Coupling of MALDI-TOF Mass Analysis to the Separation of

CNRS EP 103, 4 Place Jussieu, Bât F, Boite 45, F-75005 Paris Cedex 05, France. Extraction of biotinylated peptides by streptavidin mag- netic beads h...
0 downloads 0 Views 295KB Size
Download PDF
Anal. Chem. 1996, 68, 2122-2126

Coupling of MALDI-TOF Mass Analysis to the Separation of Biotinylated Peptides by Magnetic Streptavidin Beads S. Girault,† G. Chassaing,† J. C. Blais,‡ A. Brunot,‡ and G. Bolbach*,‡

Laboratoire de Chimie Organique et Biologique, Universite´ Pierre et Marie Curie, CNRS URA 493, 4 Place Jussieu, F-75005 Paris Cedex 05, France, and Laboratoire de Chimie Structurale Organique et Biologique, Universite´ Pierre et Marie Curie, CNRS EP 103, 4 Place Jussieu, Baˆ t F, Boite 45, F-75005 Paris Cedex 05, France

Extraction of biotinylated peptides by streptavidin magnetic beads has been directly coupled to the MALDI-TOF mass analysis. The elution of peptides from the beads is achieved by first mixing the beads with the MALDI matrix solution and removing, after a few minutes, the beads with a magnet; then, the matrix solution containing the biotinylated peptide is directly mass analyzed by MALDI. Three examples are presented to show the capabilities of this procedure to detect biotinylated peptides present at very low concentrations in complex mixtures. Detection limits of less than 100 fmol can be achieved. Such a coupling strategy is of great interest to investigate peptide/ protein interactions. Matrix-assisted laser desorption ionization time-of-flight spectrometry (MALDI-TOFMS) has become a powerful tool to determine the molecular mass of biomolecules.1 Its capability in the high-mass range and its sensitivity in the femtomole to picomole range are particularly attractive for various applications in biology and biochemistry.2 Classically, analyte molecules are mixed together with UV-absorbing molecules (matrix) and irradiation by UV laser photons leads to evaporation and partial ionization of matrix and analyte molecules. For pure analytes, a simple preparation of the target known as the dried-droplet method is particularly efficient.3 However, the presence of contaminants (solvents, buffers, salts, etc.), as generally present in biological solution, can hamper the MALDI analysis. In spite of some progress in target preparation procedures,3,4 the analysis of mixtures is still difficult, especially the detection of species in low concentration. For this reason, new strategies have been proposed by Hutchens and Yip to selectively capture a given compound from a complex biological solution.5 The so-called surface-enhanced affinity capture (SEAC), composed of singlestranded DNA covalently immobilized onto agarose beads, was used by these authors to capture lactoferrin from an infant urine sample and to detect it by MALDI after mixing the SEAC device with the matrix.5 From this pioneering work, various SEAC devices have been designed to capture antigens, antibodies, and †

CNRS URA 493. CNRS EP 103. (1) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 24, 1193A-202A. (2) Siuzdak, G. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11290-7. (3) Xiang, X.; Beavis, R. C. Rapid Commun. Mass Spectrom. 1994, 8, 199-204. (4) Vorm, O.; Roespstorff, P.; Mann, M. Anal. Chem. 1994, 66, 3281-7. (5) Hutchens, T. W.; Yip, T. T. Rapid Commun. Mass Spectrom. 1993, 7, 57680. ‡

