Analysis of the Eluted Proteins

3.7.1. Electrophoretic Mobility Shift Assay (EMSA)

1. EMSA is performed essentially as described previously (4), with minor modifications. In brief, carry out prebinding of nuclear extract (5-10 mg/mL), or respective protein fraction, to the poly(dl-dC) at 25°C for 10 min in buffer containing 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 25 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 0.05 mg/mL poly(dI-dC)poly(dI-dC).

2. Add the probe (3.5 fmol, approx 2 o 104 cpm) to the above reaction, mix, and incubate at 25°C for 20 min.

3. Add 1 ^L of 10X gel loading buffer, containing 250 mM Tris-HCl (pH 7.5), 0.2% bromophenol blue, 0.2% xylene cyanol, and 40% glycerol, to the reaction.

4. Load onto a 6% native gel (which should be prerun for 90 min at 100 V) in 0.5X nondenaturing Tris-Borate-EDTA buffer. Perform the electrophoresis at 25°C and 100 V for about 3.5 h.

5. Transfer the gel onto Whatman paper, vacuum dry, and expose to Hyperfilm (Amer-sham Pharmacia) for the desired period at -80°C and with an intensifier screen.

3.7.2. Western Blotting

Whenever a specific antibody is available, use Western blotting to detect the presence of the captured protein in the eluates. Only one-tenth (or less) of the eluted material should be used for Western blotting anlysis.

1. Protein solutions are separated first by electrophoresis in 4 to 15% gradient poly-acrylamide sodium dodecylsulfate gels, and then transferred onto a PVDF membrane in Tris-glycine-methanol buffer.

2. After the membranes are blocked for at least 1 h at room temperature with PBS, 0.05% Tween-20, 5% dry milk, the respective antibodies are added to the membranes at concentrations of 1 ^g/mL in PBS, 0.05% Tween-20, for 2 h at room temperature.

3. Anti-mouse or anti-rabbit-horseradish peroxidase-conjugated antibodies (Santa Cruz) and the Enhanced chemiluminescence kit (Pierce) are used for visualization of the immune complexes.

3.7.3. Identification of the Captured Proteins by Mass Spectrometry

1. Excise the bands from the stained/destained SDS gel, corresponding to the proteins eluted from the DNA-magnetic beads, as well as the proteins that remain on the beads after elution (see Note 16).

2. Digest these gel slices with trypsin.

3. Fractionate the mixtures on a Poros 50 R2 RP microtip.

4. Analyze the resulting peptide pools by matrix-assisted laser-desorption/ionization reflectron time-of-flight (MALDI-reTOF) MS using a BRUKER UltraFlex TOF/TOF instrument (Bruker Daltonics).

5. Take selected experimental masses (m/z) to search a nonredundant protein database (NR; National Center for Biotechnology Information, Bethesda, MD), utilizing the PeptideSearch (Matthias Mann, Southern Denmark University, Odense, Denmark) algorithm.

6. A molecular weight range twice the predicted weight should be covered, with a mass accuracy restriction better than 40 ppm, and should allow a maximum of one missed cleavage site per peptide.

7. Perform mass spectrometric sequencing of selected peptides by MALDI-TOF/TOF (MS/MS) analysis on the same prepared samples, using the UltraFlex instrument in "LIFT" mode.

8. Take fragment ion spectra to search the NR database using the MASCOT MS/MS Ion Search program (Matrix Science, London, UK) (11). Any identification thus obtained should be verified by comparing the computer-generated fragment ion series of the predicted tryptic peptide with the experimental MS/MS data.

In general, positive identifications are made on the basis of a Mascot MS/ MS score ©64 (p < 0.05) for a single peptide, or a score of ©35 for each of two or more peptides (combined score ©80). Alternatively, a Mascot MS/MS score ©35 for a single peptide should be combined with a peptide mass fingerprinting (PMF) result that yielded ©15% sequence coverage.

4. Notes

1. Incubation of NB4 cells in the hypotonic buffer causes the cells to swell. The homogenization disrupts cell membranes mechanically while the nuclei remain intact. This process can be monitored in a phase-contrast microscope. After homoge-nization, no large cell should be seen, only small dense dots representing the nuclei.

