Anal. Chem. 2005, 77, 2050-2055
Identification of Molecular Target of AMP-activated Protein Kinase Activator by Affinity Purification and Mass Spectrometry Toshiyuki Kosaka,*,†,⊥ Ryo Okuyama,*,‡,⊥ Weiyong Sun,‡ Tsuneaki Ogata,‡ Jun Harada,§ Kazushi Araki,‡ Masanori Izumi,‡ Taishi Yoshida,‡ Akira Okuno,‡ Toshihiko Fujiwara,‡ Jun Ohsumi,‡ and Kimihisa Ichikawa†
Biomedical Research Laboratories, Pharmacology and Molecular Biology Research Laboratories, and Lead Discovery Research Laboratories, Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
We show an efficient method to identify molecular targets of small molecular compounds by affinity purification and mass spectrometry. Binding proteins were isolated from target cell lysate using affinity columns, which immobilized the active and inactive compounds. All proteins bound to these affinity columns were eluted by digestion using trypsin and then were identified by mass spectrometry. The specific binding proteins to the active compound, a candidate for molecular targets, were determined by subtracting the identified proteins in an inactive compoundimmobilized affinity column from that in an active compound-immobilized affinity column. This method was applied to identification of molecular targets of D942, a furancarboxylic acid derivative, which increases glucose uptake in L6 myocytes through AMP-activated protein kinase (AMPK) activation. To elucidate the mechanism of AMPK activation by D942, affinity columns that immobilized D942 and its inactive derivative, D768, were prepared, and the binding proteins were purified from L6 cell lysate. NAD(P)H dehydrogenase [quinone] 1 (complex I), which was shown as one of the specific binding proteins to D942 by subtracting the binding proteins to D768, was partially inhibited by D942, not D768. Because inhibition of complex I activity led to a decrease in the ATP/AMP ratio, and the change in the ATP/AMP ratio triggered AMPK activation, we identified complex I as a potential protein target of AMPK activation by D942. This result shows our approach can provide crucial information about the molecular targets of small molecular compounds, especially target proteins not yet identified. Chemogenomics has recently emerged as the strategy for target and drug discovery.1 It is categorized into forward and reversed chemogenomics strategies, depending on whether the research is started from the phenotype or from the target. In * To whom correspondence should be addressed. E-mails:
[email protected] (T.K.) and
[email protected] (R.O.). † Biomedical Research Laboratories. ‡ Pharmacology and Molecular Biology Research Laboratories. § Lead Discovery Research Laboratories. ⊥ T.K. and R.O. contributed equally to this work. (1) Bredel, M.; Jacoby, E. Nat. Rev. Genet. 2004, 5, 262-275.
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forward chemogenomics, a phenotypic screening is performed in cells or organisms using compound libraries. Once biologically active compounds have been identified, efforts are directed toward identifying the gene and protein target to elucidate the mechanism of phenotype. The identified molecular targets of the active compound, which are critical for showing phenotypic effects, can be utilized in a reversed chemogenomics strategy to identify more potent compounds through specific target-based screening. Affinity purification is a widely known method for identifying the molecular targets of active compounds. A typical example of successful use of this method was the identification of a molecular target of the immunosuppressant FK506.2 Various parameters are necessary for identifying target proteins by affinity purification. It was demonstrated that the adsorption and elution conditions of proteins in compound-immobilized column chromatography have to be optimized thoroughly to identify the cellular targets of protein kinase inhibitors.3 However, it may be extremely difficult to optimize the conditions of affinity purification in the case of a compound that has a high protein binding nature, a low binding affinity to target proteins, or both. In such a case, one would encounter a high amount of nonspecific binding proteins in the affinity purification step, making it difficult to identify the specific binding proteins to active compounds. Recently, a systematic and effective strategy for identification of the specific binding proteins to compounds, fluorescent two-dimensional differential gel electrophoresis (2D-DIGE)4 combined with isotope-coded affinity tag (ICAT)5 analyses of affinity purified proteins, was reported.6 However, each 2D-DIGE and ICAT method is not always comprehensive analysis and requires relatively high amounts of samples. Here, we have reported an efficient and reliable method to identify the specific binding proteins of small molecular com(2) Harding, M. W.; Galat, A.; Uehling, D. E.; Schreiber, S. L. Nature 1989, 341, 758-760. (3) Godl, K.; Wissing, J.; Kurtenbach, A.; Habenberger, P.; Blencke, S.; Gutbrod, H.; Salassidis, K.; Stein-Gerlach, M.; Missio, A.; Cotton, M.; Daub, H. