Anti-sulfonylbenzoate Antibodies as a Tool for the Detection of Nucleotide-Binding Proteins for Functional Proteomics Lisa L. Moore, Ashley M. Fulton, Marietta L. Harrison, and Robert L. Geahlen* Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907 Received July 1, 2004
Proteins that bind ATP and GTP are important cellular components. We developed an immunological approach to selectively tag nucleotide-binding proteins based on the use of 5′-[4-(fluorosulfonyl)benzoyl]adenosine and 5′-[4-(fluorosulfonyl)benzoyl]guanosine affinity tags and an antibody against 4-(sulfonyl)benzoate. Detection follows affinity labeling, gel electrophoresis, and ester bond cleavage to expose the epitope. Trial analyses of labeled proteins from lymphoid cells identified multiple ATP-binding proteins, including chaperones, actin, kinases, an RNA splicing factor, a membrane ATPase, and ATP synthase. Keywords: 5′-[4-(fluorosulfonyl)benzoyl]adenosine • FSBA • 5′-[4-(fluorosulfonyl)benzoyl]guanosine • FSBG • ATPbinding protein • GTP-binding protein • functional proteomics • affinity labeling
Introduction Functional proteomics, the technology to separate and analyze proteins in a cell based on their function, is a fast growing field in biology and chemistry. Prior to the development of functional proteomics, proteomics was limited to the qualitative analysis of protein expression in cells.1-3 Functional proteomics uses chemical labeling,1,4-13 affinity purification,14 and structural analysis15-17 to separate or label proteins that share common characteristics, such as affinity for a probe or a common fold, and allows scientists to generalize about their functions. A variety of chemical probes are currently used in functional proteomics. Most probes consist of four moieties: a tag, a linker, a binding region, and a reactive group.1,4-8,10-13,18 Tags are the part of the probe used to detect labeled proteins, and they are chosen based on functionality: fluorescent and radiolabeled tags are useful for visualization, biotin tags are useful for separation. Probes can also be produced with several tags useful for both separation and detection. For example, a serine protease tag can be produced with a radioactive, fluorescent, or biotin tag, or it can be produced with multiple tags.4,5,7 A variety of binding and reactive groups have been developed, including a binding group that resembles phosphotyrosine to select for protein tyrosine phosphatases18 and epoxide-based binding groups that select for cysteine proteases.11 Nucleotide binding proteins, such as kinases, ATPases, and G-proteins are essential components of signaling pathways in all cells. FSBA (5′-[4-(fluorosulfonyl)benzoyl]adenosine) and FSBG (5′-[4-(fluorosulfonyl)benzoyl]guanosine) are two widely used probes of nucleotide-binding sites. Both probes contain * To whom correspondence should be addressed. E-mail: geahlen@ purdue.edu.
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a 4-(fluorosulfonyl)benzoyl group attached to adenosine or guanosine by an ester linkage. When bound by ATP- or GTPbinding proteins, the fluorosulfonyl group can react with nucleophiles at or near the binding site leading to the covalent modification of the protein.19-25 Once the protein is labeled, the bound probe can be detected by one of several methods. If the probe is radiolabeled with 13C or 3H, the labeled proteins can be detected by monitoring the incorporated radioisotope.26 Alternatively, antibodies prepared against FSBA-modified proteins can be used to detect the incorporated probe.27,28 For this approach, separate antibodies would be required to detect proteins labeled with FSBA versus FSBG and some loss of label can occur during extensive workups due to hydrolysis of the ester linkage and loss of the epitope. In this paper, we describe an alternative approach whereby antibodies are generated against proteins containing an attached sulfonylbenzoate (SB) moiety. Proteins can be detected that have been labeled with either FSBA or FSBG providing they are then treated with base to cleave the ester linkage, exposing the SB group. The immune serum was used to identify nucleotide-binding proteins present in whole cell and membrane fractions of lymphoid cells that were labeled with FSBA and separated by either 1- or 2-D gel electrophoresis. The specificity of the labeling was confirmed by the ability of an excess of natural nucleotide to inhibit labeling. Several nucleotide-binding proteins were identified by tryptic digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This technique should be useful for identifying the functionality of proteins in large-scale proteomics assays.