2122 Analytical Chemistry, Vol. 68, No. 13, July 1, 1996

metal-binding proteins by, respectively, antibodies,6,7 antigens,8 and metal ions8 immobilized onto agarose beads. The aim of this study was to develop a general methodology allowing a direct analysis of peptide/protein (enzymes, receptors, antibodies) interactions in cell culture. The peptidic substrate, ligand or antigen, must bear a photoreactive group and a biotinyl group. Photolabeling allows one to covalently bound peptide and protein. After enzymatic and chemical degradation of this complex, the photolabeled fragments (expected quantity in the low-picomole range) must be selectively extracted from the complex mixture and analyzed by MALDI mass spectrometry. Among all purification systems, affinity chromatography via the complex between biotin and streptavidin or avidin (Kd ) 10-15 M) has been widely explored.9 Using either the classical chromatography columns or magnetic beads, the elution step of the biotinyl group from the (strept)avidin is not always efficient. Most often, the advantage of streptavidin-coated magnetic beads has been demonstrated when elution from the beads was not necessary. Single-stranded DNA molecules have been purified by means of their complementary biotinylated oligonucleotide attached on streptavidin-coated magnetic beads.10 The separation of both strands occurs by a change of salinity.10 The beads can be directly analyzed by MALDI or after incubation with the matrix; the supernatant is analyzed by MALDI.11 However, in both cases the biotinylated single strand was not desorbed in MALDI.11 A method has described the removal of biotinylated DNA molecules from the beads under drastic conditions using formamide,12 which is difficult to eliminate and not always compatible with proteins (problems of aggregation, for instance). A disulfide bridge has been introduced in a biotinylated photoactive peptide to allow its recovery by just reducing this bridge, which avoids the elution of the biotinyl group from the beads. But the yield of recovery was very low because of the presence of endogeneous reductors in biological mixtures.13 In this paper, we demonstrate the validity of the methodology described above by investigating the conditions of the three last (6) Papac, D. I.; Hoyes, J.; Tomer, K. B. Protein Sci. 1994, 3, 1485-92. (7) Nelson, R. W.; Krone, J. R.; Bieber, A. L.; Williams, P. Anal. Chem. 1995, 67, 1153-8. (8) Papac, D. I.; Hoyes, J.; Tomer, K. B. Anal. Chem. 1994, 66, 2609-13. (9) Henrikson, K. P.; Allen, S. H. G.; Maloy, W. L. Anal. Biochem. 1979, 94, 366-70. (10) Wahlberg, J.; Lundeberg, J.; Hultman, T.; Uhle´n, M. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 6569-73. (11) Tang, K.; Fu, D.; Ko ¨tter, S.; Cotter, R. J.; Cantor, C. R.; Ko ¨ster, H. Nucleic Acids Res. 1995, 23, 3126-31. (12) Tong, X.; Smith, L. M. Anal. Chem. 1992, 64, 2672-7. S0003-2700(96)00043-1 CCC: $12.00

© 1996 American Chemical Society

Figure 1. Principle of the extraction of a biotinylated peptide from a mixture using streptavidin magnetic beads.

steps: (i) the conditions of a rapid and efficient extraction of biotinylated peptides (biotinylsulfone with Kd ) 10-13 M14 ) using streptavidin-coated magnetic beads, (ii) the conditions to retrieve all the trapped peptides with the regular MALDI matrix, and (iii) the detection limit for such a characterization in MALDI. EXPERIMENTAL SECTION Peptide Synthesis. The analogs of substance P (SP, ArgPro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2), [(2S,3R)Ing7] SP, [Flg8] SP, [Dip8] SP, [Pro9] SP, [biotinyl sulfone-Apa0, p-benzoylPhe8] SP, [biotinyl sulfone-Apa0, p-benzoyl-Phe8,Pro9] SP were synthesized by a standard Boc solid-phase synthetic strategy.15 A general protocol was used for the biotinylation of peptides.16 The peptides were purified by reversed-phase high-performance liquid chromatography and analyzed by MALDI mass spectrometry. For the sake of simplicity, these peptides will be referred to respectively as P1 (MW ) 1378), P2 (MW ) 1388), P3 (MW ) 1423), P4 (MW ) 1468), P5 (MW ) 1809), P6 (MW ) 1849). Magnetic Beads. Streptavidin-coupled magnetic beads (Dynabeads M-280, Dynal) were supplied as a suspension of (6-7) × 108 beads/mL (10 mg of beads/mL, bead diameter ∼2.8 µm) in a phosphate-buffered saline solution containing 0.1% BSA and 0.02% NaN3. Before use, the beads were washed twice in 50 mM Tris-HCl (pH 7.4) and 0.1% BSA to remove NaN3. These beads are reported to possess 300 pmol of biotin-binding sites/mg. The magnetic particle concentrator (Dynal AS) was used to immobilize the beads during the supernatant removal and washing steps. CNBr Digestion. A 1 mg sample of proteins (BSA or proteins of membranes from Chinese hamster ovary cells) were treated overnight with 5% 2-mercaptoethanol (v/v), 2% SDS (w/v) in 50 mM Tris-HCl (pH 8) at room temperature. They were reacted (13) Ahmed, A. R. H.; Olivier, G. W. J.; Adams, G.; Erskine, M. E.; Kinsman, R. G.; Branch, S. K.; Moss, S. H.; Notarianni, L. J.; Pouton, C. W. Biochem. J. 1992, 286, 377-82. (14) Green, N. M. In Methods in Enzymology XVIII; Mc Cormick, B., Wright, L. D., Eds.; Academic Press, Inc.: New York, 1970; pp 418-30. (15) Berlose, J. P.; Convert, O.; Brunissen, A.; Chassaing, G.; Lavielle, S. Eur. J. Biochem. 1994, 225, 827-43. (16) Lavielle, S.; Chassaing, G.; Beaujouan, J. C.; Torrens, Y.; Marquet, A.; Glowinski, J. Int. J. Pept. Protein Res. 1984, 381-7.