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 15434-15439. (4) Unlu, M.; Morgan, M. E.; Minden, J. S. Electrophoresis 1997, 18, 20712077. (5) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (6) Oda, Y.; Owa, T.; Sato, T.; Boucher, B.; Daniels, S.; Yamanaka, H.; Shinohara, Y.; Yokoi, A.; Kuromitsu, J.; Nagasu, T. Anal. Chem. 2003, 75, 2159-2165. 10.1021/ac0484631 CCC: $30.25
© 2005 American Chemical Society Published on Web 02/18/2005
pounds by a combination of affinity purification and mass spectrometry. The proteins bound to affinity columns were comprehensively identified by mass spectrometry, and then the identified proteins from the active- and inactive-immobilized affinity columns were compared to select the specific binding proteins to an active compound. This method was applied to the identification of the molecular target of D942, a novel compound we newly discovered from our chemical library, which increases glucose uptake in L6 myocytes and shows blood glucose lowering in Zucker diabetic fatty (ZDF) rats through skeletal muscle AMPactivated protein kinase (AMPK) activation. D942 did not activate AMPK directly; the mechanism of AMPK activation by D942 has not yet been elucidated. It was demonstrated that the direct protein target responsible for the AMPK activating effect of D942 was identified as NAD(P)H dehydrogenase [quinone] 1 (complex I) by our method, as described above. MATERIALS AND METHODS Preparation of D942/D768-Immobilized Affinity Column. A 1-mL portion of EAH sepharose 4B beads (Amersham) was washed several times by centrifugation at 600 rpm for 20 s with 80 mL of 0.5 M NaCl in total. Two hundred mM N-ethyl-N′-(3dimethyl aminopropyl)-carbodiimide hydrochloride (EDC) (Amersham) was prepared in double-distilled water, and the pH of the solution was adjusted to 4.5 with 1 N HCl. A 5 mM solution of D942 and its inactive derivative, D768, was prepared by adding 200 mM DMSO stock solution of each compound to doubledistilled water to give the final concentration. A 1-mL portion of resuspended beads, 1 mL of EDC, and 1 mL of each compound solution prepared above were mixed in a 15-mL conical tube and rotated at 4 °C overnight. After rotation, the compound-immobilized beads were washed with 0.1 M acetate buffer (pH 4, including 0.5 M NaCl) and 0.1 M Tris buffer (pH 8.3, including 0.5 M NaCl) three times, alternating with centrifugation at 600 rpm for 20 s. After the final washing, 375 µL of the beads and 125 µL of CelLytic M (Sigma) were packed in a plastic column and stored at 4 °C until use. Preparation of L6 Cell Lysate for Affinity Column Application. A 1-mL portion of CelLytic M was added to a 75-cm2 flask of confluent of L6 myocytes differentiated from myoblasts by decreasing supplemented fetal bovine serum (FBS) concentration from 10 to 2% over 5 days, and the myoblasts were incubated for 10 min at 4 °C. Lysed cells were collected by scraping and were transferred to a 1.5-mL tube. The cell lysate was centrifuged at 15 000 rpm for 5 min, and the supernatant was collected. The pellet was lysed again with 100 µL of CelLytic M and centrifuged as above, and the obtained supernatant was combined with the initial one. Affinity Purification. L6 cell lysate was loaded onto a D942or D768-immobilized affinity column and allowed to pass through the column by gravity. After lysate application, each column was washed with ∼3 mL of CelLytic M. Column-bound proteins were eluted with Laemmli SDS-PAGE sample buffer and used for further SDS-PAGE analysis. For mass spectrometry analysis, protein-bound columns were washed five times with 0.2× phosphate-buffered saline (PBS) to avoid dissociation of bound proteins by the usual concentrations of salts, and then the beads were collected from the packed columns using a spatula for direct application to mass spectrometry.
