Threonine Containing

Oct 30, 2012 - *Address: CEA, DSV, IBEB, SBTN, Centre de Marcoule, BP 17171, F-30207 Bagnols-sur-Cèze, France. E-mail: [email protected]...
2 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/ac

Antibody-Free Detection of Phosphoserine/Threonine Containing Peptides by Homogeneous Time-Resolved Fluorescence Béatrice Alpha-Bazin* and Eric Quéméneur§ CEA, DSV, IBEB, SBTN, Bagnols-sur-Cèze F-30207, France S Supporting Information *

ABSTRACT: Protein phosphorylation is a critical signaling mechanism in cellular regulation and stress response, and more than 95% of the phosphorylations are targeted toward Ser or Thr amino-acid residues. The classical techniques for analyzing phospho-amino acid residues use radioisotopes or sequence-specific antibodies. However, both practical and economical limitations have prevented their development, and we here propose an original approach for the detection of phospho-Ser/ Thr residues. It requires no antibody and exploits the patented homogeneous time-resolved fluorescence (HTRF) technology, in association with a 3-step chemical transformation of phospho-amino acids into fluorescent derivatives. The process involves: (i) alkaline β-elimination of the phosphorylated group, (ii) Michael addition of a bifunctional group, and then (iii) introduction of cyanin-5 as fluorescent acceptor for HTRF. The donor fluorescent moiety at the N-terminus of the phosphorylated peptide is a streptavidin europium cryptate conjugate. After its development, the detection system has been validated on synthetic peptide substrates of Chk2, a key protein kinase activated in response to DNA damage and involved in cell cycle arrest. The results showed a good correlation with known specificity profiles. Interestingly, the detection system is versatile, easy to implement, and suitable for multiple parallel analyses.

P

rotein phosphorylation acts as a biochemical on/off signaling mechanism in many cellular responses, and many diseases are related to defects in phosphorylation pathways. Protein kinases are the enzymes that catalyze the transfer of the γ-phosphate group from ATP to the hydroxyl groups of serine, threonine, or tyrosine side chains. These enzymes are important targets in drug discovery and have been the subject of a large number of studies over the last 30 years, including functionality and inhibition assays as well as activity assays. Incidentally, several kinase assay methods, involving microliter to nanoliter reactions notably for high throughput screening (HTS), have been devised, which offer a wide variety of possibilities to the scientists, depending on specific constraints, needs, or targets.1 Traditional approaches for kinase biochemistry notably include radiolabeling techniques and the use of antibodies for phosphoamino acids. Overall, they can be classified into three categories, depending on the nature of the probes: radioactive, antibody-based, and antibody-free assays. By design, radioactive assays based on 32P or 33P are robust and straightforward but depend on demanding laboratory environments. The scintillation proximity assay might be considered as the best example of a convenient homogeneous technique for radioactive assays2,3 and for monitoring phosphorylated substrate generation or inhibitor binding. Antibody-based assays have been widely associated with various fluorescence/luminescence technologies. Some examples are homogeneous time-resolved fluorescence (HTRF) from Cis Bio International,4,5 LANCE, AlphaScreen, or DELFIA from Perkin-Elmer,6,7 and fluorescence polarization.8,9 © 2012 American Chemical Society

These approaches do not require special safety procedures for handling or disposal, and the experiments are thus not only easy to implement but also environmentally and automation friendly. As shown by the chemical microarray-based ELISA method, DiscoveryDot, heterogeneous detection combined with microarray format appears well suited to HTS and kinase profiling.10 However, all these assays are limited by the availability of antibodies. This is generally not a problem in the case of phosphotyrosine since antibodies are rather independent from the nature of adjacent amino acids. Quite the opposite, antiphospho-Ser/Thr antibodies are usually sensitive to neighboring amino acids, and thus, a specific antibody is needed for almost any peptides. Among the antibody-free nonradioactive assays, B. Imperiali’s system based on a chelation enhanced fluorescence chemosensor enabled homogeneous kinase activity assays to be measured in cell lysates.11 Wang’s microarray-based kinase inhibition assay is an example of resonance light scattering of metal nanoparticle probes used as an alternative to fluorescence.12 Antibody-free assays are also largely based on the recent progress of mass spectrometry (MS). Unfortunately, phosphorylation analysis is not straightforward due to low abundance, poor ionization, and fragmentation issues (loss of neutral phosphoric acid groups). Several strategies have been developed to study phosphorylation by MS at the proteome Received: August 22, 2012 Accepted: October 17, 2012 Published: October 30, 2012 9963