with a few crystals of CNBr in 70% (v/v) aqueous formic acid. The mixture was incubated for 24 h at 23 °C in the dark, under oxygen-free nitrogen. Protein fragments were concentrated in a Speed Vac apparatus (RC 10.10, Jouan) after addition of 10 µL of glycerol and 2 × 100 µL of water. Magnetic Streptavidin Affinity Purification of the Biotinylated Peptide. In the case of a synthetic mixture of five peptides P1-P5 wherein one was biotinylated (P5), 200 µg of beads was mixed with 6 pmol of each peptide in 10 µL of 50 mM Tris-HCl (pH 7.4) and 1 mM DTT for 15 min. The beads were washed either twice with 40 µL of Tris buffer and twice with 40 µL of water or with the washing procedure described below. In the case of the biological mixture obtained after CNBr fragmentation of 1 mg of BSA or membranes, 100 µg of beads was mixed in a 1.5-mL Eppendorf tube with 100 µL of this complex mixture containing 0.5 pmol (or less) of the biotinylated peptide P6 in 100 µL of 50 mM Tris-HCl (pH 7.4), 0.02% BSA, 0.2 M NaCl, 0.2% Tween-20, and 1 mM DTT. This mixture was gently mixed at room temperature. After 30 min, 100 µL of water was added for 30 min more. Washing Procedure. After incubation and immobilization of the beads with the magnetic Concentrator, the supernatant was removed. The beads were subjected to several cycles of washing using the following buffers: two times 100 µL of 50 mM Tris-HCl (pH 7.4), 1 mM DTT, and 0.1% BSA (buffer A); 200 and 100 µL of buffer A + 0.1% SDS, 200 and 100 µL of buffer A + 1 M NaCl, 200, 100, and 50 µL of water with 1 mM DTT. Elution of the Biotinylated Peptide from the Beads. After washing, 6 µL of free biotin (potassium salt, 200 pmol) in water was added to the beads for 15 min at room temperature to saturate streptavidin sites. After a final washing of the beads with water, 2 µL of matrix was added to elute the peptides from the beads. After 15 min of incubation, 1 µL of the bead-free supernatant was deposited on the target for MALDI/TOF analysis. Mass Spectrometry. Mass spectra were acquired on an inhouse built linear TOF mass spectrometer previously described.17 (17) Blais, J. C.; Tessier, M.; Bolbach, G.; Remaud, B.; Rozes, L.; Guittard, J.; Brunot, A.; Mare´chal, E.; Tabet, J. C. Int. J. Mass Spectrom. Ion Processes 1995, 144, 131-8.