Sample Preparation for Mass Spectrometry. In-gel digestion of the proteins in the silver-stained SDS-PAGE bands with sequence-grade modified trypsin (Promega) was performed according to the procedure described by Wilm et al.7 The beads collected from the packed column after affinity purification were treated with 0.2% RapiGest (Waters) in 50 mM NH4HCO3 and then reduced with 10 mM dithiothreitol (Wako) and alkylated with 55 mM iodoacetamide (Sigma). A 1-µg portion of sequence-grade modified trypsin was added, and the enzymatic reaction was allowed to proceed overnight at 37 °C. After digestion, the supernatant was acidified below pH 2.0 by adding trifluoroacetic acid and concentrated in a vacuum centrifuge. The resulting solution was desalted with ZipTip C18 (Millipore), eluted with 70% CH3CN and 0.1% trifluoroacetic acid from ZipTip C18, dried, and redissolved in 0.05% formic acid (FA) for LC/MS/MS measurement. The samples were immediately subjected to mass spectrometry or stored in a freezer until analysis. Nano-LC/MS All experiments were carried out on a Q-TOF Ultima API mass spectrometer (Micromass) equipped with a nanoES ion source and a Waters CapLC system. The sample was analyzed in both LC/MS and data-directed acquisition mode. A fused-silica capillary (75 µm i.d. × 375 µm o.d.) was pulled using a laser puller (Shutter Instrument) to prepare a nano-ES needle. This nano-ES needle was packed with reversed-phase material (Develosil C18, particle size 3 µm, 4 cm length) and then connected to the nano-ES ion source. The samples were injected into an autosampler at a flow rate of 1 µL/min, and the peptides were eluted with a linear gradient of solvent A (0.05% FA in H2O) and solvent B (0.05% FA in CH3CN) at a flow rate of up to 200 nL/min. A gradient from 0 to 30% B over 30 or 60 min was provided using a CapLC system. Protein Identification by Database Search. Data from the LC/MS/MS measurement was processed using ProteinLynx (Micromass). Mascot (Matrix Science) software was used for protein identification. NCBInr and SwissProt databases were used for searching. 2-Deoxyglucose (2-DG) Uptake Assay in L6 Myocytes. L6 myoblasts were routinely maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 10 000 U/mL penicillin, 10 mg/mL streptomycin, and 25 µg/mL amphotericin B in a humidified incubator with 5% CO2 at 37 °C. For the 2-DG uptake assay, the cells were trypsinized and inoculated into 24well plates. L6 myoblasts were then differentiated into myocytes by decreasing supplemented the FBS concentration from 10 to 2% over 5 days. The cells were incubated with Dulbecco’s modified Eagle’s medium alpha (MEM-R) medium supplemented with 20 mM glucose and 0.1% FBS containing the indicated concentrations of test compounds after twice washing with PBS for 4 h. Then, 500 µL of PBS containing 0.4 µCi/mL [3H]2-DG and 0.4 mM 2-DG (uptake reaction buffer) was added to each well to start the uptake assay. The uptake reaction was performed for 10 min at 37 °C. The reaction was stopped by aspirating the uptake reaction buffer. The cells were lysed by adding 200 µL of 1 N NaOH after washing three times with PBS. The lysate was transferred into scintillation vials, and radioactivity was counted by a liquid scintillation counter. Since D769 was a lead compound, we initially tested for the activity (7) Wilm, M.; Shevchenko, A.; Houthaeve, T.; Breit, S.; Schweigerer, L.; Fotsis, T.; Mann, M. Nature 1996, 379, 466-469.
