Solid phase synthesis of a fully active analogue of cholecystokinin using the acid-stable Boc-Phe (p-CH2) SO3H as a substitute for Boc-Tyr(SO3H) in CCK8

Substitution of the -OSO3H group in the sulfated-tyrosine by the non-hydrolyzable-CH2SO3H group was the first described modification of the sulfate ester that does not affect CCK8 activity. In addition to its capacity to mimic the sulfated tyrosine residue, the amino acid Phe(p-CH2SO3Na) was shown to be stable in acidic media, including HF containing mixtures. The synthesis of Boc-Phe(p-CH2SO3Na)-OH in racemic and resolved forms and its introduction into the sequence of CCK8 by solid phase using standard Boc/benzyl synthesis conditions and BOP as coupling reagent is now reported. The two CCK8 analogues containing the L- or the D-Phe(p-CH2SO3Na) residue, obtained in satisfactory yields, were separated by HPLC and the stereochemistry of Phe(p-CH2SO3Na) residue in each peptide was established by NMR spectroscopy and confirmed by a separate solid phase synthesis in which the pure L isomer was used. Both CCK8 analogues displayed high affinities for peripheral and central receptors (KI approximately 1 nM) and proved to be full agonists in the stimulation of pancreatic amylase secretion. The "stabilized-CCK8 peptide", easily prepared by solid phase, could replace the native peptide in biochemical and pharmacological studies. Moreover the modified amino acid Phe (p-CH2SO3Na) could also be used in solid phase synthesis to prepare a wide variety of CCK analogues and more generally, peptides analogues containing the acid-labile O-sulfated tyrosine.     

Intrinsic photoaffinity labeling probes for cholecystokinin (CCK)-gastrin family receptors. D-Tyr-Gly-[Nle28,31,pNO2-Phe33)CCK-26-33)

Attempts to biochemically characterize the pancreatic cholecystokinin (CCK) receptor by affinity labeling have utilized either 125I-Bolton-Hunter-CCK-33 ("long" probes) or decapeptide analogues of the carboxyl terminus of CCK ("short" probes), and covalent attachment via the amino-terminal regions of these probes. The long probe has identified a protein of Mr = 80,000 while "shorter" probes, which have their site of cross-linking closer to the receptor binding region of the probes, have labeled a distinct protein of Mr = 85,000-95,000. To extend and complement these observations, we have designed and synthesized a new probe for the CCK receptor which incorporates a photolabile p-nitrophenylalanine moiety within the theoretical receptor-binding region of the hormone, as its carboxyl-terminal residue. This "intrinsic" photoaffinity labeling probe has been shown to possess full biological activity, with potency and efficacy in stimulating amylase secretion by dispersed rat pancreatic acini similar to that of CCK-8 (CCK-26-33). When iodinated oxidatively, this probe binds rapidly, in a temperature-dependent, reversible, saturable, specific, high affinity manner to enriched pancreatic plasma membranes. In this work, we have used this probe to specifically label the CCK binding site on rat pancreatic plasma membranes. The Mr = 85,000-95,000 protein previously identified with amino-terminal cross-linking of short probes appears to be the protein labeled with this reagent as well. This provides strong evidence that this pancreatic plasma membrane protein contains the CCK-binding domain of the CCK receptor. This intrinsic photoaffinity labeling probe should be quite useful for the characterization of the active site of this receptor and for other CCK and gastrin receptors in many species.     