dx.doi.org/10.1021/ac3021505 | Anal. Chem. 2012, 84, 9963−9970

Analytical Chemistry

Article

scale level. Among them, enrichment of phosphopeptides using immobilized metal affinity chromatography13−15 or metal oxide affinity chromatography16,17 are widely used. Alternatively, a functional group can be chemically introduced in order to improve phosphopeptide enrichment,18−23 MS detection,24−27 and quantitation.20,22,28−30 They are all based on the general principle first described by Simpson in 197231 and adapted to a picomolar sensitivity by Meyer et al. in 1986.32 The generic chemical procedure involves an alkaline β-elimination of the phosphoryl group, followed by the subsequent Michael addition at the dehydro site. An original and complementary approach for the modification of serine/threonine phosphopeptides is presented here. It combines both the Simpson’s chemical process and the homogeneous time-resolved fluorescence technology,5 HTRF. The latter relies on fluorescence resonance energy transfer (FRET) between a europium cryptate, as HTRF donor, and cyanin-5 (Cy5), as a secondary photon acceptor.33−35 The peptide substrates display a biotin residue attached to their Nterminus that enables standard labeling using the Eu cryptatestreptavidin conjugate. The presence of the HTRF acceptor and donor in close proximity gives rise to a FRET signal after excitation at 337 nm. Moreover, the long fluorescence lifetime of europium enables a time-resolved analysis of the FRET signal. Homogeneous HTRF assays for kinases have already been developed,36 relying on the use of a specific peptide substrate and of the corresponding phospho-specific antibody. The assay was also adapted to the analysis of the human Cds1/ Chk2 kinase with a peptide derived from cell division cycle 25 homologue C (Cdc25C).37 Chk2 is a highly conserved DNA checkpoint kinase that plays a critical role in the DNA damage response transduction pathway.38−40 Once activated, this effector Ser/Thr-kinase phosphorylates numerous substrates including the tumor suppressors tumor protein 53 (p53),41 Breast cancer 1 (Brca1),42 and the Cdc25 phosphatases involved in the cell cycle arrest: cell division cycle 25 homologue A (Cdc25A) on Ser12343,44 and Cdc25C on Ser216.45,46 Interestingly, the study of human Chk2 has been facilitated by the fact that recombinant protein can be purified as a phosphorylated protein. The development of the HTRF method was motivated by the need to study a large number of hChk2 peptide substrates. Our results showed a perfect correlation with those obtained previously by a classical method using [γ-32P]-ATP47.

Table 1. List of Model Peptides Used for the Optimization of Chemical Steps

a b

peptidea

amino acid sequence

use

bK6RpS bK6RpT bP14D bY14L

KRPpSQR KRpTITR PALKRSHSDSLDHD YCLLPGTLVFSDFL

pSer model peptideb pTer model peptideb unphosphorylated control peptidec thiol-containing control peptide

“b” in the peptide name means that the peptide is biotinylated. Molloy 2001.48 cPeptide derived from Cdc25A sequence.

Chemical Modifications (β-Elimination/Michael Addition). Phosphorylated Biotinylated Peptide. The reactions were performed in a one-step procedure derived from previously reported conditions.18,21,22,49 For 3 nmoles of peptide, 75 μL of a 220 mM solution of Ba(OH)2 and 2.2 μL of 1.95 M dithiothreitol (DTT) were added and filled up to 100 μL with ultrapure water. The Eppendorf tubes were closed under a N2 atmosphere and then incubated for 5 h at 37 °C with mild shaking. Control tubes corresponded to 3 nmoles of peptide in 100 μL of H2O incubated in the same conditions. Chk2 Reaction Products. The reactions were carried out in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes, pH 7.4) containing 13.5 mM MgCl2, 1 mM DTT, 5 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and 1 mM Na3VO4. All reactions were performed at RT for 30 min using 50 ng of rGST-hChk2SF9, 3 nmoles of biotinylated peptide solution, and 90 μM ATP in a total volume of 40 μL. After the addition of 16.5 μmoles Ba(OH)2 and 4.2 μmoles DTT, the volume was adjusted to 200 μL with water and processed as described above. For each peptide, a “(no enzyme)” control tube, obtained by omitting rGST-hChk2SF9 kinase addition at the phosphorylation step, was treated in the same conditions. Product Recovery. Ammonium Sulfate Precipitation. One mole of ammonium sulfate per mole of barium hydroxide was added to each tube (from a 5 M (NH4)2SO4 stock solution) for the chemical reactions with synthetic phosphopeptides, and 10 mols per mole was added after the kinase reaction. The tubes were centrifuged at 16 000g for 5 min at RT, and the supernatants (95% vol) were recovered. Acetone Precipitation. The reaction mixture was neutralized with 20% acetic acid. A 13.3 equivalent volume of ice-cold acetone was added to each tube which was then incubated overnight at −20 °C. The pellets were recovered by centrifugation at 16 000g for 15 min at 4 °C and then washed with 200 μL of ice-cold acetone. The pellets were solubilized either in 100 μL of 50 mM Hepes (pH 7.5) or in 100 μL of H2O for a subsequent ammonium sulfate precipitation. Coupling of Cy5. A 100 molar excess of Tris (2-carboxy ethylphosphine) hydrochloride (TCEP) over the peptide was added to the reaction mixture, i.e., 1 μL of a 16 mM aqueous TCEP solution added to each 25 μL reaction mixture containing 6.5 μM peptide. The tubes were incubated for 10 min at RT under a N2 atmosphere. A 12 molar excess of Cy5mal (solution in DMF) was then added and left to incubate in the dark, under N2 atmosphere, for 2−3 h at RT, either with or without refrigeration overnight at 4 °C. The concentration of Cy5-mal solutions was measured spectrophotometrically; ε650 nm = 250 000 L·mol−1·cm−1 (ε, extinction coefficient). The reactions were quenched by the addition of 40 mols of cysteine per mole of peptide.