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of AMPK activation and glucose uptake stimulation in L6 cells, the maximum increase of 2-DG uptake achieved by D769 treatment was defined as 100%, and the concentration of each compound giving a half increase of the maximum was defined as EC50. Immunoblot of Phospho-AMPK and Phospho-acetyl-CoA Carboxylase (ACC). L6 myoblasts were inoculated into 6-well plates and then differentiated as above. The cells were incubated with each compound for 4 h unless otherwise indicated, described above in the 2-DG uptake assay part. The incubation was terminated by aspirating the medium, and then Laemmli SDSPAGE sample buffer was immediately added to the cells at a volume of 200 µL per well. The cells were completely lysed by repetitive pipetting through wide-bore tips. After sonication and clarification by quick centrifugation, the lysate was subjected to 4-15% gradient SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated in blocking solution (20 mM Tris pH 7.5, 500 mM NaCl, 5% skim milk, 0.1% Tween 20) for 1 h at room temperature. The blocked membrane was immunoblotted with antibodies against phosphopeptides corresponding to residues surrounding Thr-172 of human AMPK R-subunit or Ser-79 of rat ACC (Cell Signaling) and then secondary antibodies conjugated to horseradish peroxidase. Bands were visualized on X-ray films by enhanced chemiluminescence (Amersham). In Vivo Experiments. Female Zucker diabetic fatty (ZDF) rats were maintained under a 12-h light/12-h dark cycle and had free access to water and MR-DBT diet (Nihon Nosan) supplemented with 10% lard. Blood glucose levels were measured from tail neck samples, and the animals with blood glucose levels above 350 mg/dL were selected. Those rats were divided into four groups (n ) 5) to achieve as close as possible an average blood glucose and body weight among the groups. D942 was orally given by bolus administration at doses of 10, 30, and 100 mg/kg of body weight. A vehicle (0.5% carboxymethylcellulose) was given to a control group. The rats were fasted immediately after administration. Blood samples were taken from the tail vein 30 min before, and 2, 4, 6, and 8 h after administration. Plasma was obtained from each blood sample by centrifugation (11 000 rpm, 5 min). Glucose concentrations of all plasma samples were analyzed. In western analysis, indicated doses of 5-aminoimidazole-4-carboxamide 1-β-ribofuranoside (AICAR) or D942 was orally administrated and the soleus muscle was dissected 4 h after administration. The muscle samples were lysed with a 3× volume of Laemmli SDS-PAGE sample buffer and subjected to SDS-PAGE and the following immunoblots of phospho-AMPK and phospho-ACC as described above. Preparation of Skeletal Muscle Mitochondria Fraction from ZDF Rats. The ZDF rats were sacrificed, and the soleus muscle was quickly excised. The excised soleus muscle was homogenized in 10 mL of sucrose buffer (250 mM sucrose, 20 mM Tris-HCl pH 7.4, 1 mM EDTA) by repetitive strokes with a glass-Teflon homogenizer. The homogenate was centrifuged at 3000 rpm for 10 min, and the supernatant was collected. The supernatant was centrifuged at 10 000 rpm for 10 min, and the pellet was resuspended in 100 µL of assay buffer (20 mM TrisHCl, pH 7.4, 2 mM MgCl2, 1 mM EDTA). This resuspended 2052
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mitochondrial fraction was frozen by liquid nitrogen and stored at -80 °C until use. Mitochondrial Complex I Assay. A 400-µL portion of 130 µM Coenzyme Q in assay buffer and 0.8 µL of compound stock solution prepared in DMSO were added to a cuvette to give the final concentration of the test compound. The same volume of DMSO was added to the control, then 10 µL of mitochondrial fraction was added, vortexed, and preincubated for 10 min at room temperature. The mitochondrial complex I assay was initiated by adding 400 µL of 200 µM NADH in assay buffer to the reaction mixture. Absorbance at 340 nm was measured immediately and 10 min after initiation with reference absorbance at 425 nm, and the decrease in the absorbance value during a 10-min reaction period was calculated. Complex I activity in the presence of the test compound was calculated by dividing the decrease in absorbance by that in the control. RESULTS AND DISCUSSION AMPK activation by D942. As shown in Figure 1A, we found that D769, a furancarboxylic acid derivative, dose-dependently increased glucose uptake after 4 h incubation in L6 myocytes. Western blot analysis using phospho-specific antibodies showed that D769 increased phosphorylation of threonine 172 of the AMPK R subunit, which is known to lead to activation of the kinase.8 Serine 79 of acetyl-CoA carboxylase (ACC), a representative substrate of AMPK,9 was also strongly phosphorylated