Environment and mobility of a series of fluorescent reporters at the amino terminus of structurally related peptide agonists and antagonists bound to the cholecystokinin receptor

Fluorescence is a powerful biophysical tool for the analysis of the structure and dynamics of proteins. Here, we have developed two series of new fluorescent probes of the cholecystokinin (CCK) receptor, representing structurally related peptide agonists and antagonists. Each ligand had one of three distinct fluorophores (Alexa(488), nitrobenzoxadiazolyl, or acrylodan) incorporated in analogous positions at the amino terminus just outside the hormone's pharmacophore. All of the probes bound to the CCK receptor specifically and with high affinity, and intracellular calcium signaling studies showed the chemically modified peptides to be fully biologically active. Quenching by iodide and measurement of fluorescence spectra, anisotropy, and lifetimes were used to characterize the response of the fluorescence of the probe in the peptide-receptor complex for agonists and antagonists. All three fluorescence indicators provided the same insights into differences in the environment of the same indicator in the analogous position for agonist and antagonist peptides bound to the CCK receptor. Each agonist had its fluorescence quenched more easily and showed lower anisotropy (higher mobility of the probe) and shorter lifetime than the analogous antagonist. Treatment of agonist-occupied receptors with a non-hydrolyzable GTP analogue shifted the receptor into its inactive low affinity state and increased probe fluorescence lifetimes toward values observed with antagonist probes. These data are consistent with a molecular conformational change associated with receptor activation that causes the amino terminus of the ligand (situated above transmembrane segment six) to move away from its somewhat protected environment and toward the aqueous milieu.     

Establishment of a new short, protease-resistant, affinity labeling reagent for the cholecystokinin receptor

Proteolytic degradation of radioligands is an important source of artifact in affinity labeling of receptor proteins. To complement our previous characterization of the pancreatic acinar cell cholecystokinin (CCK) receptor, we synthesized D-Tyr-Gly[(Nle28,31)CCK-26-33]. The amino terminal D-enantiomer of tyrosine provided a site for oxidative iodination, a free amino group for cross-linking, and rendered the peptide resistant to aminopeptidases. The decapeptide was oxidatively iodinated and purified by reverse-phase HPLC to 2,000 Ci/mmol, to yield a probe which was equal in potency and efficacy to CCK-8, and which bound to rat pancreatic membranes in a rapid, reversible, temperature-dependent, specific, saturable and high affinity manner. This probe was resistant to aminopeptidase degradation, and maintained its ability to bind to receptor after incubation with pancreatic membranes or dispersed cells. Affinity labeling of pancreatic membranes with this analogue identified an Mr = 85,000-95,000 molecule. This analogue offers several advantages over existing probes and should be useful for future studies of this and other CCK receptors.     

Synthetic probes for the alpha-factor receptor

The binding of the tridecapeptide yeast mating pheromone, alpha-factor, to its receptor represents an excellent model for the investigation of peptide hormone-receptor interactions. In this paper we present a number of strategies to probe the binding site of the alpha-factor receptor, and discuss the synthesis of probes containing radioactive and affinity tags. Preferential acylation of the alpha- or epsilon-amine in [Nle12]-alpha-factor was accomplished using 3-[3,5-diiodo-4-hydroxyphenyl] propanoic acid hydroxysuccinimide ester (diiodo Bolton-Hunter reagent). At pH 8.0 in a N-N-dimethylformamide/water mixture the ratio of epsilon- to alpha-acylation was 2.15 to 1, whereas at pH 6.5 in a 1,2-dimethoxyethane/water mixture alpha-acylation was favored by more than 3 to 1. The product distribution was found to depend on pH, organic cosolvent, and the ratio of organic solvent and aqueous buffer. Product distributions were followed using analytical high performance liquid chromatography and the products were characterized enzymatically and by mass spectrometry. Citraconic anhydride preferentially alpha-acylated [Nle12]-alpha-factor and served as a temporary masking group during the synthesis of epsilon-Bolton-Hunter acylated pheromone. Biotin or diiodo Bolton-Hunter reagents were also directly incorporated into [Nle12]-alpha-factor or Lys[Nle12]-alpha-factor during peptide synthesis. The peptides were assembled on a chloromethyl polystyrene resin or on a (phenylacetamido)methyl resin, and cleaved using anhydrous hydrogen fluoride (HF). Probes were inserted on amino groups either prior (biotin) or subsequent (Bolton-Hunter reagent) to HF cleavage. The biological activity of the synthetic peptides was characterized using growth arrest assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peptide mapping of the insulin-binding site of the 130-kDa subunit of the insulin receptor by means of a novel cleavable radioactive photoprobe