■

MATERIALS AND METHODS Chemicals and Biochemicals. Unless otherwise stated, the chemicals used in this study were obtained from SigmaAldrich-Fluka (St Louis, USA). CH3CN, trifluoroacetic acid (TFA), and N,N-Dimethylfomamide (DMF) Rectapurfrom VWR International Inc. were of high performance liquid chromatography (HPLC) grade. The biotinylated peptides and phosphopeptides (Table 148 and Table S-1, Supporting Information) were all purchased from Neosystem (Strasbourg, France), the biotinylated CHKtide excepted which was obtained from Upstate. Cy5-maleimide (Cy5-mal) was purchased from GE Healthcare Life Sciences. The conjugate europium cryptate-streptavidin (SA-K) was purchased from Cis Bio International (Bagnols/Cèze, France). The N-terminal glutathione S-transferase (GST) human Chk2 fusion protein expressed in SF9 insect cells, rGST-hChk2SF9, was produced and purified in the Service.37 9964

dx.doi.org/10.1021/ac3021505 | Anal. Chem. 2012, 84, 9963−9970

Analytical Chemistry

Article

Figure 1. Principle of the antibody-free detection of phospho-Ser/Thr containing peptide by HTRF. The method is based on the replacement of the phosphate group by a fluorescent moiety. The chemical transformation is achieved by alkaline β-elimination and then Michael addition of DTT. Cyanin 5, the fluorescence acceptor (λem of 665 nm), is coupled to the thiol group via Cy5-maleimide. Conjugation of the HTRF donor europiumcryptate (λexc of 337 nm) is achieved via the N-terminally biotinylated peptide and a streptavidin conjugate. The Eu(K) signal at 620 nm serves as an internal control, and the ratio of counts at 665 nm to the counts at 620 nm is recorded as HTRF signal. Symbols: ●, biotin; Ⓟ, phosphate; sun shape in the lower position, Europium cryptate Eu(K); large X shape, streptavidin (SA); sun shape in the upper position, Cyanin 5.

HTRF Detection. The assay was performed in black 96 well microtiter plates (Corning). Two microliters of each coupling reaction was diluted into 540 μL of “reading buffer” containing 50 mM Hepes (pH 7.5), 0.1% (w/v) bovine serum albumin (BSA), and 0.4 M potassium fluoride. Fifty microliters of each diluted sample was pipetted into the corresponding wells in duplicate. SA-K was added according to the supplier’s recommendations, in a final volume of 100 μL, and the microplates were then incubated at RT before signal detection. Detection control tubes were used to validate the experiments (Supporting Information). Fluorescence was recorded on a dedicated Rubystar HTRF reader (BMG Labtechnologies, Germany), with excitation at 337 nm and simultaneous signal collection at 665 and 620 nm. The time-resolved settings were set at 50 μs for the delay and 400 μs integration time. HTRF data were processed as explained (Supporting Information). In the detection system development part, delta F1 values were associated with a “(SA-K + Cy5) control” tube used as negative control. In the application part, delta R2 values were obtained by the use of the “(no enzyme)” control tube associated with each peptide as negative control. Determination of Peptide Concentration. The concentrations of biotinylated peptides were determined via a biotin assay,50 using streptavidin-fluorescein isothiocyanate (SAFITC) (Perbio, France). D-biotin standards and the samples were all diluted in 50 mM Hepes buffer (pH 7.5) containing 0.1% BSA. Fifty microliters was distributed into a clear microtiter plate in duplicate. After addition of 150 μL of SA− FITC working dilution and a 5−10 min incubation, fluorescence was measured on a Labtech FLUOstar Optima reader (BMG Labtechnologies, Germany), λexc (excitation wavelength) of 485 nm, and λem (emission wavelength) of 520 nm. HPLC Analysis. After the recovery step, the reaction products were separated by reverse phase-HPLC (C-18 Lichrospher, Merck) using an Agilent Series 1100 apparatus

piloted by HPCHEM software. A linear gradient from 0 to 30% B at 1.2% B/min was used at a flow rate of 1 mL/min. Solvent A was 0.1% TFA in water, and solvent B was 80% CH3CN and 0.075% TFA in water. Absorbance was monitored at 210 nm.