Materials and Methods Affinity Probes. FSBA was purchased from Sigma (St. Louis, MO). 4-(fluorosulfonyl)benzoic acid (SB) and 4-(fluorosulfonyl)10.1021/pr0498943 CCC: $27.50
2004 American Chemical Society
Detection of Nucleotide-Binding Proteins
benzoyl chloride were purchased from Aldrich (Milwaukee, WI). The preparation of guanosine hydrochloride and its conversion to FSBG by reaction with 4-(fluorosulfonoyl)benzoyl chloride was as described previously.19 Preparation of the Anti-sulfonoylbenzoate Antibody. For preparation of the antigen for immunization, 15 mg keyhole limpet hemocyanin (KLH) (Sigma, St. Louis, MO) (5 mg/mL) was reacted with 10 mM 4-(fluorosulfonyl)benzoic acid in phosphate-buffered saline (PBS), 10% DMSO for 2 h at 37 °C. The mixture was then dialyzed overnight at 4 °C against PBS. Antibodies were prepared by Lampire Biological Laboratories (Pipersville, PA) according to their normal protocol. A rabbit was injected with 1 mg of the SB-modified KLH (SB-KLH) in complete Freund’s adjuvant on day 1, and then injected with 1 mg injections of SB-KLH in incomplete Freund’s adjuvant on days 7, 14, 28, 56, and 84. A preimmune bleed occurred on day 0, test bleeds on days 42 and 70, with the production bleed on day 98. The specificity and titer of the antibody was determined by an ELISA assay using 96-well plates coated with 1.25 µg of SBmodified bovine serum albumin (SB-BSA), SB-modified ovalbumin (SB-OVAL), ovalbumin (OVAL), KLH or BSA. SB-BSA and SB-OVAL were prepared by reaction of BSA or ovalbumin (Sigma, St. Louis, MO) with 4-(fluorosulfonyl)benzoic acid as described above for the preparation of SB-KLH. Bound antibody was detected using a secondary, goat anti-rabbit antibody coupled to horseradish peroxidase and a premixed liquid substrate (3,3′,5,5′-tetramethylbenzidine) (Sigma, St. Louis, MO). Preparation of Cell Lysates and Membrane Fractions. Human DG75 B cells, human Jurkat T cells and Lck-deficient Jurkat T cells (JCaM1) were cultured in RPMI 1640 medium (Gibco/Invitrogen, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum, 50 µM 2-mercaptoethanol, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 IU/mL penicillin G and 100 µg/mL streptomycin. Cells were used in the log phase of growth at a density of 5 × 106 - 1 × 106/mL. For the preparation of detergent lysates, DG75 cells were lysed in 1% Triton X-100, 50 mM tris-(hydroxymethyl)-aminomethane hydrochloride (Tris/HCl), pH 8.0, 100 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM sodium orthovanadate, 1 µg/mL leupeptin, and 1 µg/mL aprotinin for 15 min on ice. Nuclei were removed by centrifugation at 14 500 × g for 10 min in a Microfuge 18 centrifuge with a fixed angle rotor (Beckman Coulter, Fullerton, CA). For the preparation of crude membrane fractions, DG75, Jurkat or JCaM1 cells were swollen in hypotonic buffer (5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5, 1 mM MgCl2, 0.5 mM sodium orthovanadate, 1 µg/mL leupeptin, and 1 µg/mL aprotinin) for 10 min on ice, followed by homogenization using a Dounce homogenizer. Nuclei were removed by centrifugation at 460 × g for 3 min in a Microfuge. Membranes were then pelleted by centrifugation at 200 000 × g for 30 min in a Beckman Optima TLX ultracentrifuge with a TLA 100.2 rotor (Beckman Coulter, Fullerton, CA). The membrane fraction was resuspended by incubation in 5 mM HEPES, pH 7.5, 1% Triton-X 100 for 30 min. Insoluble particulate matter was removed by centrifugation at 75 × g for 10 min (Microfuge). All buffer components were purchased from Sigma (St. Louis, MO). Labeling by FSBA or FSBG. Glutamate dehydrogenase (GDH) (Sigma, St. Louis, MO) (0.3 mg/mL) in PBS, 10% DMF was incubated with 0.5 mM FSBA or FSBG for 20 min at 37 °C.