A radioactive photoaffinity probe for the insulin receptor was prepared by derivatizing insulin at its B29 lysine with a novel crosslinking reagent having a cleavable azo linkage. Insulin receptors purified from human placental membranes were photoaffinity labeled with this probe. The photolabeled receptor was treated with dithionite to cleave the azo linkage, thereby removing the insulin ligand and transferring the radioactivity to the receptor protein. The radioactive labeled subunit was isolated and digested with elastase for peptide mapping and separation by high performance liquid chromatography. Results obtained indicated that it will be feasible to use this new photoaffinity probe to obtain radioactive peptides representing the insulin-binding site(s) on the receptor subunit.     

Synthesis and biological evaluation of cyclic and branched peptide analogues as ligands for cholecystokinin type 1 receptor

A library of cyclic CCK8 analogues, containing unnatural amino acids in the peptide sequence, is prepared using solid-phase synthesis. The structure of these cyclic peptides is based on a previously synthesised compound, cyclo-CCK8, selective for CCK(1) receptor. Structure-activity investigations are performed by evaluating the binding properties of the new analogues. In particular, the binding ability of the cyclic CCK8 analogues is tested by nuclear medicine studies on cell line transfected with CCK(1) receptor. Compounds named cyclo-A4-cyclo-A7 show binding constant in the range 6.0-8.0 microM, with an improved affinity over the previous described cyclo-CCK8, but almost comparable IC(50) values among new analogues towards CCK(1) were obtained.     

Distinct molecular mechanisms for agonist peptide binding to types A and B cholecystokinin receptors demonstrated using fluorescence spectroscopy

Fluorescence spectroscopy provides a direct method for evaluating the environment of a fluorescent ligand bound to its receptor. We utilized this methodology to determine the environment of Alexa within a cholecystokinin (CCK)-like probe (Alexa488-Gly-[(Nle(28,31))CCK-26-33]; CCK-8 probe) bound to the type A CCK receptor (Harikumar, K. G., Pinon, D. L., Wessels, W. S., Prendergast, F. G., and Miller, L. J. (2002) J. Biol. Chem. 277, 18552-18560). Here, we study this probe at the type B CCK receptor and develop another probe with its fluorophore closer to the carboxyl-terminal pharmacophore of type B receptor ligands (Alexa488-Trp-Nle-Asp-Phe-NH2; CCK-4 probe). Both probes bound to type B CCK receptors in a saturable and specific manner and represented full agonists. Similar to the type A receptor, at the type B receptor these probes exhibited shorter lifetimes and lower anisotropy when the receptor was in the active conformation than when it was shifted to its inactive, G protein-uncoupled state using guanosine 5'-[beta,gamma-imido]-triphosphate trisodium salt. Absolute values for lifetime and anisotropy were lower for the CCK-8 probe bound to the type B receptor than for this probe bound to the type A receptor, and Alexa fluorescence was more easily quenched by iodide at the type B receptor. This represents the first direct evidence that, despite having identical affinities for binding and potencies for activating type A and B receptors, CCK is docked via distinct mechanisms, with the amino terminus more exposed to the aqueous milieu when bound to the type B CCK receptor than to the type A CCK receptor. Of interest, despite this difference in binding, activation of both receptors results in analogous direction of movement of the fluorescent indicator probes.     

Evidence for a direct interaction between the penultimate aspartic acid of cholecystokinin and histidine 207, located in the second extracellular loop of the cholecystokinin B receptor.