2. Slow addition of high salt buffer to the nuclear suspension prevents dissociation of the chromatin through local excess of salt leading to lysis of the nuclei and increased viscosity. Blood cells are particularly sensitive to exposure to high salt, and frequently the extract becomes viscous. This problem can be solved by short sonication (2 min at 50% duty cycle and output control 3 on a Branson Sonifier 450) without any effect on the protein quality.

3. During this dialysis step, a white protein precipitate is formed, which is removed after the dialysis by centrifugation for 20 min at 15,000g. In our experience, this precipitation is not specific. An overall protein loss occurs owing to the drop in salt from 0.42 to 0.15 M.

4. One of the major reasons for fractionating nuclear proteins prior to DNA-affinity capture is to reduce the binding of nonspecific proteins. We conducted experiments on direct binding of crude nuclear extract to specific oligo DNA-beads (AP.1, NFkB, Sp.1) under the condition established in EMSA (3). The profiles of the proteins eluted from these DNA-beads looked nearly identical, regardless of the difference in DNA sequences attached, suggestive of a high degree of nonspecific binding. Several major bands from the gel containing such proteins were excised and identified by MS as mostly nonspecific RNA or DNA binding proteins, some with a preference for binding to free DNA ends (PARP, DNA-dependent protein kinase, Ku protein, splicing factors, DNA helicase, and so on) Thus, under conditions in which specific binding is observed in EMSA, most of the DNA binding sites on the beads are occupied nonspecifically.

5. Ion exchangers (DEAE-cellulose, BioRex70) or heparin-sepharose, phenyl-sephar-ose, and gel-filtration columns (Sephacryl S300) have been used as a prefraction-ation step for affinity purification of nucleic acid binding proteins (7,8). However, we chose to fractionate nuclear extracts on P11 phosphocellulose as it provides a unique combination of ion exchange and affinity properties. P11 chromatography is a simple, popular procedure that has been used as a first step in many proven transcription factor purification schemes; it allows enrichment of multiple factors, cofactors, and corepressors in separate fractions (2,9,10). In this way, parallel affinity captures of several different factors, each from a separate P11-fraction but all derived from a single batch of nuclear extract, can be performed.

6. For best results, the instructions for preparation of the P11 resin from Whatman that come with the product must be followed strictly. The P11 column chromatography can be run with FPLC equipment (Amersham Pharmacia Biotech); the flow rate must not exceed 0.4 mL/min to avoid pressure buildup.

7. It should be noted that PCR-based concatamerization reactions are sequence specific and must be carefully optimized first in pilot experiments. For example, for GABP-binding concatamers, the PCR conditions were: 95°C for 2 min followed by 14 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 3 min; for PURa: 95°C for 2 min followed by 9 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 3 min; for AP.1: 92°C for 2 min followed by 7 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 3 min (Fig. 1); for PU.1: 92°C for 2 min followed by 14 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 3 min; for RARa: 95°C for 2 min followed by 9 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 5 min.

8. The efficiency of concatamer binding to the M280 beads depends on the length of the DNA. According to the manufacturer's specifcations, binding of 1000 bp DNA is on the order of 2 to 5 ^g/mg beads; lower and higher molecular weight (DNA)n bind at up to approx 12 ^g/mg.

9. Effect of salt: we observed a striking correlation between the salt concentration at which a given protein eluted from the P11 column and the stability of the DNAprotein complex in the presence of salt as measured by EMSA. Proteins eluting at

<300 mM NaCl formed less stable complexes with DNA than proteins that eluted at ©500 mM. For example, formation of the GABP-DNA complex was adversely affected by the presence of 500 mM compared with 50 mM KCl, whereas high salt had no such destabilizing effect on the PU.1-DNA (3). In some cases (e.g., AP.1 and PURa), the presence of high salt caused a slight change in the mobility of the DNA-protein complexes, but the DNA binding specificity was preserved, as confirmed in subsequent capturing experiments.

10. The effect of Mg2+ on DNA binding must also be optimized a priori in an EMSA. Low Mg2+ concentrations would reduce the probability for degradation of DNA concatamers, attached to the beads, by endogeneous nucleases. We noticed that five chosen proteins bound equally well to their cognate DNA in the presence or absence of Mg2+ (3). Consequently, all further DNA captures could be performed in the absence of magnesium.