research articles Proteins in cell lysates or membrane preparations were incubated at a concentration of 2.5 mg/mL with 0.5 mM analogue in 10% DMSO (FSBA) or DMF (FSBG) for 30 min at 30 °C unless noted otherwise. In some reactions, MgATP or MgGTP was included at a final concentration of 10 mM. Reactions were terminated by the addition of 5% 2-mercaptoethanol. Protein concentrations were determined by the BCA assay (Pierce, Rockford, IL). Separation and Detection of Proteins. Proteins (1/100 of the amount of protein found in 1 mL of initial sample) were separated in one dimension by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)29 at 6 mAmp for 18 h on 16 × 18 cm gels. Alternatively, proteins (40-60 µg) were separated in two dimensions by a combination of isoelectric focusing (IEF) in 1.5 mm × 8 cm tubes and SDS-PAGE.30 For IEF, ampholytes (BioRad, Hercules, CA) in the pH range 3-10 were used and proteins were separated by electrophoresis at 400 V for 19 h. Silver staining was accomplished using the ProteoSilver Kit available from Sigma (St. Louis, MO). For immunoblotting, proteins were transferred to polyvinylidene fluoride (PVDF)-membranes (BioRad, Hercules, CA) for 70 min at 4 °C at 1 amp in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). Membranes were treated with or without 0.2 M NaOH for 30 min at room temperature prior to Western blotting with the anti-SB antibody. For immunoblotting, membranes were first blocked by incubation in 5% nonfat dried milk in 0.1% NP-40, 50 mM Tris/HCl, pH 7.2, 150 mM NaCl for 1 h at 4 °C prior to addition of the anti-SB antisera at a 1:1000fold dilution. Membranes were then washed once in TBS buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl) for 10 min, twice in TBS containing 0.1% NP-40 for 10 min and once in 20 mM Tris/ HCl, pH 7.5, 500 mM NaCl for 15 min. The membrane was then incubated with a 1:3000-fold dilution of secondary goat antirabbit antibody linked to horseradish peroxidase (Sigma, St. Louis, MO) in blocking buffer for 45 min, and the washes were repeated. Reactive proteins were visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL). The migration position of Lck was determined using 3A5 mouse monoclonal anti-Lck antibody from Santa Cruz Biotechnology (Santa Cruz, CA). Identification of Labeled Proteins. Images of silver stained gels and membranes that had been immunoblotted using the anti-SB antibody were scanned using an Epson 3170 PHOTO scanner, imported into Adobe Photoshop and aligned using prestained molecular weight markers (BioRad, Hercules, CA) and the prominent protein spots visible in both gels. The pH gradient was measured directly from a master gel run under the same conditions. Once aligned, proteins were chosen for digestion if they were distinct spots in both the silver stained and immunoblot images. In-gel protein digestion, peptide enrichment, and placement of samples onto a MALDI target was accomplished by the Investigator ProPrep Station from Genomic Solutions (Ann Arbor, MI), an auto-digester and autospotter. After tryptic digestion, the peptides were enriched by adsorption to C18-resin using a ZipTip (Millipore) and eluted with 60% acetonitrile, 0.2% formic acid, and 2 mg/mL cyano4-hydroxycinnamic acid. The resulting solutions were spotted directly onto a wax-coated MALDI target. The peptides were analyzed using a Voyager-DE Pro MALDI-time-of-flight mass spectrometer from Applied Biosystems (Framingham, MA) with the following settings: positive-ion, delayed-extraction mode (120 ns) with 500 Da low-mass gate, 0.005% guide wire, 20 kV accelerating voltage, 72% grid voltage, and 1.12 mirror voltage Journal of Proteome Research • Vol. 3, No. 6, 2004 1185
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ratio. Spectra were calibrated to tryptic peptides (m/z ) 515.33, 842.51, and 2211.10) and matrix peak (m/z ) 568.12) using the Data Explorer Program, v. 4.0.0.0 (Applied Biosystems, Foster City, CA). Isotopic peaks were removed and the baseline was set so that the total number of peaks was between 50 and 100. Peaks that remained were analyzed using PeptIdent set to search human proteins in the ExPASY and TrEMBL databases with the following settings: mass tolerance of ( 100 ppm, ( 1 pI unit, ( 20% molecular weight, two missed cleavages, alkylation of cysteine, and oxidation of the methionine. Approximate molecular weight and pI were determined from the position of the protein on the 2-D gel. Resulting matches were analyzed based on their ability to explain the major m/z peaks that appeared in the spectra. Proteins that could explain less than half of the peaks in the spectrum were eliminated to minimize the number of false positive identifications.