Recently, we reported that the mutation of His(207) to Phe located in the second extracellular loop of the cholecystokinin B receptor strongly affected cholecystokinin (CCK) binding (Silvente-Poirot, S., Escrieut, C., and Wank, S. A. (1998) Mol. Pharmacol. 54, 364-371). To characterize the functional group in CCK that interacts with His(207), we first substituted His(207) to Ala. This mutation decreased the affinity and the potency of CCK to produce total inositol phosphates 302-fold and 456-fold without affecting the expression of the mutant receptor. The screening of L-alanine-modified CCK peptides to bind and activate the wild type and mutant receptors allowed the identification of the interaction of the C-terminal Asp(8) of CCK with His(207). The H207A-CCKBR mutant, unlike the wild type receptor, was insensitive to substitution of Asp(8) of CCK to other amino acid residues. This interaction was further confirmed by mutating His(207) to Asp. The affinity of CCK for the H207D-CCKBR mutant was 100-fold lower than for the H207A-CCKBR mutant, consistent with an electrostatic repulsion between the negative charges of the two interacting aspartic acids. Peptides with neutral amino acids in position eight of CCK reversed this effect and displayed a gain of affinity for the H207D mutant compared with CCK. To date, this is the first report concerning the identification of a direct contact point between the CCKB receptor and CCK.     

Refinement of the structure of the ligand-occupied cholecystokinin receptor using a photolabile amino-terminal probe

Affinity labeling is a powerful tool to establish spatial approximations between photolabile residues within a ligand and its receptor. Here, we have utilized a cholecystokinin (CCK) analogue with a photolabile benzoylphenylalanine (Bpa) sited in position 24, adjacent to the pharmacophoric domain of this hormone (positions 27-33). This probe was a fully efficacious agonist that bound to the CCK receptor saturably and with high affinity (K(i) = 8.9 +/- 1.1 nm). It covalently labeled the CCK receptor either within the amino terminus (between Asn(10) and Lys(37)) or within the third extracellular loop (Glu(345)), as demonstrated by proteolytic peptide mapping, deglycosylation, micropurification, and Edman degradation sequencing. Truncation of the receptor to eliminate residues 1-30 had no detrimental effect on CCK binding, stimulated signaling, or affinity labeling through a residue within the pharmacophore (Bpa(29)) but resulted in elimination of the covalent attachment of the Bpa(24) probe to the receptor. Thus, the distal amino terminus of the CCK receptor resides above the docked ligand, compressing the portion of the peptide extending beyond its pharmacophore toward the receptor core. Exposure of wild type and truncated receptor constructs to extracellular trypsin damaged the truncated construct but not the wild type receptor, suggesting that this domain also may play a protective role. Use of these additional insights into molecular approximations provided key constraints for molecular modeling of the peptide-receptor complex, supporting the counterclockwise organization of the transmembrane helical domains.     

A Study of the Cerebral Cortex Cholecystokinin Receptor Using Two Radiolabelled Probes: Evidence for a Common CCK 8 and CCK 4 Cholecystokinin Receptor Binding Site

Abstract: This study was directed at the issue of whether or not subpopulations of cholecystokinin (CCK) receptors exist within the CNS. This was achieved through the use of two radiolabelled probes, namely [¹²⁵I] Bolton-Hunter (BH) CCK 8 and [³H]pentagastrin (Boc-β-Ala CCK 4), in comparative studies under identical conditions. Both probes bound with high affinity to the mouse cerebral cortical CCK receptor binding site with apparent equilibrium dissociation constants (K_D) of 1.9 nM and 1.4 nM for [³H]pentagastrin and [¹²⁵I]BH CCK 8, respectively. The maximal binding capacity was 1.05 and 1.15 pmol/g weight for the tritium and iodinated probes, respectively. Hill analysis yielded Hill numbers close to unity, suggesting the absence of more than one binding site and the lack of cooperativity of CCK receptor binding. Kinetic studies revealed binding site homogeneity in that no evidence of multiphasic dissociation curves was seen. Computerised analysis of displacement binding data using LIGAND established that both radiolabelled probes bound to a single site, with the one-site model providing the best fit of the data. Similar rank orders of potency were obtained for various fragments of CCK 8 in competing for the CCK receptor, labelled with either probe. Both CCK 8 and CCK 4 bound with roughly equinanomolar affinity. These studies demonstrate that both CCK 8 and its shorter C-terminal fragment CCK 4 bind to a single class of high-affinity binding site, with as yet no evidence of CNS CCK receptor multiplicity.     

