue that is labelled with the fluorophore. At Amersham Biosciences a considerable number of peptides have been labelled at the N-terminus. For example, peptide YY (PYY) can be labelled with fluorescein, Cy TM 3 or Cy5 with maintenance of full binding activity, whereas similar labellings of substance P result in a ten-fold decrease. In the case of [Asp 1, Phe 8 ]angiotensin II, N-terminal labelling results in a 500-fold decrease in binding activity. The most convenient labelling strategies usually involve addition of the fluorophore to an appropriate amino acid residue side-chain (e.g. lysine) or N-terminal labelling. These fluorescently labelled peptides can be utilized in assays, which may be configured in either hetereogeneous or homogeneous formats such as fluorescence polarization or FRET. August 2000, Issue No. 7 p1

2 eadseeker homogeneous imaging system N-terminal labelling of peptides still attached to the solid phase Most peptides are prepared by solid phase peptide synthesis, therefore labelling of the peptide while still attached to the solid phase is a very convenient method for labelling with fluorophores (1-3). An activated form (eg. N-hydroxysuccinimide [NHS] ester) of the fluorophore is coupled to the peptide while still attached to the solid phase with all the reactive side-chains protected and only the N-terminal amino group free. The crude peptide is deprotected and cleaved from the resin, followed typically by HPC purification and characterization of the labelled product. The main advantages of this method are that it is very simple and uncoupled materials are readily washed away. The disadvantages are that a substantial excess of the activated fluorophore is usually required to drive the reaction to completion and labelling at the N-terminus may affect the binding activity. Typically, the coupling reaction will be performed using an anhydrous solvent such as dimethyl sulphoxide (DMSO), dimethylformamide (DMF) or dimethyl acetamide, often in the presence of a base such as N,N-diisopropylethylamine. Example protocol 1: Coupling of Cy3B to peptide E9K 1 Add Cy3B NHS ester (5 mg) in anhydrous DMSO (0.5 ml) to peptide E9K (EpYINQSVPK) on resin (15 mg, 0.55 mmol/g) in a 1 ml capacity Sarstedt TM vial together with a triangular magnetic stirrer bar. 2 Add N,N-diisopropylethylamine (10 µl) and stir overnight at room temperature in the dark. 3 Remove the magnetic stirrer bar, make up the volume to 1 ml with DMSO and spin in a microfuge (1 min at rpm). 4 Wash the resin with DMSO (1 x 1 ml), then methanol (4 x 1 ml), and dry in vacuo for 1 h. 5 Deprotect and cleave the peptide from the resin with 0.5 ml trifluoroacetic acid (TFA): water mixture (95:5) for 2 h on ice. 6 Filter off the resin (eg. using a small plug of glass wool in a pasteur pipette) into ice-cold diethyl ether (30 ml) in a centrifuge tube. Centrifuge at 3000 g for 5 min, wash the crude precipitate once with cold diethyl ether (30 ml), centrifuge again and dry in vacuo. 7 Dissolve the crude product in water (1.0 ml) and purify by HPC on a Vydac TM protein and peptide C18 column (25 cm x 10 mm) as 2 x 0.5 ml injections, using a gradient from water containing 0.1%TFA to acetonitrile: water (60:40) containing 0.1%TFA over 30 min at a flow rate of 4 ml/min. 8 Collect the appropriately coloured peak. Note that the retention time of the dye labelled peptide will be longer than that of the unlabelled peptide. 