Results and Discussion Characterization of Immune Serum. Rabbits were immunized against KLH that had been modified in vitro by reaction with 4-(fluorosulfonyl)benzoic acid to generate antisera that could specifically recognize an immobilized SB moiety. An ELISA assay was used to characterize the antisera for the presence of antibodies specific for SB-labeled proteins using immobilized BSA- and SB-modified BSA as antigens. KLH, the hapten carrier, was included as a positive control. As shown in Figure 1A, the serum contained antibodies that reacted against the carrier KLH as expected, and also recognized SBBSA, but not unmodified BSA. Immunoreactivity could be detected at a dilution of antisera of 1:100 000. Similarly, the antibodies also recognized SB-modified ovalbumin, but not ovalbumin itself (Figure 1B). These data indicate that the antiserum contains antibodies that specifically recognize proteins containing a covalently attached sulfonylbenzoyl moiety. We reacted GDH separately with each affinity probe to determine if the antibodies could recognize proteins modified with FSBA or FSBG on Western blots. GDH was chosen for its ability to bind both ATP and GTP.19,21 Labeled proteins were subjected to SDS-PAGE and transferred to PVDF-membranes, which were then treated with or without NaOH. As shown in Figure 1C, the antibody recognized GDH modified with either FSBA or FSBG, but only after the PVDF membrane was treated with 0.2 M NaOH to cleave the ester bond, releasing the nucleoside and revealing the SB epitope. Thus, these antibodies should be useful for the detection of proteins labeled in a reaction with any ligand containing an attached 4-(fluorosulfonyl)benzoyl group that can be readily cleaved following protein separation. Labeling Specificity. Both FSBA and FSBG have been used extensively as probes for ATP- and GTP-binding sites, respectively. However, incubation of proteins with high concentrations of either can lead to the nonspecific incorporation of probe. Inclusion of an excess of the natural ligand will protect proteins from being modified by occupying the nucleotide binding site and preventing the binding of the probe, but will have no significant effect on the nonspecific incorporation of probe. To examine labeling specificity, we incubated a membrane fraction isolated from DG75 B cells with 0.5 mM FSBA in the presence of increasing concentrations of ATP. As shown in Figure 2A, inclusion of ATP effectively prevented the incorporation of FSBA into membrane proteins, indicating that 1186
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Figure 1. Specifity of anti-SB antibody for SB-labeled proteins. (A) a 96-well plate coated with 1.25 µg of KLH, SB-BSA or BSA was incubated with the indicated dilutions of rabbit antisera. Binding was detected by an end point colorimetric assay using TMB as a substrate for goat anti-rabbit antibody conjugated to horseradish peroxidase. (B) the ELISA assay was repeated using plates coated with 1.25 µg of KLH, SB-BSA, BSA, SB-OVAL, or OVAL and increasing dilutions of antisera. For each group of four antigens, the antisera was diluted 102, 103, 104 or 105-fold (from left to right). (C) GDH labeled with or without FSBA or FSBG as indicated was transferred from SDS-polyacrylamide gels to a PVDF membrane. The membrane either remained untreated (-) or was treated with 0.2 M NaOH for 30 min (+), and then immunoblotted with anti-SB antisera (1:1000 dilution).
the probe was selectively reacting with ATP-binding proteins under the conditions of our assay. To further standardize labeling conditions, we reacted DG75 B cell lysates at various protein concentrations with 0.5 mM FSBA in the presence or absence of an excess of ATP. Even at relatively high total protein concentrations (2.5 mg/mL), labeling with FSBA could be effectively prevented by inclusion of an excess of ATP (Figure 2B). Similarly, labeling of proteins with FSBG could be prevented by inclusion of an excess of GTP (Figure 2C). Consequently, proteins present within crude mixtures that react with the probes represent genuine nucleotide-binding proteins. Finally, to confirm our ability to label a known ATP-binding protein present within a mixture of proteins, we examined the ability of the antisera to detect the protein-tyrosine kinase Lck. Membrane fractions were prepared from LSTRA T cells,
Detection of Nucleotide-Binding Proteins
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Figure 2. Optimization of labeling. (A) A membrane fraction isolated from DG75 cells was labeled with 0.5 mM FSBA where indicated in the presence of increasing concentrations of ATP. Covalent modification of proteins was determined by Western blotting of NaOHtreated membranes with the anti-SB antisera. (B) Whole cell lysates, prepared in buffer containing 1% Triton X-100, were diluted to the final protein concentration indicted and then labeled by incubation with 0.5 mM FSBA in the presence or absence of 10 mM ATP. Immunoblotting was performed after transfer of labeled proteins to a PVDF membrane and treatment of the membrane with 0.2 M NaOH for 30 min. (C) The membrane fraction from DG75 cells was treated in the same manner as described above in panel A, except the labeling was carried out using 0.5 mm FSBG in the presence or absence of 10 mM GTP.