by the treatment of D769, confirming that AMPK was enzymatically active. Because AMPK activation causes an increase in the glucose uptake in skeletal muscle,10,11 these data reveal that this compound works as an activator of AMPK and the subsequent uptake of glucose in L6 myocytes. From the screening of chemical derivatives of D769, we identified D942, a more potent activating agent of AMPK and glucose uptake (Figure 1B). Since AMPK activation and increased glucose uptake in skeletal muscle in vivo are expected to decrease blood glucose levels and ameliorate diabetes,12 we administrated D942 to ZDF rats. D942 significantly decreased blood glucose concentrations, as compared to vehicletreated animals at 2, 4, and 8 h after oral administration at doses of 30 and 100 mg/kg. In these rats and at the same doses, D942 stimulated phosphorylation of skeletal muscle AMPK 4 h after administration (Figure 1C). Therefore, D942 was an effective and potent AMPK activating agent working both in vitro and in vivo. Although AICAR, a direct AMPK activator,13 also showed an increase in AMPK phosphorylation in skeletal muscle in vivo after oral administration, D942 did not directly activate recombinant AMPK in enzyme assay (data not shown), suggesting that the direct molecular target of D942 is other than AMPK. Identification of the Binding Proteins to D942. To elucidate the mechanism of AMPK activation by D942, we prepared (8) Hawley, S. A.; Davison, M.; Woods, A.; Davies, S. P.; Beri, R. K.; Carling, D.; Hardie, D. G. J. Biol. Chem. 1996, 271, 27879-27887. (9) Haystead, T. A.; Moore, F.; Cohen, P.; Hardie, D. G. Eur. J. Biochem. 1990, 187, 199-205. (10) Bergeron, R.; Russell, R. R., 3rd; Young, L. H.; Ren, J. M.; Marcucci, M.; Lee, A.; Shulman, G. I. Am. J. Physiol. 1999, 276 (5 Pt 1), E938-944. (11) Kurth-Kraczek, E. J.; Hirshman, M. F.; Goodyear, L. J.; Winder, W. W. Diabetes 1999, 48, 1667-1671. (12) Bergeron, R.; Previs, S. F.; Cline, G. W.; Perret, P.; Russell, R. R., 3rd; Young, L. H.; Shulman, G. I. Diabetes 2001, 50, 1076-1082. (13) Sullivan, J. E.; Brocklehurst, K. J.; Marley, A. E.; Carey, F.; Carling, D.; Beri, R. K. FEBS Lett. 1994, 353, 33-36.
Figure 1. (A) Discovery of D769 as a novel AMPK activator in L6 myocytes. Left: dose-dependent stimulation of glucose uptake by D769. Right: time-dependent increase of phospho-AMPK (active form of the enzyme) and phospho-ACC (substrate for AMPK) by D769. (B) Identification of D942 as a potent AMPK activator and D768 as an inactive derivative from D769 analogues. Left: effects of D769, D768, and D942 on glucose uptake and ACC phosphorylation. Right: EC50 values and maximum increase of glucose uptake, and chemical structures of D769, D942, and D768. (C) Blood glucose lowering (left panel) and skeletal muscle AMPK activation (right panel) by the treatment of D942 in ZDF rats.
the affinity columns of immobilized D942 and its inactive derivative, D768 (Figure 1B), for identifying the primary binding proteins to D942. In the structure-activity relationship of the furancarboxylic acid derivatives, we found that pharmacological activity remained if the furancarboxylic acid moiety was substituted by salicylic acid or phenyl methyl ether (data not shown). Therefore, the furancarboxylic acid moiety was not critical for the effects of glucose uptake stimulation and AMPK activation, so this moiety was considered to be independent of interacting with target proteins. Therefore, the carboxylic acid of D942 and D768 was covalently coupled to the Sepharose beads to prepare the affinity columns. After affinity purification from L6 cell lysate using D942and D768-immobilized columns, the proteins bound to each column were analyzed by SDS-PAGE first. However, as shown in Figure 2, the profiles of the silver-stained protein bands observed in SDS-PAGE did not show significant differences, so the specific binding proteins to D942 could not be identified. Because of the complexity of the nonspecific binding proteins to the compounds and Sepharose beads, the resolution of SDSPAGE analysis might not have been enough to identify the specific binding proteins to D942. Competitive protein elution by the compound from an affinity column is effective in reducing nonspecific binding proteins, but the poor solubility of D942 in buffer made it difficult to apply in this analysis. It may be possible to distinguish the specific binding proteins in 2D-PAGE analysis, which is a powerful tool to isolate protein mixtures; however, relatively high amounts of samples are required for 2D-PAGE
Figure 2. Affinity purification of proteins using D942- and D768immobilized affinity columns. Lane 1, eluents from D942-conjugated affinity column; lane 2, eluents from D768-conjugated affinity column; and lane 3, molecular weight marker.