9 yophilize the purified product, or store as aliquots at -20 o C. 10 Characterize the product appropriately (e.g. mass spectrometry). Notes a) This protocol has been designed for small quantities (15 mg) of resin. abelling reactions involving smaller quantities of resin will be more difficult to manipulate, and yields may be reduced. b) The addition of small quantities of other components (e.g. thioanisole, 1,2-ethanedithiol) to the deprotection/ cleavage mixture may be necessary for peptide sequences which include sulphur containing amino acids. c) Depending on the peptide sequence being labelled, problems may be encountered solubilizing the peptide in water alone, the addition of some acetic acid or DMSO are possibilities that should be considered. d) Select a HPC gradient appropriate for the labelled peptide being purified. Solution labelling An alternative for single labelling of peptides is to perform the labelling in solution. The principle advantage of this method is that it is suitable for small quantities of peptide (e.g. 1 mg or less). The coupling reactions can be performed in buffered aqueous solution, or in solvents such as DMF or DMSO. The labelling process is straightforward if the peptide contains only one reactive amino group for coupling with the activated ester of the fluorophore. This is commonly the N-terminal amino acid residue of the peptide. August 2000, Issue No. 7 p2

3 eadseeker homogeneous imaging system However, if the N-terminal amino group is blocked, for example by acetylation, reaction of the activated ester of the fluorophore may occur with the ε-amino group of a lysine side-chain. If the peptide sequence contains more than one reactive amino group, it may be possible to distinguish between them using differential reactivity (e.g. by careful control of ph (4) ). However it is likely that the many possible labelling products would have to be separated by chromatography. An alternative approach is to design a synthetic strategy in which the other reactive amino groups are protected, leaving only one amino group free for reaction with activated ester of the fluoroprobe. Example protocol 2: N-terminal labelling of (D-ser 2 )- leucine-enkephalin with Cy5 1 Dissolve Cy5 NHS ester (1.26 mg) in DMSO (500 µl), and add this to (D-ser 2 )-leucine-enkephalin acetate (YSGFT, 1.0 mg) followed by DMSO (500 µl) in a 1 ml capacity Sarstedt vial. 2 Add triethylamine (20 µl) to the solution, and roll the reaction mixture overnight in the dark at room temperature. 3 Purify the crude product by HPC on a Vydac protein and peptide C18 column (25 cm x 10 mm) as 2 x 500 µl injections, using a gradient from water containing 0.1% TFA to acetonitrile:water (70:30) containing 0.1% TFA over 30 min at a flow rate of 4 ml/min. 4 Collect the appropriately coloured peak. Note that the retention time of the dye labelled peptide will be longer than that of the unlabelled peptide. 5 yophilize the purified product, or store as aliquots at -20 o C. 6 Characterize the product appropriately (e.g. mass spectrometry). Notes a) The ratio of dye to peptide was 1.1 to 1. b) Select a HPC gradient appropriate for the labelled peptide being purified. abelling with two fluorophores Multiple homogeneous FRET based applications are under development for use with EADseeker. Assays configured in this format require the use of donor and acceptor fluorophores. For binding event assays such as antibody-antigen (immunoassays), protein-dna interactions and DNA hybridization, each binding partner is labelled. However, in the case of substrates for monitoring protease activity, the peptide substrate has to be labelled with two fluorophores. For example, where the donor and acceptor are Cy3/Cy3B and Cy5 respectively, FRET occurs between the pair of fluorophores. Upon cleavage of the substrate by the proteolytic enzyme, FRET no longer occurs, so there is a decrease in the observed fluorescence emission at the Cy5 wavelength. However, a signal increase format is possible, if the acceptor is a quencher. The commonly employed donor and quencher pair has been 5-(2'-aminoethyl)aminonaphthalene sulphonic acid (EDANS) and 4-(4'-dimethyl-aminobenzenazo)benzoic acid (DABCY) (5). Amersham Biosciences has developed quenchers* based on the cyanine dye family which have all of the advantages of fluoropobes, but are non-fluorescent. Suitable donor and quencher pairs are Cy3/Cy3B and