Analytical Chemistry, Vol. 68, No. 13, July 1, 1996

2123

Briefly, the light of a pulsed nitrogen laser (VSL 337ND, Laser Science Inc.; λ ) 337 nm, pulse duration 3 ns, energy 250 µJ) is focused onto the target (laser spot ∼180 µm in diameter on the target). The laser energy is adjusted using a circular neutral filter (Raynard Corp.), and the secondary ion acceleration voltage is in the 20-24-kV range. The ion detector is a Venetian blind secondary electron multiplier (Thorn-EMI, EM 643). The output signal was digitized by a digital oscilloscope (Lecroy 9450), and the data were transferred to a Mac II ci computer for mass spectra summation, mass calibration, and storage. Sinapinic acid (Aldrich Chemical) and R-cyano-4-hydroxycinnamic acid (Sigma Chemical) were used as matrices with respective concentrations 10-1 and 5 × 10-2 M in acetonitrile/H2O (4:1, v/v). As mentioned above, 1 µL of the bead-free supernatant (matrix and biotinylated peptide) was deposited onto a stainless steel substrate (5 mm in diameter; cleaned by sonification for 15 min in 1:1:1 formic acid/ethanol/water). RESULTS AND DISCUSSION Preliminary studies were conducted to analyze a pure biotinylated peptide trapped onto streptavidin beads. The direct MALDI analysis of beads is possible and the peptide is detected (in the picomole range) as well as streptavidin (mainly as a monomer). However, the homogeneity of the target is low. For this reason, we tried a direct elution of the peptide by mixing the beads in an acidic solution. The simplest way, i.e., beads in matrix solution, was found to be more efficient than a two-step procedure: beads in an acidic solution (H2O/TFA 0.1% or HCl in the pH range 1-4) and mixture of the supernatant with the matrix, the beads being trapped on the walls of the Eppendorf tube with a magnet. Elution time efficiency was also studied. Fast elution, typically less than 3 min was found to be sufficient to take off the peptide, in the case of saturation of the streptavidin sites by biotinylated peptide. In the experiments reported below (nonsaturation), an elution time of 15 min was chosen; we verified after this time that no peptide could be further released from the beads by subsequent elution in the matrix solution. In addition, the intensities of the peptide peak with and without streptavidin beads are similar for the same peptide concentration on the target and the same laser energy; these results confirm that all the peptide trapped on the beads was released. When the total number of peptide molecules is much lower than the expected number of available biotin sites on the streptavidin beads, this method allows a concentration of the peptide since, after fixation onto the beads, all the peptides are then released in the 2-µL matrix solution. Under these conditions we have found that saturation of the unoccupied sites by free biotin for 15 min is more efficient to get a complete release of the peptide in the matrix solution compared to untreated beads. This fact is likely connected to the cooperative effects observed for the dissociation of the biotinylated peptide/(strept)avidin complex.18 Furthermore, in the present study we have used a biotinyl sulfone-substituted peptide, in order to enhance the dissociation of the peptide/(strept)avidin complex and then reduce the incubation time. These preliminary results define a general procedure to analyze biotinylated peptide by MALDI, as depicted in Figure 1. For a (18) Lavielle, S.; Chassaing, G.; Marquet, A. Biochim. Biophys. Acta 1983, 759, 270-7.

2124 Analytical Chemistry, Vol. 68, No. 13, July 1, 1996

Figure 2. Extraction of a biotinylated peptide from a peptide mixture. (a) Positive MALDI mass spectrum of a (P1-P5) peptide mixture; 1 µL of the peptide solution (1 pmol of each peptide/µL) and 1 µL of the matrix (R-cyano-4-hydroxycinnamic acid, 5 × 10-2 M in acetonitrile/H2O, 4:1 v/v) were mixed directly on the target. Laser energy 4.5 µJ/pulse. Homogeneous target. (b) Positive MALDI mass spectrum of 1 µL of the solution resulting from the elution of beads in sinapinic acid (2 µL of 10-1 M in acetonitrile/H2O, 4:1 v/v) (step 6 in Figure 1). Laser energy 7.5 µJ/pulse. Homogeneous target. (b′) Positive MALDI mass spectrum of 1 µL of the supernatant and 1 µL of the matrix (R-cyano-4-hydroxycinnamic acid, 5 × 10-2 M in acetonitrile/H2O, 4:1 v/v) directly mixed on the target (step 2 in Figure 1). Laser energy 4.5 µJ/pulse. Homogeneous target. (c) Positive MALDI mass spectrum of 1 µL of the solution resulting from the elution of beads in sinapinic acid (2 µL of 10-1 M in acetonitrile/H2O, 4:1 v/v); this operation corresponds to the step 6 in Figure 1 with the washing procedure. Laser energy 7.5 µJ/pulse. Homogeneous target.