Acid-labile anchoring linkages for solid phase synthesis of C-terminal asparagine peptides using the Fmoc strategy

Two acid-labile substituted benzylamine type anchoring linkages, 4-benzoxy-2,6-dimethoxybenzylamine and 2-benzoxy-4,6-dimethoxybenzylamine, for solid phase synthesis of peptide amides were prepared. The Na-9-fluorenylmethyloxycarbonyl (Fmoc) amino acids could be easily attached to the resins with DCC/HOBt (loading 0.5-0.6 mmol/g resin). After final removal of the Na-protecting groups, treatment with TFA (50-95%) yielded amino acid and peptide amides in high purity. As we could show for the synthesis of thymulin (FTS, pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn), these two resins with anchoring linkages are well suited for the synthesis of C-terminal Asn peptides using protected aspartic acid derivative as starting material.     

Concise site-specific synthesis of DTPA-peptide conjugates: application to imaging probes for the chemokine receptor CXCR4

Diethylenetriaminepentaacetic acid (DTPA) is a useful chelating agent for radionuclides such as (68)Ga, (99m)Tc and (111)In, which are applicable to nuclear medicine imaging. In this study, we established a facile synthetic protocol for the production of mono-DTPA-conjugated peptide probes. A novel monoreactive DTPA precursor reagent was synthesized in two steps using the chemistry of the o-nitrobenzenesulfonyl (Ns) protecting group, and under mild conditions this DTPA precursor was incorporated onto an N(ε)-bromoacetylated Lys of a protected peptide resin. The site-specific DTPA conjugation was facilitated by using a highly acid-labile 4-methyltrityl (Mtt) protecting group for the target site of the bioactive peptide during the solid-phase synthesis. A combination of both techniques yielded peptides with disulfide bonds, such as octreotide and polyphemusin II-derived CXCR4 antagonists. DTPA-peptide conjugates were purified in a single step following cleavage from the resin and disulfide bond formation. This site-specific on-resin construction strategy was used for the design and synthesis of a novel In-DTPA-labeled CXCR4 antagonist, which exhibited highly potent inhibitory activity against SDF-1-CXCR4 binding.     

A new hybrid resin for stepwise screening of peptide libraries combined with single bead Edman sequencing

A library system was developed for the discovery of bioactive peptides. Library synthesis and peptide sequencing was performed on a solid support while the screening for bioactivity was done with peptides in solution. The peptides were synthesized by split and mix, one-bead–one-peptide library synthesis, using a Tentagel S-NH₂ solid support with a loading of approximately 100 pmol/bead. The major part of the peptide was connected to the support by a single acid-labile linker and a minor part of the peptide was acid-stabile attached to the polymer. The percentage of acid-stabile attached peptides could easily be controlled during modification of the amino functionalities of the resin at the start of the process. The cleavage rate of the acid-labile attached peptide from the resin depends on the composition of the cleavage mixture. When cleavage conditions were carefully controlled, a three-step partial cleavage protocol allowed for convergent bioactivity screening on peptide libraries using only one type of acid-labile linker. The partial cleavage and convergent screening procedure was repeated three times, after which the bead containing the bioactive peptide was sequenced. As such a bead still contained acid-stabile attached peptide, the Edman sequencing was straightforward and repetitive yields were excellent because the immobilized peptide was not washed out. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Affinity labeling of a novel cholecystokinin-binding protein in rat pancreatic plasmalemma using new short probes for the receptor