Cy5Q or Cy5 and Cy7Q. The general synthetic approach, after the selection of an appropriate substrate sequence for the enzyme under investigation, is coupling of a substantial excess of the activated ester of either the donor or acceptor/quencher to the free N-terminus of the protected peptide while still bound to the resin. After deprotection/cleavage from the resin and purification of the peptide labelled with one of the FRET pair, the second of the FRET pair (either donor or acceptor/ quencher) is coupled to the C-terminus, followed by purification and characterization of the desired substrate. The sequence should ideally have a lysine residue at or close to the C-terminus for convenient coupling with an approximately ten-fold excess of the NHS ester of the second donor/acceptor/quencher of the FRET pair. The example protocol below describes the synthesis of a substrate (YVADAPVK) for the enzyme Asp-N, labelled with Cy3B at the N-terminus and Cy5 at the C-terminus. Example protocol 3: Synthesis of FRET substrate for the enzyme, Asp-N 1 Add Cy3B NHS ester (10 mg) in anhydrous DMSO (1.0 ml) to peptide (YVADAPVK) on resin (30 mg) in a 1 ml capacity Sarstedt vial, together with a triangular magnetic stirrer bar and N,N-diisopropylethylamine (40 µl). August 2000, Issue No. 7 p3

4 eadseeker homogeneous imaging system 2 Stir overnight at room temperature in the dark. 3 Remove the stirrer bar, transfer the reaction mixture to a small sintered glass funnel. 4 Wash with DMSO (3 x 2 ml), methanol (3 x 2 ml) and dichloromethane (2 x 2 ml), and then dry in vacuo for 1 h. 5 Deprotect and cleave the peptide from the resin using 2 ml trifluoroacetic acid (TFA): triisopropylsilane: water mixture (95:2.5:2.5) for 90 min at room temperature. 6 Filter off the resin (e.g. using a small plug of glass wool in a pasteur pipette) into ice-cold diethyl ether (30 ml) in a centrifuge tube. Centrifuge at 3000 g for 5 min, wash the crude precipitate three times with cold diethyl ether (30 ml), and finally dry in vacuo. 7 Dissolve the crude product in the absolute minimum quantity of DMSO followed by the addition of water (700 µl). Purify by HPC on a Vydac protein and peptide C18 column (25 cm x 10 mm) as four 200 µl injections, using a gradient from acetonitrile:water (5:95) containing 0.1%TFA to 30% water + 70% acetonitrile:water (70:30) containing 0.1%TFA over 25 min at a flow rate of 2 ml/min. 8 Collect the appropriately coloured peak. Note that the retention time of the dye labelled peptide will be longer than that of the unlabelled peptide. 9 yophilize the purified product. 10 Characterize the purified product with a single donor/ acceptor/quencher appropriately (e.g. mass spectrometry). 11 Dissolve the lyophilized material (0.78 mg) in DMSO (150 µl) add to Cy5 NHS ester (2 mg) in DMSO (100 µl) together with diisopropylethylamine (25 µl) in a 0.4 ml capacity Sarstedt vial. Total volume is 275 µl. 12 Roll the vial overnight in the dark at room temperature. 13 Purify the crude dual labelled product by HPC using a Vydac protein and peptide C18 column (25 cm x 10 mm) as four injections, using a gradient from acetonitrile:water (5:95) containing 0.1%TFA to acetonitrile:water (70:30) containing 0.1%TFA over 25 min at a flow rate of 2 ml/min. 14 Collect the appropriately coloured peak, note that the retention time of the dual labelled peptide will be longer than that of the singly labelled peptide. 