■

RESULTS AND DISCUSSION Presentation of the System. The principle of the antibody-free phosphopeptide detection by HTRF, depicted in Figure 1, involves N-terminally biotinylated peptides and the widely used chemical conversion of phospho-Ser/Thr residues19,21,23 into suitable fluorescent derivatives. DTT was selected as the nucleophilic reagent in the Michael addition in order to introduce the thiol group as useful for the acceptor coupling step. Cyanine 5 (Cy5) was selected as the acceptor group for two major reasons; its absorption spectrum displays a favorable overlap with the emission spectrum of europium, and a highly reactive maleimide-activated reagent is commercially available. The reagents for the HTRF technology are also commercially available, in particular the europium cryptatestreptavidin conjugate. Our goal was to demonstrate that a user-friendly assay can be designed and that satisfactory sensitivity and specificity can be reached after technical optimization. Optimization of the Chemical Derivatization Steps. Several factors impacted the efficiency of the chemical steps and the recovery of modified peptides. Primary and secondary hydroxyl groups, for serine and threonine, respectively, are known to react differently during β-elimination or Michael addition reaction.49 The conditions were thus experimentally adjusted using bK6RpS, bK6RpT, and bP14D model peptides (where bK6R is biotinylated K6R peptide, pS is phosphoserine, and pT is phosphothreonine; Table 1). The chemical reactions were evaluated by high performance liquid chromatography on reverse-phase C18 column. β-Elimination Reaction (Step 1 in Scheme S-1, Supporting Information). The influences of the alkaline conditions and the reaction time were tested (Figure 2). The 9965

dx.doi.org/10.1021/ac3021505 | Anal. Chem. 2012, 84, 9963−9970

Analytical Chemistry

Article

Figure 2. HPLC analysis of the Ba(OH)2 role on β-elimination reaction. The reactions were carried out for 1.5 h at 37 °C on either bK6RpS (A), bK6RpT (B), or bP14D (C) in the presence of various molar excesses of Ba(OH)2: 366-fold (dotted line); 5500-fold (solid line). (D) HPLC analysis of the β-elimination reaction performed on bK6RpT for a 3 h incubation time at 37 °C under a N2 atmosphere.

best results were obtained in the presence of 5500 molar excess of Ba(OH)2. The conversion of bK6RpS (Figure 2A) to its corresponding dehydro-alanyl derivative to near completion required 1.5 h of incubation time with this excess reagent. For bK6RpT (Figure. 2B), a slower conversion rate was observed, which thus required longer reaction time. A further improvement of the transformation yield was obtained by removing oxygen during the reaction, for example, by performing the reaction under N2 atmosphere for 3 h (Figure 2D). For both bK6RpS and bK6RpT, higher temperatures, higher concentrations of Ba(OH)2, or longer incubation times all resulted in the formation of byproducts (data not shown). The best compromise was then set at 5500 molar excess of Ba(OH)2 and 3 h of reaction time at 37 °C under N2 atmosphere. As a control reaction, the unphosphorylated peptide bP14D (Figure 2C) was not modified under these harsh conditions. β-Elimination/Addition Reactions (Scheme S-1, Supporting Information). DTT was selected as a bifunctional nucleophile in the Michael addition reaction to enable the introduction of a thiol group in place of the former phosphate group. In a first stage, the best conditions for DTT (molar excess and reaction time) were optimized in a one-step procedure, carried out with all the reagents added to the reaction tube from the beginning. The conversion was assessed by C-18 RP-HPLC; the results obtained with peptide bK6RpT are reported in Figure 3. No reaction was observed in the presence of the 55 000 molar excess of DTT, the conditions suggested by Byford,49 whereas an efficient conversion occurred in the presence of 1400-fold molar excess of DTT. Thus, an excess of reducing agent might inhibit the β-elimination reaction. A minor side reaction product (peak 3) but nearly half of the peptide could be converted into the thiol-containing derivative (peak 2) after a 5 h reaction time at 37 °C. The β-elimination/addition reactions could also be carried out in a two-step procedure by consecutive addition of the reagents. We monitored the impact of withdrawing excess barium on the subsequent reaction. The transformation rates were estimated by HPLC to be about 55%, for the all-in-the-pot procedure, and in the two-step procedure to be about 80% or 40% when Ba(OH)2 was or was not removed, respectively

Figure 3. HPLC analysis of a one-step procedure for β-elimination/ Michael addition reactions. Six nmoles of bK6RpT were incubated for either 1.5 h (A), 3 h (B), or 5 h (C), in the presence of two different molar excesses of DTT: 1400 (solid line) or 50 000 (dotted line). All reactions were performed in 200 μL of final volume, with 5500 molar excess of Ba(OH)2. Peaks 1, 2, and 3 correspond to the native peptide, to its thiol derivative, and to a side reaction product, respectively. The percentage for each species was estimated from their relative peak heights in the presence of 1400 molar excess DTT.