Previous biochemical characterizations of the cholecystokinin (CCK) receptor have used the "long" probe 125I-Bolton-Hunter-CCK-33 since it was the only CCK analogue with high affinity and high specific radioactivity which possessed an amino group available for chemical cross-linking. These studies have consistently identified a major binding protein of approximately 81 kilodaltons and have identified several minor proteins which were obtained under different cross-linking conditions and in different laboratories. Because the receptor-binding region of CCK-33 (carboxyl-terminal heptapeptide) is so far removed from the radiolabel and from available amino groups (positions 1 and 11), this probe carries potential for proteolytic cleavage of label from receptor and for labeling "near neighbors" instead of the binding site. We therefore designed two "short" probes for the CCK receptor. 125I-Bolton-Hunter-Lys-Gly-CCK-8 has an epsilon-amino group available for cross-linking. 125I-Tyr-[Thr28,Nle31]CCK-25-33 has an alpha-amino group for cross-linking and has the major advantage of being labeled by oxidative means, unique for CCK derivatives. Both radioiodinated decapeptides were purified by reverse-phase high pressure liquid chromatography to yield specific radioactivity of 2,000 Ci/mmol; demonstrated saturable, specific, and high affinity binding to rat pancreatic plasma membranes; and retained full biological activity to stimulate amylase secretion. Using a variety of cross-linking methods, these probes each identified the same Mr = 85,000-95,000 protein in rat pancreatic plasmalemma, and CCK-8 competed for this labeling in a concentration-dependent manner (IC50 = 1 nM). No change in apparent mobility of this band was observed under reducing or nonreducing conditions, suggesting lack of covalent attachment to other subunits. The Mr = 85,000-95,000 species migrated differently on sodium dodecyl sulfate gels than any of the components previously identified using 125I-Bolton-Hunter-CCK-33, confirming the novel nature of this binding protein. These short probes should be very useful for further characterization of CCK receptors on this and other tissues.     

Fractionation of immunoreactive cholecystokinin by adsorption to QUSO or talc.

The technique of adsorption of peptides containing basic amino acids to surfaces of silica or talc has been extended to distinguish between the basic precursor peptides, cholecystokinin (CCK33) and its variant (CCK39), and their COOH-terminal 12 and 8 amino acid fragments (CCK12 and CCK8) in plasma and tissue extracts. CCK39 and CCK33 are quantitatively adsorbed from 2 ml of solution by 5 mg QUSO G32 or 25 mg talc. The adsorbed basic peptides can be completely eluted from QUSO but not from talc by 0.1N HC1. CCK12 and CCK8 are not detectably adsorbed by either QUSO or talc. The method is simple, inexpensive and is suitable for rapid handling of multiple samples.     

Multiple neurotransmitter receptors in the brain: amines, adenosine, and cholecystokinin

Radioligand binding studies of neurotransmitter receptors have provided discrimination at the molecular level, permitting the differentiation of multiple receptor subtypes for several biogenic amines. Using this paradigm we have labeled two distinct receptors each for cholecystokinin (CCK) and for adenosine. Adenosine receptors were labeled in brain with [3H]N6-cyclohexyladenosine (3H-CHA) and [3H]1,3-diethyl-8-phenylxanthine (3H-DP). The adenosine receptor labeled by 3H-CHA appears to be an A1 site, associated with reduction of adenylate cyclase activity, while 3H-DP sites resemble A2 receptors linked to adenylate cyclase enhancement. Cholecystokinin-33 labeled by the Bolton-Hunter procedure with 125I(125I-BH-CCK) labels different receptors in brain and pancreas. The pancreatic receptor does not react with CCK derivatives of fewer than eight amino acids, while the brain receptor does recognize pentagastrin, the carboxyl-terminal five amino acids of CCK. The "brain type" CCK receptor may normally interact with CCK-4, the carboxyl-terminal tetrapeptide of CCK, recently identified as a unique neuropeptide highly concentrated in the brain. CCK-8, the other major molecular form of CCK, may be the endogenous ligand for the "pancreatic type" receptor.     