15 yophilize the purified product, or store as aliquots at -20 o C. 16 Characterize the final dual labelled FRET substrate appropriately (e.g. mass spectrometry). Notes a) This protocol has been designed for small quantities (30 mg) of resin. abelling reactions involving smaller quantities of resin will be more difficult to manipulate and yields may be reduced. b) The addition of small quantities of other components (eg. thioanisole, 1,2-ethanedithiol) to the deprotection/ cleavage mixture may be necessary for peptide sequences which include sulphur containing amino acids. c) Select HPC gradients appropriate for the labelled peptides being purified. d) For the second labelling reaction, a ten-fold excess of the labelling reaction is typically used. e) Careful selection of the peptide sequence used for the protease under study may be required. Biological labelling service Amersham Biosciences is also offering EADseeker collaborators exclusive access to a fluorescence labelling service. This is a dedicated custom labelling service for the preparation of CyDye labelled products, typically peptides, proteins and DNA. EADseeker collaborators are able to take advantage of our expertise in custom synthesis and labelling, assay design and exclusive CyDye reagents in order to achieve optimal substrate or ligand labelling. Techniques available include: specific positional labelling of peptides (mono-labelled and FRET pairs), specific functional group labelling of proteins and site-specific labelling of oligonucleotides. Please contact your local area representative for more details. August 2000, Issue No. 7 p4

5 eadseeker homogeneous imaging system Conclusion It is possible to conveniently label peptides with activated CyDye derivatives employing standard methods typically used for preparing fluorescently labelled peptides. Conditions need to be carefully optimized for the peptide sequence being investigated. References 1. WU, P., BRASSEUR, M. and SCHINDER, U. A high-throughput STAT binding assay using fluorescence polarization. Anal. Biochem., 249(1), (1997). 2. YNCH, B.A., OIACONO, K.A., TIONG, C.., ADAMS, S.E. and MACNEI, I.A. A fluorescence polarization based Src-SH2 binding assay. Anal. Biochem., 247(1), (1997). 3. WEBER, P.J.A., BADER, J.E., FOKERS, G. and BECK-SICKINGER, A.G. A fast and inexpensive method for N-terminal fluorescein labelling of peptides. Bioorg. Med. Chem. ett., 8(6), (1998). 4. TOTA, M.A., DANIE, S., SIROTINA, A., MAZINA, K.E., FONG, T. M., ONG- MORE, J. and STRADER, C.D. Characterisation of a fluorescent substance P analog. Biochem., 33(44), (1994). 5. WANG, G.T., MATAYOSHI, E., HUFFAKER, H.J. and KRAFFT, G.A. Tetrahedron ett., 31, (1990). The article contains example protocols. It is the user's responsibility to design and optimize protocols that are appropriate for the peptide sequences that are being labelled. These example protocols utilize chemicals that may be hazardous, and these operations should only be performed by appropriately qualified and trained professionals. For further information, contact Amersham Biosciences' Technology Transfer Support Groups. Europe: Phone no. +44 (0) Fax no. +44 (0) Voic +44 (0) e:mail leadseeker@eu.amershambiosciences.com North America: Phone no IMAGE US ( ) Fax no Voic IMAGE US ( ) e:mail leadseeker.us@am.amershambiosciences.com Japan: Phone no Fax no August 2000, Issue No. 7 p5

6 EADseeker is covered by PCT application number WO 98/ CyDye or portion thereof is manufactured under licence from Carnegie Mellon University, US Patent Numbers 5,268,486 and 5,569,587 and Patent Application number WO 99/ * Patent Application number PCT/GB 99/ Cy, CyDye and EADseeker are trademarks of Amersham Biosciences or it's subsidiaries. Amersham is a trademark of Nycomed Amersham plc.vydac is a trademark of The Separations Group, Inc. Sarstedt is a trademark of Sarstedt Inc.All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them. A copy of these terms and conditions is available on request. Amersham Biosciences UK imited 2000 All rights reserved. Amersham Biosciences AB, Björkgatan 30, SE Uppsala, Sweden. Amersham Biosciences Inc., 800 Centennial Avenue, Piscataway, New Jersey 08855, USA. Amersham Biosciences UK imited, Amersham Place, ittle Chalfont, Bucks, England HP7 9NA. Amersham Biosciences Europe GmbH, Amersham Biosciences Europe GmbH, Munzinger Strasse 9, D-79111, Freiburg, Germany S1452/00/07