(Figure S-1, Supporting Information). In this latter condition, a large fraction of the peptide was lost during the barium precipitation step. Moreover, the peaks identified as reaction products in this approach were at different retention times in the HPLC profile compared with those formed after the two other types of reaction. The overall transformation rate was 9966

dx.doi.org/10.1021/ac3021505 | Anal. Chem. 2012, 84, 9963−9970

Analytical Chemistry

Article

Figure 4. Influence of reaction time and pH on Cy5 coupling. The reaction time was tested at pH 7.5 on several peptides: bY14L, as positive control, and bP14D, as negative control on panel A; bK6RpSrec, bK6RpTrec, and bP14DTrec, as peptides recovered from the chemical modification media on panel B. The following reaction times were tested: 1 h (black bars), 2 h (dark gray bars), 2.75 h (light gray bars), 2.75 h + overnight at 4 °C (polkadot bars), 5 h + overnight at 4 °C (hatched bars). Panel C displays the comparative results obtained for bY14L, bP14D, and bK6RpTrec after Cy5mal coupling for 2.75 h either at pH 7.5 (light gray bars) or at pH 6.0 (lined horizontal bars).

(NH 4 ) 2 SO 4 over Ba(OH) 2 for reaction on indirectly phosphorylated peptide, i.e., after a kinase reaction. The relative volumes of acetone over sample and the pH, which could both affect the precipitation efficiency, were also evaluated. A good recovery was observed with up to 13 volumes of acetone over that of the sample, whatever the pH. For instance, the recovery of bK6RpS exceeded ∼90%, whenever it was or was not subjected to the chemical reactions, at the two ratios of 10 to 13 volumes of acetone over the sample. However, it rapidly decreased to 39% or 20% at ratios of 8 or 3. Regarding pH, it could be noticed that the yield was equivalent under all the conditions tested, i.e., neutral, acidic, and basic buffer. The order for addition of the two agents (i.e., ammonium sulfate then acetone or the opposite) was tested on bK6RpT and bK6RpS, just after they had undergone βelimination/Michael addition reactions. The results were similar whatever the order (data not shown). Coupling of the Fluorophor. The free sulfhydryl group incorporated into the peptide via the addition of DTT can react with a maleimide-activated fluorophor, Cy5-mal. It is one of the most commonly used conjugation methods due to both its efficiency and site specificity.51 The coupling efficiency was estimated using HTRF detection. The signal obtained for the positive control bY14L (Table 1) increased with the amount of added Cy5-mal (Figure 4A). The signal did not significantly change between 1 and 5 h reaction time at room temperature (RT) but largely increased following an overnight incubation at 4 °C. As expected, the negative control bP14D did not give rise

considered as satisfactory in the simplest one-step procedure that was then kept in the final process. The final reaction product (peak 2) was characterized by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The 56 Da increment with regard to the initial molecular weight of the peptide confirmed the elimination of the phosphate group and addition of DTT (data not shown). Product Recovery. The reaction with Cy5-maleimide (Cy5mal) requires intermediate purification of the thiol-containing peptide (removal of excess DTT that would interfere, pH buffer change, ...). In order to handle multiple samples simultaneously, the use of chromatography purification was deliberately avoided. Dialysis was also not considered due to the low molecular weight of the peptides. Purification by selective precipitation was therefore preferred, involving two complementary agents: (NH4)2SO4 in order to remove excess barium and acetone for peptides (DTT is soluble in acetone and is then eliminated in the supernatant phase). A fluorescent assay for biotin was used to monitor the peptide recovery (see Materials and Methods section). This assay is very sensitive to the sample composition and especially to the pH. However, tests performed with samples contaminated with up to 50% of DTT showed no impact on the assay. On the contrary, ammonium sulfate was shown to interfere with the biotin assay. To avoid the presence of residual salts and potential pH issues, an equal molarity of (NH4)2SO4 and Ba(OH)2 was used for the direct chemical derivatization and only 10-fold excess of 9967

dx.doi.org/10.1021/ac3021505 | Anal. Chem. 2012, 84, 9963−9970

Analytical Chemistry

Article

Figure 5. HTRF analysis of peptides phosphorylated in vitro by rGST-hChk2SF9. The fluorescence data were processed from photon counts at 620 and 665 nm, using the following formulas: R = (cps665 nm/cps620 nm) × 104 and Delta R2 = (Rsample − Rno enzyme). The experimental conditions were the following ones. (A) For peptides derived from known in vivo substrates of Chk2: 1 = “blind 12X” (see text) with coupling for 2 h at RT plus ON 4 °C before cysteine addition (vertical hyphen bars); 2 = Cy5-mal “blind 12X” with coupling for 2 h at RT plus ON 4 °C no cysteine addition (white bars); 3 = Cy5-mal 6-fold molar excess with coupling for 3 h at RT no cysteine addition (tiled bars); 4 = Cy5-mal 6-fold molar excess with coupling for 3 h at RT + ON 4 °C before cysteine addition (gray bars); 5 = Cy5-mal 6-fold molar excess with coupling for 3 h at RT + ON 4 °C no cysteine addition (black bars). (B) For peptides derived from Chk2 consensus substrate sequence: 1 = chemistry 4 h/Cy5-mal 12-fold molar excess with coupling for 3 h at RT before cysteine addition (white bars); 2 = chemistry 5 h/Cy5-mal 12-fold molar excess with coupling for 3 h at RT before cysteine addition (black bars); 3 = 1 with overnight revelation (tiled bars); 4 = 2 with overnight revelation (vertical hyphen bars).