pure biotinylated peptide, the ensemble of experiments done at various peptide concentrations show that the limit detection is less than 100 fmol. Extraction of a Biotinylated Peptide from a Peptide Mixture. An equimolar solution of five peptides (P1-P5) was mixed with streptavidin beads. Only the peptide P5 was biotinylated. The MALDI mass spectrum of the initial peptide mixture is shown in Figure 2a. After incubation (15 min), the beads were trapped by a magnet (step 2 in Figure 1) and the supernatant was removed. Then, the beads were treated as depicted in steps 3-5 (Figure 1). The MALDI mass spectrum corresponding to step 6 (elution of trapped peptides in the matrix solution) is shown in Figure 2b: the most abundant ion was that of the biotinylated peptide P5, appearing as the protonated molecule. Contrary to what was expected, P3 and P4 protonated molecules are also present in this spectrum, even though their relative abundances are very low with respect to that of P5. The MALDI analysis of the supernatant (1 µL of the supernatant was mixed with 1 µL of sinapinic solution on the target) shows only the nonbiotinylated

Figure 3. Extraction of a biotinylated peptide (P6) from a solution containing CNBr-cleaved BSA fragments. (a) Positive MALDI mass spectrum of the supernatant (step 2 in Figure 1); 1 µL of the supernatant and 1 µL of the matrix (sinapinic acid 10-1 M in acetonitrile/H2O, 4:1 v/v) were mixed directly on the target. Laser energy 11 µJ/pulse. Target roughly homogeneous. (b) Positive MALDI mass spectrum of 1 µL of the solution resulting from the elution of the beads (step 6 + washing procedure) in R-cyano-4-hydroxycinnamic acid (2 µL of 5 × 10-2 M in acetonitrile/H2O, 4:1 v/v). Laser energy 4.5 µJ/pulse. Homogeneous target.

peptides (P1-P4), indicating an efficient trapping of the biotinylated peptide P5. In addition, their relative intensities (Figure 2b′) are different from what was expected from the equimolar solution (Figure 1a). Lower intensity peaks associated with the peptides P3 and P4 were observed in good agreement with the appearance of these peaks in the beads eluent mass spectrum (Figure 2b). This indicates that nonselective adsorption of peptides P3 and P4 onto the streptavidin beads takes place when the beads are washed only with Tris buffer and water before the elution step. Such an effect represents a major drawback of the presented method. A washing procedure between steps 2 and 3 was investigated to eliminate nonspecific binding to the streptavidin beads; As shown in Figure 2c, the washing procedure described in the Experimental Section (two times 100 µL of 50 mM Tris-HCl (pH 7.4), 1 mM DTT, and 0.1% BSA (buffer A); 200 and 100 µL of buffer A + 0.1% SDS, 200 and 100 µL of buffer A + 1 M NaCl, 200, 100, and 50 µL of water with 1 mM DTT) proves to be very efficient, as only the biotinylated peptide was observed. This washing procedure thus eliminates nonselective adsorption on the streptavidin beads. It should be noted that using magnetic beads makes the washing procedure more efficient than using agarose beads. The spectrum in Figure 2c was obtained using only a small fraction of the beads (10 µg) corresponding to ∼150 fmol of P5 on the target. Extraction of a Biotinylated Peptide from a Mixture of CNBr-Cleaved BSA. BSA is generally used for coating test tubes used in a biological assay to avoid adsorption of peptides and proteins. A solution containing BSA (1 mg) previously cleaved by CNBr digestion and 500 fmol of the biotinylated P6 was mixed with streptavidin beads. In the initial solution (BSA fragments and biotinylated peptide) the molecular ratio (BSA fragments/ P6) can be estimated to be ∼3 × 105. These conditions cannot