Figure 3. Lck is labeled by FSBA. Membrane fractions from LSTRA or JCaM1 cells were diluted to equal protein concentrations and incubated with 0.5 mM FSBA for 30 min. Labeled proteins were then separated by 2-D gel electrophoresis and imunoblotted using anti-SB or anti-Lck antibodies with (+) or without (-) treatment with 0.2 M NaOH for 30 min.
which overexpress Lck, and JCaM1 T cells, which do not express Lck. The protein mixture was treated with FSBA, separated by 2-D gel electrophoresis and transferred to PVDF membranes for immunoblotting. A portion of the blot is shown in Figure 3. A protein that co-localized with Lck, as determined by Western blotting with an anti-Lck antibody, was labeled in the membrane fraction prepared from LSTRA cells. This protein was not present in the membrane fraction isolated from Lck-deficient JCaM1 cells. As a further show of antibody specificity, the labeled protein was only detected after treatment of the membrane with base to expose the SB epitope. This approach should be generally useful for the detection of protein kinases since all classical protein kinases possess, like Lck, a Lys in their ATP-binding sites that is covalently modified by FSBA.31 Labeling and Identification of Nucleotide-Binding Proteins. There is considerable interest in the analysis of distinct subsets of the total proteome. To determine if the antisera would be useful for the large-scale identification of ATP-binding proteins
Figure 4. Labeling of ATP-binding proteins in a crude membrane fraction. Proteins present in the membrane fraction isolated from DG75 B cells were incubated with 0.5 mM FSBA in the absence (upper panel) or presence (lower panel) of 10 mM ATP, separated by 2-D gel electrophoresis, transferred to PVDF membranes and detected by immunoblotting with anti-SB antibodies.
present within a heterogeneous mixture, we labeled proteins in crude extracts and separated the mixture by 2-D gel electrophoresis. Proteins present in either detergent lysates or membrane preparations from DG75 B cells were incubated with FSBA under the conditions defined above that yield the specific modification of ATP-binding proteins. Modified proteins were separated using 2-D gel electrophoresis and detected within the gel by silver staining or transferred to a PVDF membrane, which was treated with NaOH and immunorevealed with the anti-SB antiserum. Upward of 100 distinct spots were observed Journal of Proteome Research • Vol. 3, No. 6, 2004 1187
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Table 1. Proteins Identified from DG75 Cells by Labeling with FSBA
IDa
apparent MW (kDa)
calcd MW (kDa)
apparent pI
calcd pI
protein
AC no.
ATP binding
subcellular localization
Heterogeneous nuclear ribonucleo-protein Ub Serine/threonine-protein kinase 10c Stress-70 proteind
Q00839
Y
O94804
Y
P38646
Y
Heat shock cognate 71 kDa proteine T-complex protein 1, ζ subunitf LMW Splice isoform of Kininogeng
P11142
Y
nuclear, binds to double- and singleribonucleosome stranded DNA and RNA unknown specific substrates are unknown mitochondrial chaperone; involved in molecular and cellular aging unknown chaperone
P40227
Y
cytoplasmic
P01042
N
secreted
1
110
90
6
5.76
1
110
112
6
6.52
3, 20
72
69
6
5.44
4, 18
72
71
6
5.37
6
55
58
6.5
6.24
9
50
48
6.5
6.29
12
42
42
6
5.29
Actinh
P60709
Y
cytoplasmic
16
100
100
6
5.14
P55072
Y
17
75
70
6
5.01
P11021
Y
nuclear and cytoplasmic ER lumen
19 21
70 60
70 58
6 6
5.2 5.24
P13796 P10809
N Y
22
50
52
6 (5.00)
5.00
Transitional endoplasmic reticulum ATPasei 78 kDa glucose-regulated proteinj L-plastink 60 kDa heat shock proteinl ATP synthase β chainm
P06576
Y
cytoplasmic mitochondrial matrix mitochondria
function
molecular chaperone folding of actin and tubulin inhibitor of thiol protease and the aggregation of thrombocytes cytoskeletal protein involved in various types of cell motility important in Golgi and tER formation protein assembly actin-bundling protein folding of mitochondrial proteins produces ATP from ADP. The β chain is the catalytic subunit
a ID numbers indicate the sample. Numbers 1-15 are from the FSBA-labeled membrane fraction of DG75 cells; 16-30 are from the FSBA-labeled whole cell lysate. The references for protein function and localization are as follows: b34,37, c35, d38,39, e40,41, f 42, g43, h44, i41,45, j46, k47, l48, m49.