1 by convention (recommendation of the supplier). Various ratios of biotinylated peptide over SA-K were tested, ranging from 32/1 to 1/1 (Figure S-2A, Supporting Information). The best signals were obtained for the 4/1 and 8/1 ratios, due to the lower amount of peptide and to the deficit of streptavidin at high ratio. Another set of experiments was carried out by testing a fixed amount of biotinylated peptide with varying SAK, over a range from 8/0.25 to 8/2 (Figure S-2B, Supporting Information). Excess of SA-K (lowest ratio) gave rise to weaker signals due to a high emission at 620 nm. The best result was obtained at the 8/1 ratio. In all the conditions tested, the dynamic transfer was found to be negligible. The specificity of the fluorescence signal was attested by the signal increase in correlation with the tested reaction volume.

to any HTRF signal. Model peptides recovered from the derivatization reactions were also assayed (Figure 4B). Satisfactory results were obtained for bK6RpTrec and bK6RpSrec (where rec is recovered) after a 5 h incubation and, as expected, bP14Drec stayed negative. The coupling at pH 6, compared with that at pH 7.5, led to weaker values (Figure 4C). Final Setup of the HTRF Assay. The HTRF assay was developed in black 96-well plate format. Thanks to the high affinity of streptavidin,52,53 the signal is largely specific for the peptide, but a nonspecific fluorescent signal, called the dynamic transfer signal, may also occur. It results from the colliding of free Cy5 and SA-K, and it is therefore strongly dependent on their respective concentrations. The conditions leading to the best compromise have to be selected. The dilution of SA-K generating a HTRF signal at about 25 000 cps at 620 nm equals 9968

dx.doi.org/10.1021/ac3021505 | Anal. Chem. 2012, 84, 9963−9970

Analytical Chemistry

Article

DTT Contaminant. After the recovery step, unreacted DTT might compete with the peptide for the maleimide coupling reaction. The choice of precipitation steps was dictated by the objective of performing parallel treatments of multiple samples. Satisfactory results were obtained that could however be improved. Centrifugation using spin column might be an efficient alternative. Side Reaction at Serine and/or Threonine. During βelimination, an undesired dehydration reaction might occur at the level of free hydroxyl group of serine and threonine.27 This reaction largely depends on experimental parameters such as OH− concentration, temperature, reaction time, and peptide composition. This would lead to illegitimate incorporation of Cy5 and to nonspecific FRET signals. In our system, the use of the “unphosphorylated control tube”, “no enzyme”, as negative control enables any possible side reaction to be taken into consideration. Processing the fluorescence data to get delta R2 prevented the risk for side reaction signal. Side Reaction at Other Reacting Groups. In the case of a peptide sequence containing a thiol group or any amino acid residue susceptible to being modified during the chemical reactions, an upstream protection step must be included in the procedure. Rigorous controls to take into account the potential labeling of nonphosphorylated residues should be planned and considered in the data processing.

Performance of the Whole System. Obviously, the global performance depends mainly on the concentration of phosphopeptide and on the level of phosphorylation. The system was first tested on fully phosphorylated peptides at concentrations varying from 0.6 to 60 μM (i.e., 60 to 6000 pmoles in the 100 μL reaction volume). The results of HTRF detection (in the above-defined conditions) indicate that the chemical derivatization was detectable for reactions performed on 100% phosphothreonine peptide at 6 μM (0.6 nmoles). The feasibility of the chemical reactions depending on the phosphorylation rate was also evaluated. The experiment was carried out by diluting model phosphopeptides with a solution of nonphosphorylated peptide (bP14D), in order to keep the biotin concentration constant. The corresponding solutions corresponded to 5%, 10%, 20%, and 100% phosphorylation, respectively (data not shown). The results showed that the system works properly for as low as 5% in the case of the pSer model peptide. Application to the Analysis of Chk2 Substrate Peptides. To investigate the applicability of the approach in the context of kinase characterization, several Chk2 kinase substrate mimics were analyzed (Table S-1, Supporting Information). Chk2 was used in the form of rGST-Chk2SF9, a recombinant fusion protein expressed in Sf9 insect cells, previously shown to be autoactivated.37 The peptides bK23R, bG12L, and bP14D, deriving, respectively, from Cdc25C (AC P30307) or CdC25A (AC P30304) were first tested. The first one, bK23R, corresponds to the commercial biotin conjugated CHKtide, a substrate peptide for routine screening for Chk2 activity which we already assayed in a first generation of HTRF assay.37 CHKtide was introduced as positive control, for the kinase reaction, and its phosphorylated form, bK23RpS, was introduced as control for the chemical reaction. The yield of in vitro phosphorylation is certainly far from 100%, generating partially phosphorylated peptides. This explains the low signals observed, much lower than those previously obtained for the fully phosphorylated peptides. Positive responses were nevertheless obtained for the three peptides (Figure 5A). Delta R2 varied from 100 to 720 for bK23R and from 100 to 300 for bG12L, a shorter sequence than bK23R. No signal was obtained in the case of a “blind 12X” experiment with bP14D, where the coupling step was directly performed without checking the peptide concentration. A FRET signal was obtained by taking into account the recovery yield and lowering the theoretical Cy5-mal molar excess to 6-fold. The system was then applied to a set of peptides previously selected as putative Chk2 substrate on the basis of a consensus substrate sequence of Chk247,54 and of their involvement in the DNA damage process. Noteworthily, these peptides were already used in a [γ32P]ATP assay.39 The detection system detected phosphorylation for Cdc25C, Brca1, Xrcc9 (X-ray repair cross-complementing protein 9), and Smc3 (structural maintenance of chromosomes 3) biotinylated peptides (Figure 5B). The results correlated well with those obtained in the radioactive assay and similarly failed in detecting any phosphorylation for the p53-derived peptide. It has indeed been shown that p53 recruitment as substrate occurs via remote docking sites,55 a structure the peptide cannot reproduce. Analysis of Side Reactions. Side reactions in the chemical process were reported before (e.g., Figures 3 and 4). They had to be characterized since they might lead to either false positive or false negative HTRF signals.