Figure 4. Extraction of a biotinylated peptide (P6) in solution with CNBr-cleaved membrane proteins of CHO cells. (a) Positive MALDI mass spectrum of the supernatant (step 2 in Figure 1); 1 µL of the supernatant mixed to 10 µL of the matrix (R-cyano-4-hydroxycinnamic acid, 5 × 10-2 M in acetonitrile/H2O, 4:1 v/v) was deposited on the target. Laser energy 11 µJ/pulse. Homogeneous target. (b) Positive MALDI mass spectrum of 1 µL of the solution resulting from the elution of the beads (step 6 + washing procedure) in R-cyano-4-hydroxycinnamic acid (2 µL of 5 × 10-2 M in acetonitrile/H2O, 4:1 v/v). Laser energy 4.5 µJ/pulse. Target roughly homogeneous.

allow the detection of the peptide by regular analysis of this solution by MALDI, as shown in the Figure 3a. As previously noted, the direct application of steps 2-6 was not efficient to detect only the biotinylated peptide. To eliminate most of nonselective adsorption of BSA fragments onto the beads, Tween, NaCl, and BSA had to be added in the incubation solution, and the washing procedure described above was again necessary (Figure 3b). However, it was not possible to completely eliminate the nonspecific adsorption of different BSA fragments. The target was homogeneous and the detection of the peptide (∼125 fmol on the target) allows one to expect a detection limit from the signal-tonoise ratio below 10 fmol. Extraction of a Biotinylated Peptide from CNBr-Cleaved Membrane Proteins from Chinese Hamster Ovary Cells. In order to subsequently investigate the interactions of a peptide with its receptor embedded in membranes from CHO cells and to map the binding site, we have used the streptavidin beads to isolate biotinylated peptide diluted in a biological solution and to subsequently detect it with MALDI. To simulate such a situation, we mixed membrane proteins of CHO cells (1 mg), previously cleaved by CNBr, with 500 fmol of peptide P6 and streptavidin beads (100 µg). Direct MALDI analysis of the mixture (protein fragments and P6) shows a very broad distribution of peptides in the mass range 1000-8000 Da (Figure 4a). Among such a distribution peptide P6 cannot be obviously detected. But, by applying steps 2-6 with the washing procedure, peptide P6 is then easily observed in MALDI (Figure 4b) without contamination due to nonspecific adsorption on the beads (250 fmol of P6 was expected on the target). This result shows that strong nonspecific adsorption, as expected from highly hydrophobic membrane protein fragments, can be eliminated from the washing procedure described above. Analytical Chemistry, Vol. 68, No. 13, July 1, 1996

2125

CONCLUSION These experiments highlight the potential of streptavidincoated magnetic beads for extracting biotinylated peptides in low concentration from complex mixtures and further characterization in MALDI with (i) minimal sample manipulation, thus avoiding loss of biological material, and (ii) a high detection sensitivity (detection limit ∼10 fmol). This purification technique coupled to MALDI is of interest for investigation of the binding site between any biotinylated ligand (peptides, oligonucleotides) and a protein. The strategy developed in this study allows one to replace the classical HPLC purification of proteolytic photolabeled fragments19 by a simple and efficient one-step process, which is well adapted to the study of the interaction between peptides and hydrophobic proteins. The results presented in this paper on the extraction of biotinylated substance P highlight that this strategy can be carried out directly in cell culture. The determination of

the NK-1 receptor (a G protein-coupled seven transmembrane receptor) regions involved in substance P recognition is currently under investigation by using this strategy. Nomenclature: Indanylglycine, Ing; fluorenylglycine, Flg; diphenylalanine, Dip; amino pentanoic acid, Apa; benzyloxycarbonyl, Boc; bovine serum albumin, BSA; sodium dodecyl sulfate, SDS; cyanogen bromide, CNBr; dithiothreitol, DTT; Chinese hamster ovary, CHO.

(19) Dennis, M.; Giraudat, J.; Kotzyba-Hibert, F.; Goeldner, M.; Hirth, C.; Chang, J.; Lazure, C.; Chre´tien, M.; Changeux, J. P. Biochemistry 1988, 27, 234657.

AC960043R

2126

Analytical Chemistry, Vol. 68, No. 13, July 1, 1996

ACKNOWLEDGMENT S.G. gratefully acknowledges the IFSBM (Institut de Formation Supe´rieure Biome´dicale) and Rhoˆne Poulenc Rorer for their financial support. Received for review January 16, 1996. Accepted March 22, 1996.X

X

Abstract published in Advance ACS Abstracts, May 15, 1996.