11. As nonspecific protein binding appears to be the major obstacle for successful transcription factor affinity capture, the problem could be reduced to the adequate removal of such interfering proteins. Conceptually, a suitable medium could be an immobilized DNA sequence that binds many or all of the nonspecific proteins but not the specific one. We envisioned that this could be readily accomplished by using DNA ligands consisting of minimally modified binding sites, for instance, by one or more point mutations, just enough to abolish specific factor binding in vitro. The mutated DNA sequences should be derived from pilot competition EMSA experiments performed in the presence of an excess of unlabeled, mutant DNA probes. Point mutations that fully abolish competition will be then selected. Preclearing of protein solutions with beads loaded with such DNA is referred to as negative selection.

12. Based on our previously reported results (3), we propose the following general, empiric rules for future DNA affinity capture and identification of transcription factors by MS, as illustrated in Fig. 2.

a. After initial fractionation of nuclear extract on a P11 column, it is critical to determine whether the protein of interest will form a stable complex with its target DNA in the presence of at least 500 mM salt. This is generally the case for all proteins that elute from P11 at or higher than 500 mM salt (e.g., AP.1, PURa, PU.1, and others).

b. Binding affinities may also be known from prior molecular characterization of the promoter of interest.

c. Affinity and salt tolerance, often directly linked, are the primary determinants of the order in which positive and negative selections are then carried out: "negative/positive" for high-affinity, high-salt binders; "positive/negative/ positive" for low-affinity, low-salt binding transcription factors.

d. The addition of a positive step prior to the negative selections serves a dual purpose: (1) the protein mass is drastically reduced, and (2) protein eluates are very concentrated, allowing a further microcapture format. This is not possible for high-affinity binders, as approximately 1 M salt is required for elution, in most cases resulting in some degree of denaturation, which complicates both assaying and subsequent affinity captures. Consequently, the first positive selection must be omitted. This can be compensated, in part, by the option to include 0.5 M salt throughout the entire selection process, thereby also increasing capturing stringency, albeit by a different mechanism. Both options appear to be mutually exclusive.

e. Regardless of the selection sequence, the number of negative selections on mutant DNA-magnetic beads always depends on the abundance of the transcription factors in the respective fraction. Low-abundant species require more nuclear extract and proportionally more rounds of negative selection than the higher abundant ones, for example, one round for GABP and AP.1, two for PURa, three for PU.1, but more than three for RARa (Fig. 2). All capturing schemes end with a positive selection, including extensive washing of the beads with E. coli competitor ds- and ssDNA.

f. Final elution is done with 0.5 MNaCl for low-affinity binders, or by boiling in Laemmli gel-loading buffer for the others, followed by gel electrophoresis and identification by the mass spectrometric method of choice.

g. The purification scheme generates proteins in amounts and form that are readily compatible with MALDI TOF-based peptide mass fingerprinting.

13. Poly(dI:dC) competitor at a concentration of 0.1 mg/mL, already high, is insufficient to prevent the unwanted nonspecific DNA-protein interactions in binding to DNA-magnetic beads. We established experimentally that nonspecific protein binding is significantly reduced in the presence of double-stranded oligodI:dC and does not visibly increase when concatamerized DNA instead of oligonucleotide is used as affinity ligand on the beads (3). From the premise that several major, non-specifically binding proteins (e.g., DNA-PK, Ku autoantigen, PARP) have high affinities for DNA breaks or ends, we reasoned that the resultant background could be reduced by using (1) beads with DNA affinity ligands of a lower ends-to-binding site ratio and, conversely, (2) competitor DNA of a higher ends-to-weight ratio. The latter option was implemented by including a 30-bp long, synthetic double-stranded oligo(dI:dC) in the binding reaction, in addition to the standard polymeric competitor (6). An 0.1 mg/mL oligo(dI:dC) concentration was empirically found to be the highest that did not visibly interfere with protein-DNA binding in EMSA. As for the first option, long (©1000 bp) 5'-biotinylated double-stranded concatamers of specific oligonucleotides that are produced in a self-priming PCR effectively increase ligand density on the beads without added DNA ends.

14. We prepare single-stranded E. coli DNA by heating double-stranded genomic DNA in boiling water for 20 min and quick chilling on ice.

15. Silver staining of the sodium dodecylsulfate gels prior to mass spectrometric analysis is not advisable as silver binds to traces of competitor DNA left in the final preparations.

16. Clean work while handling the samples and processing the gel slices (wearing gloves, using clean glassware and tools) is imperative for successful identification since the mass spectrometry can detect the presence of protein contaminants, particularly keratins.


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