■

CONCLUSIONS Assessment of phosphorylation is a major challenge to analytical chemists and drug discoverers. Although MS methods have shown their potential, the need for robust, affordable, and straightforward methods is still there. Our approach meets a large part of these criteria and furthermore affords automation. The proposed system does not require radioactivity or antibody and targets pSer/pThr-containing peptides. The treatment of multiple samples simultaneously could be of great use for large in vitro studies of kinase substrates or kinases themselves.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Address: CEA, DSV, IBEB, SBTN, Centre de Marcoule, BP 17171, F-30207 Bagnols-sur-Cèze, France. E-mail: beatrice. [email protected]. Tel: +33-466-791-915. Present Address §

CEA, Direction des sciences du vivant, F-92265 Fontenay-auxroses Cedex 05, France. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Pablo Hö cht for technical assistance in the development of the detection system and also Alain Dedieu and Isabelle Dany for mass spectroscopy analyses.

■

REFERENCES

(1) Ma, H.; Deacon, S.; Horiuchi, K. Expert Opin. Drug Discovery 2008, 3, 607−621. (2) Bosworth, N.; Towers, P. Nature 1989, 341, 167−168.

9969

dx.doi.org/10.1021/ac3021505 | Anal. Chem. 2012, 84, 9963−9970

Analytical Chemistry

Article

(3) Cook, N. Drug Discovery Today 1996, 1, 287−294. (4) Bazin, H.; Trinquet, E.; Mathis, G. J. Biotechnol. 2002, 82, 233− 250. (5) Mathis, G. Clin. Chem. 1995, 41, 1391−1397. (6) Greg, W.; Chantal, I.; Liliana, P.; Philippe, R.; Roger, B. Curr. Med. Chem. 2004, 11, 721−730. (7) Hemmila, I. I. J. Biomol. Screening 1999, 4, 303−308. (8) Gaudet, E. A.; Huang, K. S.; Zhang, Y.; Huang, W.; Mark, D.; Sportsman, J. R. J. Biomol. Screening 2003, 8, 164−175. (9) Seethala, R.; Menzel, R. Anal. Biochem. 1998, 255, 257−262. (10) Horiuchi, K. Y.; Wang, Y.; Diamond, S. L.; Ma, H. J. Biomol. Screening 2006, 11, 48−56. (11) Shults, M. D.; Janes, K. A.; Lauffenburger, D. A.; Imperiali, B. Nat. Methods 2005, 2, 277−283. (12) Sun, L.; Liu, D.; Wang, Z. Anal. Chem. 2007, 79, 773−777. (13) Andersson, L.; Porath, J. Anal. Biochem. 1986, 154, 250−254. (14) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Nat. Biotechnol. 2002, 20, 301−305. (15) Thingholm, T. E.; Jensen, O. N. Methods Mol. Biol. 2009, 527, 47−56. (16) Kweon, H. K.; Hakansson, K. J. Proteome Res. 2008, 7, 749−755. (17) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen, T. J. Mol. Cell. Proteomics 2005, 4, 873−886. (18) Amoresano, A.; Marino, G.; Cirulli, C.; Quemeneur, E. Eur. J. Mass Spectrom. (Chichester, Engl.) 2004, 10, 401−412. (19) Brittain, S. M.; Ficarro, S. B.; Brock, A.; Peters, E. C. Nat. Biotechnol. 2005, 23, 463−468. (20) Goshe, M. B.; Conrads, T. P.; Panisko, E. A.; Angell, N. H.; Veenstra, T. D.; Smith, R. D. Anal. Chem. 2001, 73, 2578−2586. (21) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379− 382. (22) Qian, W. J.; Goshe, M. B.; Camp, D. G., 2nd; Yu, L. R.; Tang, K.; Smith, R. D. Anal. Chem. 2003, 75, 5441−5450. (23) Zhou, H.; Watts, J. D.; Aebersold, R. Nat. Biotechnol. 