in each FSBA-labeled sample, consistent with the presence of a large number of ATP-binding proteins in eukaryotic cells (Figures 4 and 5). The inclusion of an excess of ATP in the reaction prevented the labeling of nearly all proteins as illustrated in Figure 4 for studies on the membrane fraction. Images of the gels and blots were then merged and aligned as shown in the example in Figure 5 for proteins present in detergent lysates. A small sample of 15 overlapping spots were excised from each silver stained gel, trypsinized and the resulting peptides were analyzed by mass spectrometry. Overall, 14 proteins were identified from analyses of the FSBA labeled samples, including two proteins that were identified from both the detergent lysate and the membrane fraction (Table 1). The majority were ATP-binding proteins, consistent with their covalent modification by FSBA. The class of proteins most frequently identified were chaperone proteins, which are abundant ATP-binding proteins involved in protein folding. The chaperones listed here function in a large number of pathways, including those controlling apoptosis, T and B cell signaling and protein degradation.32,33 Other abundant ATP-binding proteins identified were heterogeneous nuclear ribonucleoprotein U, which contains both RNA and ATP-binding domains and is involved in processing of RNA transcripts;34 the cytoskeletal protein, actin, whose polymerization is regulated by ATP; the transitional endoplasmic reticulum ATPase (TERA, also known as valosin-containing protein or VCP), which is ubiquitous and makes up approximately 1% of the protein in cell types where it has been measured;32 and the catalytic subunit of mitochondrial ATP synthase. A single protein kinase, serine/threonine kinase 10 or STK10, which is also known as lymphocyte oriented kinase or LOK, was identified by this assay. STK10 is expressed primarily in lymphocytes and is related to the polo-like kinase kinases.35,36 The lack of additional protein kinases identified in this relatively small sample is most likely a function of the small number of 1188
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spots analyzed and the relatively low levels at which they are expressed. Many labeled proteins were present in too small of a concentration to be identified by silver staining. In addition, many labeled proteins could not be isolated as discrete spots due to the overwhelming concentration of unlabeled protein migrating at similar positions. These problems could be addressed by using larger samples and bigger 2-D gels with greater resolving power, by further subcellular fractionation techniques or by enriching the labeled proteins prior to separation. It should also be possible to enrich selectively the labeled peptides by immunoaffinity chromatography for their eventual analysis and identification by MS/MS approaches. Two of the identified proteins do not have an ATP binding site and most likely are present as artifacts of the separation protocol that was used. One is L-plastin, an abundant actinbinding protein not known to possess a nucleotide-binding site. The second was low molecular weight kininogen, which also is not known to have any association with ATP and is a serum protein. It is possible that this protein is present as a contaminant from the cell culture medium. Conclusions. These studies demonstrate the feasibility and practicality of using FSBA and FSBG as chemical probes in conjunction with an anti-SB antibody for the analysis of nucleotide-binding proteins in the proteome. This approach requires little sample preparation and uses no radioisotopes. Additionally, the labeling is covalent, which assists in detection and could be used to define both the binding protein and the specific binding site. The applications of this approach are limited by imagination alone, including monitoring of ATP- and GTP-binding proteins in various cells types, in various subcellular compartments or macromolecular complexes and how they change in response to various stimuli. The SB antibody, in particular, should be useful for monitoring proteins modified by reaction with any probe containing a 4-(fluorosulfonyl)benzoyl moiety. The synthesis of such probes is expected to
Detection of Nucleotide-Binding Proteins
Figure 5. Alignment of silver stained and SB-labeled proteins from detergent lysates from DG75 cells. Identical samples containing the detergent-soluble lysate of DG75 cells were diluted to a final concentration of 2.5 mg/mL and treated with 0.5 mM FSBA for 30 min. The samples were run on two separate gels, which were then silver stained (A) or transferred to PVDF, treated with NaOH, and immunoblotted with the anti-SB antibody (B). The gels were then aligned (color added) according to molecular weight, and prominent protein spots (C).
be straightforward since 4-(fluorosulfonyl)benzoyl chloride is readily available and the coupling through the formation of an ester bond is possible when the binding moiety contains an available hydroxyl group.
Acknowledgment. This work was supported by National Institutes of Health Grant CA37372 awarded by the National Cancer Institute. References (1) Adam, G. C.; Sorensen, E. J.; Cravatt, B. F. Mol. Cell. Proteomics 2002, 1, 781-790. (2) Banks, R. E.; Dunn, M. J.; Hochstrasser, D. F.; Sanchez, J. C.; Blackstock, W.; Pappin, D. J.; Selby, P. J. Lancet 2000, 356, 17491756. (3) Werner, T. Mass Spectrom. Rev. 2004, 23, 25-33.