analysis. Here, we identified all trapped proteins to these columns using mass spectrometry, and then the specific binding proteins to D942 were determined by subtracting D768-binding proteins from D942-binding proteins. The beads collected from the packed columns were digested with trypsin, and the resulting peptide mixtures were analyzed by nano-LC/MS/MS. The nano-LC/MS chromatograms of the tryptic digest of D768- and D942-binding proteins are shown in Figure 3. The nano-LC/MS/MS experiment of each sample was carried out three times, and proteins identified Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
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Table 1. Mass Measurements Obtained by Nano-LC/MS/MS of the Tryptic Digest of D942 Binding Proteins and Their Assignment to the Protein Identified by Peptide-Sequence Tagging Using Mascot obs ions (m/z)
charge
assigned sequencea
residue no.
calcd M
∆ (Da)
obs fragment ions
437.22 550.32 479.76
2 2 2
(R)NDITGEPK(D) (R)LSPDIVAEQK(K) (R)VQVLEGWK(K)
53-60 80-89 201-208
872.42 1098.59 957.53
0.01 0.03 -0.02
y1, y2, y4, y5, y6, b2 y1, y2, y3 - H2O, y4, y5, y6, y7, y8, y9, y82+, b2 y1, y2, y3, y4, y5, y6, y7, y7 - NH3, b2
a
Residues before and after the sequences of tryptic peptides are indicated in parentheses.
Figure 3. Base peak intensity chromatograms obtained by nanoLC/Q-TOF MS of the tryptic digests of (A) D768 and (B) D942 binding proteins.
as the significant hits in a Mascot search were listed. In the case of proteins identified by one peptide, MS/MS spectra were checked if the assignment of fragment ions by Mascot was not random. In consequence, 97 and 90 proteins were identified from D942- and D768-binding proteins, respectively. After subtracting D768-binding proteins, there were nine proteins as candidates for the specific binding proteins to D942 as follows: NAD(P)H dehydrogenase [quinone] 1 (complex I), stathmin, endoplasmin precursor, fascin, 26S proteasome regulatory subunit 6B, telomerase-binding protein p23, dihydropyrimidinase related protein2, desmoyokin, and vat1 protein. Among these proteins that bound to D942 but not to D768, mitochondrial complex I is the most likely to be a pharmacologically relevant D942-binding protein. This is because AMPK is directly activated by AMP,14 and a blockade of complex I is expected to decrease the cellular ATP level and increase the AMP level through inhibition of electron transport, causing AMPK activation.15 The other eight proteins seemed not to be functionally linked to AMPK activation, although some of those proteins have not been fully elucidated with regard to their physiological function. Before biological assay of complex I, the reproducibility of complex I detection as the specific binding proteins to D942 was examined using a mitochondrial fraction in L6 cells, because complex I is located in the mitochondoria. In affinity purification from L6 cell mitochondrial fraction, three peptides derived from complex I (the doubly charged ions at m/z (14) Moore, F.; Weekes, J.; Hardie, D. G. Eur. J. Biochem. 1991, 199, 691697. (15) Hayashi, T.; Hirshman, M. F.; Fujii, N.; Habinowski, S. A.; Witters, L. A.; Goodyear, L. J. Diabetes 2000, 49, 527-531.
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Figure 4. Nano-ES Q-TOF mass spectra of the tryptic peptides of D942 and D768 binding proteins. Doubly charged protonated molecules of tryptic peptides of mitochondrial complex I (peaks marked with arrow) were observed at m/z 550.3 (LSPDIVAEQK; A, lower), 479.7 (VQVLEGWK; B, lower), and 437.2 (NDITGEPK; C, lower) in nano-LC/MS of D942 binding proteins. These ions were not observed in the corresponding nano-LC/MS scans (A, B, and C, upper) of D768 binding proteins. Because a peak at m/z 430.2 (C, lower) was a singly charged ion, this peak should not be a peptide.