2001, 19, 375−378. (24) Amoresano, A.; Monti, G.; Cirulli, C.; Marino, G. Rapid Commun. Mass Spectrom. 2006, 20, 1400−1404. (25) Chen, M.; Su, X.; Yang, J.; Jenkins, C. M.; Cedars, A. M.; Gross, R. W. Anal. Chem. 2010, 82, 163−171. (26) Li, W.; Boykins, R. A.; Backlund, P. S.; Wang, G.; Chen, H. C. Anal. Chem. 2002, 74, 5701−5710. (27) McLachlin, D. T.; Chait, B. T. Anal. Chem. 2003, 75, 6826− 6836. (28) Amoresano, A.; Cirulli, C.; Monti, G.; Quemeneur, E.; Marino, G. Methods Mol. Biol. 2009, 527, 173−190. (29) Blackburn, K.; Goshe, M. B. Briefings Funct. Genomics Proteomics 2009, 8, 90−103. (30) Tsumoto, H.; Ra, M.; Samejima, K.; Taguchi, R.; Kohda, K. Rapid Commun. Mass Spectrom. 2008, 22, 965−972. (31) Simpson, D. L.; Hranisavljevic, J.; Davidson, E. A. Biochemistry 1972, 11, 1849−1856. (32) Meyer, H. E.; Hoffmann-Posorske, E.; Korte, H.; Heilmeyer, L. M., Jr. FEBS Lett. 1986, 204, 61−66. (33) Alpha, B.; Lehn, J. M.; Mathis, G. Angew. Chem., Int. Ed. Engl. 1987, 26, 266−267. (34) Förster, T. Ann. Phys. 1948, 2, 55−75. (35) Stryer, L. Annu. Rev. Biochem. 1978, 47, 819−846. (36) Jia, Y.; Quinn, C. M.; Gagnon, A. I.; Talanian, R. Anal. Biochem. 2006, 356, 273−281. (37) Gabant, G.; Lorphelin, A.; Nozerand, N.; Marchetti, C.; Bellanger, L.; Dedieu, A.; Quemeneur, E.; Alpha-Bazin, B. J. Mol. Biol. 2008, 380, 489−503. (38) Abraham, R. T. Genes Dev. 2001, 15, 2177−2196. (39) Bartek, J.; Falck, J.; Lukas, J. Nat. Rev. Mol. Cell Biol. 2001, 2, 877−886. (40) Melo, J.; Toczyski, D. Curr. Opin. Cell Biol. 2002, 14, 237−245. (41) Bartek, J.; Lukas, J. Cancer Cell 2003, 3, 421−429.

(42) Lee, J. S.; Collins, K. M.; Brown, A. L.; Lee, C. H.; Chung, J. H. Nature 2000, 404, 201−204. (43) Falck, J.; Mailand, N.; Syljuasen, R. G.; Bartek, J.; Lukas, J. Nature 2001, 410, 842−847. (44) Falck, J.; Petrini, J. H.; Williams, B. R.; Lukas, J.; Bartek, J. Nat. Genet. 2002, 30, 290−294. (45) Blasina, A.; Price, B. D.; Turenne, G. A.; McGowan, C. H. Curr. Biol. 1999, 9, 1135−1138. (46) Matsuoka, S.; Huang, M.; Elledge, S. J. Science 1998, 282, 1893− 1897. (47) Seo, G. J.; Kim, S. E.; Lee, Y. M.; Lee, J. W.; Lee, J. R.; Hahn, M. J.; Kim, S. T. Biochem. Biophys. Res. Commun. 2003, 304, 339−343. (48) Molloy, M. P.; Andrews, P. C. Anal. Chem. 2001, 73, 5387− 5394. (49) Byford, M. F. Biochem. J. 1991, 280 (Pt 1), 261−265. (50) Rao, S. V.; Anderson, K. W.; Bachas, L. G. Bioconjugate Chem. 1997, 8, 94−98. (51) Kim, Y.; Ho, S. O.; Gassman, N. R.; Korlann, Y.; Landorf, E. V.; Collart, F. R.; Weiss, S. Bioconjugate Chem. 2008, 19, 786−791. (52) Green, N. M. Methods Enzymol. 1990, 184, 51−67. (53) Wilchek, M.; Bayer, E. A. Biomol. Eng. 1999, 16, 1−4. (54) O’Neill, T.; Giarratani, L.; Chen, P.; Iyer, L.; Lee, C. H.; Bobiak, M.; Kanai, F.; Zhou, B. B.; Chung, J. H.; Rathbun, G. A. J. Biol. Chem. 2002, 277, 16102−16115. (55) Craig, A.; Scott, M.; Burch, L.; Smith, G.; Ball, K.; Hupp, T. EMBO Rep. 2003, 4, 787−792.

9970

dx.doi.org/10.1021/ac3021505 | Anal. Chem. 2012, 84, 9963−9970