research articles (4) Patricelli, M. P.; Giang, D. K.; Stamp, L. M.; Burbaum, J. J. Proteomics 2001, 1, 1067-1071. (5) Kidd, D.; Liu, Y.; Cravatt, B. F. Biochemistry 2001, 40, 4005-4015. (6) Adam, G. C.; Cravatt, B. F.; Sorensen, E. J. Chem. Biol. 2001, 8, 81-95. (7) Adam, G. C.; Sorensen, E. J.; Cravatt, B. F. Mol. Cell. Proteomics 2002, 1, 828-835. (8) Adam, G. C.; Sorensen, E. J.; Cravatt, B. F. Nat. Biotechnol. 2002, 20, 805-809. (9) Jessani, N.; Liu, Y.; Humphrey, M.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10335-10340. (10) Greenbaum, D.; Baruch, A.; Hayrapetian, L.; Darula, Z.; Burlingame, A.; Medzihradszky, K. F.; Bogyo, M. Mol. Cell. Proteomics 2002, 1, 60-68. (11) Greenbaum, D.; Medzihradszky, K. F.; Burlingame, A.; Bogyo, M. Chem. Biol. 2000, 7, 569-581. (12) Greenbaum, D. C.; Arnold, W. D.; Lu, F.; Hayrapetian, L.; Baruch, A.; Krumrine, J.; Toba, S.; Chehade, K.; Bromme, D.; Kuntz, I. D.; Bogyo, M. Chem. Biol. 2002, 9, 1085-1094. (13) Liu, Y.; Patricelli, M. P.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14694-14699. (14) Bartnicki, D., et al. Novagen Innovations 2004, 19, 6-7. (15) Yee, A.; Pardee, K.; Christendat, D.; Savchenko, A.; Edwards, A. M.; Arrowsmith, C. H. Acc. Chem. Res. 2003, 36, 183-189. (16) Weir, M.; Swindells, M.; Overington, J. Trends Biotechnol. 2001, 19, S61-66. (17) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. Science 2002, 298, 1912-1934. (18) Lo, L. C.; Pang, T. L.; Kuo, C. H.; Chiang, Y. L.; Wang, H. Y.; Lin, J. J. J. Proteome Res. 2002, 1, 35-40. (19) Pal, P. K.; Reischer, R. J.; Wechter, W. J.; Colman, R. F. J. Biol. Chem. 1978, 253, 6644-6646. (20) Pal, P. K.; Colman, R. F. Biochemistry 1979, 18, 838-845. (21) Pal, P. K.; Wechter, W. J.; Colman, R. F. Biochemistry 1975, 14, 707-715. (22) Bennett, J. S.; Colman, R. F.; Colman, R. W. J. Biol. Chem. 1978, 253, 7346-7354. (23) Esch, F. S.; Allison, W. S. J. Biol. Chem. 1978, 253, 6100-6106. (24) Pettigrew, D. W., and C. Frieden J. Biol. Chem. 1978, 253, 36233627. (25) Wyatt, J. L.; Colman, R. F. Biochemistry 1977, 16, 1333-1342. (26) Esch, F. S.; Allison, W. S. Anal. Biochem. 1978, 84, 642-645. (27) Anostario, M., Jr.; Harrison, M. L.; Geahlen, R. L. Anal. Biochem. 1990, 190, 60-65. (28) Parker, P. J. FEBS Lett. 1993, 334, 347-350. (29) Laemmli, U. K. Nature 1970, 227, 680-685. (30) Celis, J. E.; Ratz, G.; Basse, B.; Lauridsen, J. B.; Celis, A.; Jensen, N. A.; Gromov, P. In Cell Biology: A Laboratory Handbook; Celis, J. E., Carter, N., Hunter, T., Shotton, D., Simons, K., Small, J. V., Eds.; Academic Press: New York; 1994, pp 375-385. (31) Xu, B.; English, J. M.; Wilsbacher, J. L.; Stippec, S.; Goldsmith, E. J.; Cobb, M. H. J. Biol. Chem. 2000, 275, 16795-16801. (32) Wang, Q.; Song, C.; Li, C. H. J. Struct. Biol. 2004, 146, 44-57. (33) Carty, S. M.; Greenleaf, A. L. Mol. Cell. Proteomics 2002, 1, 598610. (34) Fackelmayer, F. O.; Richter, A. Biochemistry 1994, 33, 1041610422. (35) Kuramochi, S.; Moriguchi, T.; Kuida, K.; Endo, J.; Semba, K.; Nishida, E.; Karasuyama, H. J. Biol. Chem. 1997, 272, 2267922684. (36) Endo, J.; Toyama-Sorimachi, N.; Taya, C.; Kuramochi-Miyagawa, S.; Nagata, K.; Kuida, K.; Takashi, T.; Yonekawa, H.; Yoshizawa, Y.; Miyasaka, N.; Karasuyama, H. FEBS Lett. 2000, 468, 234-238. (37) Fackelmayer, F. O.; Richter, A. Biochim. Biophys. Acta 1994, 1217, 232-234. (38) Li, G. C.; Li, L.; Liu, R. Y.; Rehman, M.; Lee, W. M. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 2036-2040. (39) Bhattacharyya, T.; Karnezis, A. N.; Murphy, S. P.; Hoang, T.; Freeman, B. C.; Phillips, B.; Morimoto, R. I. J. Biol. Chem. 1995, 270, 1705-1710. (40) Dworniczak, B.; Mirault, M. E. Nucleic Acids Res. 1987, 15, 51815197. (41) Strausberg, R. L.; Feingold, E. A.; Grouse, L. H.; Derge, J. G.; Klausner, R. D.; Collins, F. S.; Wagner, L.; Shenmen, C. M.; Schuler, G. D.; Altschul, S. F.; Zeeberg, B.; Buetow, K. H.; Schaefer, C. F.; Bhat, N. K.; Hopkins, R. F.; Jordan, H.; Moore, T.; Max, S. I.; Wang, J.; Hsieh, F.; Diatchenko, L.; Marusina, K.; Farmer, A. A.; Rubin, G. M.; Hong, L.; Stapleton, M.; Soares, M. B.; Bonaldo, M. F.; Casavant, T. L.; Scheetz, T. E.; Brownstein, M. J.; Usdin, T. B.; Toshiyuki, S.; Carninci, P.; Prange, C.; Raha, S. S.; Loquellano, N. A.; Peters, G. J.; Abramson, R. D.; Mullahy, S. J.; Bosak, S. A.;
Journal of Proteome Research • Vol. 3, No. 6, 2004 1189
research articles McEwan, P. J.; McKernan, K. J.; Malek, J. A.; Gunaratne, P. H.; Richards, S.; Worley, K. C.; Hale, S.; Garcia, A. M.; Gay, L. J.; Hulyk, S. W.; Villalon, D. K.; Muzny, D. M.; Sodergren, E. J.; Lu, X.; Gibbs, R. A.; Fahey, J.; Helton, E.; Ketteman, M.; Madan, A.; Rodrigues, S.; Sanchez, A.; Whiting, M.; Young, A. C.; Shevchenko, Y.; Bouffard, G. G.; Blakesley, R. W.; Touchman, J. W.; Green, E. D.; Dickson, M. C.; Rodriguez, A. C.; Grimwood, J.; Schmutz, J.; Myers, R. M.; Butterfield, Y. S.; Krzywinski, M. I.; Skalska, U.; Smailus, D. E.; Schnerch, A.; Schein, J. E.; Jones, S. J.; Marra, M. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16899-16903. (42) Li, W. Z.; Lin, P.; Frydman, J.; Boal, T. R.; Cardillo, T. S.; Richard, L. M.; Toth, D.; Lichtman, M. A.; Hartl, F. U.; Sherman, F. J. Biol. Chem. 1994, 269, 18616-18622. (43) Takagaki, Y.; Kitamura, N.; Nakanishi, S. J. Biol. Chem. 1985, 260, 8601-8609. (44) Ohmori, H.; Toyama, S. J. Cell Biol. 1992, 116, 933-941.
1190
Journal of Proteome Research • Vol. 3, No. 6, 2004
Moore et al. (45) Hu, R. M.; Han, Z. G.; Song, H. D.; Peng, Y. D.; Huang, Q. H.; Ren, S. X.; Gu, Y. J.; Huang, C. H.; Li, Y. B.; Jiang, C. L.; Fu, G.; Zhang, Q. H.; Gu, B. W.; Dai, M.; Mao, Y. F.; Gao, G. F.; Rong, R.; Ye, M.; Zhou, J.; Xu, S. H.; Gu, J.; Shi, J. X.; Jin, W. R.; Zhang, C. K.; Wu, T. M.; Huang, G. Y.; Chen, Z.; Chen, M. D.; Chen, J. L. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9543-9548. (46) Ting, J.; Lee, A. S. DNA 1988, 7, 275-286. (47) Lin, C. S.; Park, T.; Chen, Z. P.; Leavitt, J. J. Biol. Chem. 1993, 268, 2781-2792. (48) Venner, T. J.; Singh, B.; Gupta, R. S. DNA Cell Biol. 1990, 9, 545552. (49) Neckelmann, N.; Warner, C. K.; Chung, A.; Kudoh, J.; Minoshima, S.; Fukuyama, R.; Maekawa, M.; Shimizu, Y.; Shimizu, N.; Liu, J. D. Genomics 1989, 5, 829-843.
PR0498943