550.32, 479.76, and 437.22) were observed in nano-LC/MS of the tryptic digest of D942 binding proteins, but not in that of D768 binding proteins (Figure 4). The results of the protein identification are summarized in Table 1. When analyzing affinity purified proteins from L6 whole cell lyaste, only one peptide derived from complex I was observed in the nano-LC/MS of the tryptic digest of D942 binding proteins. Therefore, the identity of complex I as a specific binding protein to D942 was confirmed with certainly. The molecular weight of complex I protein identified in this study is ∼30 kDa. It was detectable by SDS-PAGE analysis, but no significant difference was observed in SDS-PAGE analysis between D942 and D768 binding proteins. A high amount of nonspecific binding proteins made it difficult to identify complex I protein as specific binding protein to D942 by conventional SDS-
Figure 6. (A) Partial inhibition of mitochondrial complex I activity by D942. Left: absorbance of NADH at 340 nm (425 nm as reference) reduced as complex I enzyme assay proceeded. This reduction of absorbance was slowed by addition of D942 in a dose-dependent manner. Right: dose-response curve of complex I inhibition by D942. (B) Correlation between glucose uptake stimulating effect and mitochondrial complex I inhibitory activity among various D769 analogues.
Figure 5. Nano-ES MS/MS spectra of tryptic peptides of complex I (A) m/z 550.3 (LSPDIVAEQK), (B) m/z 479.7 (VQVLEGWK), and (C) m/z 437.2 (NDITGEPK) shown in Figure 4.
PAGE analysis. Our approach based on comprehensively identification of proteins bound to affinity columns by mass spectrometry could identify the specific binding proteins in such a case. Inhibitory Effects of D942 on Complex I Activity. Since severe respiration inhibition of complex I must be toxic for cells and animals,16,17 D942 was speculated to inhibit complex I only partially at pharmacological doses because the compound showed no cytotoxicity or animal toxicity for doses achieving maximum AMPK activating effect. To test this hypothesis, we prepared the mitochondrial fraction from the skeletal muscle of ZDF rats, measured mitochondrial complex I activity, and assessed the inhibitory effect of D942 on the enzyme activity. As expected, D942 inhibited complex I activity in a dose-dependent manner, and the inhibition was partial surrounding the doses causing stimulation of glucose uptake in L6 myocytes (Figure 6A). Regarding structurally related compounds (D768, 930, 769, 833, 847), the potency of the glucose uptake stimulating effect and complex I inhibitory activity was very well correlated in each compound (Figure 6B). These data strongly suggest that the direct action point of the furancarboxylic acid derivatives responsible for the common AMPK activating effect and glucose uptake stimulating activity was mitochondrial complex I. (16) Holm, G. Exp. Cell Res. 1967, 48, 334-349. (17) Rotenberg, Y. S. Bull. Exp. Biol. Med. 1975, 77, 783-785. (18) Yao, X.; Afonso, C.; Fenselau, C. J. Proteome Res. 2003, 2, 147-152. (19) Wang, Y. K.; Ma, Z.; Quinn, D. F.; Fu, E. W. Anal. Chem. 2001, 73, 37423750.
CONCULSIONS AND FURTHER PERSPECTIVES We described the method of identification of the molecular target of D942 by affinity purification and mass spectrometry. All proteins bound to affinity columns were eluted by digestion with trypsin and analyzed by mass spectrometry. By subtracting proteins identified in the control columns, candidates of the specific binding proteins to D942 were selected. Complex I, one of the specific binding proteins to D942, was proved as a molecular target for AMPK activation by D942. This approach would be effective and useful for identifying specific binding proteins, even in cases in which no significant changes are observed when comparing purified proteins between active and inactive compoundimmobilized affinity columns by conventional SDS-PAGE analysis. However, quantitative information of the binding proteins to the affinity columns could not be obtained by this method. It is probable that the target proteins of the active compound slightly exhibit nonspecific binding to Sepharose or the inactive compound. If the amount of nonspecific binding is sufficient to identify a protein by mass spectrometry, the target protein is not extracted as a specific binding protein to the active compound. Therefore, quantitative analysis of binding proteins is important for identifying specific binding proteins to the active compound in such a case. Enzymatic stable isotope labeling of peptides, the 18O-postlabeling method, for quantitative protein analysis by mass spectrometry has recently been reported18,19 and may be applicable for obtaining quantitative information of binding proteins to affinity columns. Further investigations to apply this method to global target protein identification are now in progress. Received for review December 9, 2004. Accepted January 11, 2005 AC0484631
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