Immunoreactive cholecystokinin in human and rat plasma: correlation of pancreatic secretion in response to CCK

Immunoreactive cholecystokinin (CCK) levels in human and rat plasma are described using a radioimmunoassay specific for the biologically active sulfated end of CCK. This assay detected significant changes in plasma cholecystokinin levels during intrajejunal administration of amino acids and intravenous infusions of CCK-8 which were followed by increased pancreatic secretion. In humans, the concentration (pg/ml) of plasma cholecystokinin increased from 10.8 to 18.9 following intrajejunal amino acid instillation and from 15.4 to 31.1 during CCK infusion, while pancreatic trypsin secretion increased more than 15 fold. Ingestion of a test meal also caused a rapid and significant elevation (P less than 0.05) in both plasma CCK (14.5-21.7 pg/ml) and gastrin (50-160 pg/ml) levels. In the rat, an injection of 46 ng of CCK-8 produced a 300% increase in immunoreactive plasma CCK levels (2 min) and caused peak pancreatic protein secretion within 5 min; 4 fold lower doses (11.5 ng) elevated plasma CCK by 38% and pancreatic protein secretion to a small but significant extent. The ability of this assay to detect various forms of sulfated CCK in human plasma was also determined. Following gel chromatography on Sephadex G-50, at least three different immunoreactive peaks were found in plasma from fasted subjects and after intrajejunal amino acid stimulation. While the lower molecular weight CCK peptides (CCK-8 and CCK-12) were detected in plasma from both fasted and stimulated subjects, the larger form (CCK-33) was only present in measurable concentrations after amino acid infusion. The simultaneous measurement of increased plasma CCK levels and pancreatic secretion and the changes in the distribution of CCK peptides following amino acid infusion provides strong support that this assay detects physiologically relevant changes in biologically active CCK peptides.     

Synthesis of acid-cleavable light isotope-coded affinity tags (ICAT-L) for potential use in proteomic expression profiling analysis

A convenient synthesis of some homologous light isotope-coded affinity tags (ICAT-L) containing an acid-labile moiety between the affinity component biotin and an electrophilic polar linker is described. These light ICAT reagents give smooth mass spectral signals in tandem mass spectrometry (MS/MS) analyses of some commercially available cysteine-containing peptides. However, these ICAT molecules are designed for use in identification and relative quantification of whole or partially purified cellular and tissue proteomes. Since the biotin moiety can be readily cleaved off the reagent after mass tagging, undesired residual fragmentation patterns caused by biotin of derived peptides, as normally observed using biotin-containing ICAT reagents, are effectively eliminated. This strategy should enhance peptide sequence coverage significantly which, in turn, should result in improving the quality of data obtained during data-dependent peptide mass and tandem mass spectral analysis of whole proteomes.     

Use of a nitrotryptophan-containing peptide for photoaffinity labeling the pancreatic cholecystokinin receptor

We report the preparation and characterization of a new type of intrinsic photoaffinity labeling probe, on the basis of the incorporation of a photolabile nitrotryptophan into a biologically relevant domain of a peptide. The model system used was the pancreatic cholecystokinin (CCK) receptor, previously affinity labeled with a variety of probes. Those studies have suggested that an Mr = 85,000-95,000 protein is more likely to be labeled as the site of covalent attachment approaches the receptor-binding domain of this hormone. Indeed, CCK has a Trp in the center of its receptor-binding region, and replacement of that residue with 6-nitrotryptophan resulted in a photolabile probe which affinity labeled the same Mr = 85,000-95,000 pancreatic membrane protein. This probe, 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Trp30)CCK-26-33], was synthesized by solid-phase and solution techniques and characterized by mass spectrometry. Following oxidative iodination, it was purified on HPLC to 2000 Ci/mmol. Binding to pancreatic membranes was rapid, temperature dependent, reversible, saturable, and specific and was with high affinity (Kd = 3 nM). While its binding affinity was only 3-fold lower than that of native CCK-8, this probe was 70-fold less potent than native hormone in stimulating amylase secretion (EC50 = 1 nM) and equally efficacious to native hormone. Despite the slight decrease in affinity, this probe demonstrated a high relative efficiency of covalent labeling of the Mr = 85,000-95,000 protein. This confirms that the Mr = 85,000-95,000 protein represents the hormone-binding subunit of the CCK receptor and demonstrates the utility of this type of photoaffinity labeling probe.(ABSTRACT TRUNCATED AT 250 WORDS)