Name: profiling of methyltransferases and other s-adenosyl-l-homocysteine-binding proteins by capture compound mass spectrometry (ccms)
Uploaded: 2010-12-20T16:20:00
Duration: 17 min 12 s
Description: Watch this Scientific Journal Video about Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS at JoVE.com

This work was supported by the Human Frontier Science Program Organization (HFSP Award 2007, RGP0058/2007-C). We thank Prof. Richard Roberts for initiating the project and for fruitful discussions.

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Thomas Lenz, Olivia Grabner, Mirko Glinski, Mathias Dreger, and Hubert Kosterare employed by caprotec bioanalytics GmbH, Inc that produces some of the reagents used in this article.

The following precautions and comments may be useful when following the described protocol: a) A major advantage of CCs lies in the formation of a covalent bond between the CC and the MTase, as this permits subsequent stringent washing conditions. The covalent crosslink is achieved by a photoreaction triggered by UV light (310 nm max.). Normal overhead light contains only a small fraction of UV, however, protect the SAH-CC from longer exposure to overhead or even sun light up to the controlled and cooled irradiation in the caproBox. b) The biological samples from which the MTases are to be isolated may contain proteins prone to denaturation, consequently it is mandatory to keep the samples cool and to avoid frothing at all times. c) The caproBox cools the samples to 0-4 &deg;C, the lamps emitting the UV light, however, also emit heat. Therefore it is necessary to briefly centrifuge the vials before irradiation, so proteins adhering to the caps or vial walls cannot form precipitation seeds. d) If re-suspension of the magnetic beads (e.g. in the wash solutions) is not possible by hand, shortly apply ultrasound by placing the samples into an ultrasound bath. e) Freshly prepare the 0.2% TFA/60 %ACN solution. We found that otherwise the captured proteins may not be cleaved from the beads. f) The final analysis of captured proteins is carried out by LC-MS/MS. Mass spectrometry is a highly sensitive method. It is necessary to use exclusively LC-MS grade reagents in the final steps (step 2.11 and further). Avoid contamination of the experiments by external protein sources, e.g. keratin originating from dust or from the experimenter. Particularly during the final digestion steps, it is recommended to pay attention to a clean work space, to wear gloves and a lab coat and possibly a hair net or ideally perform the final steps under a clean bench. Prepare the 50 mM ammonium bicarbonate buffer used for tryptic digest in LC MS grade water, filter through 0.22 &mu;m filter, aliquot, store at -20&deg;C, and use each aliquot only once to avoid contaminations. The trypsin solution (sequencing grade, Roche, prepare a 0.5 &mu;g/&mu;l solution by adding 1 mM HCl to lyophilized trypsin) can be stored at 4&deg;C for several weeks. g) To obtain reliable mass spectra it is essential to have a stable spray in ESI-MS/MS analysis. h) For other LC-MS/MS systems than the one used in the present study, measurement parameters and peptide identification algorithms must be adjusted individually.
The following modifications are possible with respect to the described protocol: a) Alternatively to releasing the proteins from the beads by 60% ACN/0.2% TFA (step 2.11), the proteins can be directly tryptically digested within a bead suspension (same volumes as in step 5.1) or, for SDS-PAGE, the proteins can be released by suspending and heating the beads collected after step 2.9 to 95&deg;C for 10 min in SDS sample buffer (both, the whole suspension or only the supernatant can be loaded into the gel pocket). We found that the beads slowly release small amounts of polymer into the aqueous tryptic digest solution during on-bead tryptic digest even after several aqueous wash steps. The polymer contamination interferes with MS peptide identification and can be washed off the beads using 80% ACN (at least three times). After the 80% ACN wash steps, the beads should be washed once with water prior to on-bead tryptic digest. b) Western blots using streptavidin-horseraddish peroxidase and ECL substrate can also be used to visualize successful crosslinking of the biotin containing CC to the proteins. Therefore, either the gel obtained after step 3.2 can be blotted or, because the sensitivity is about 10-fold higher than silver staining of gels, 10 &mu;l samples after step 2.7 may also be analyzed by western blot. Mind that in the latter case endogeneously biotinylated proteins will also be detected besides the proteins artificially biotinylated by the SAH-CC. c) We found that it depends on the special system (lysate, addressed target proteins, selectivity and reactivity function of he CC), whether the "off-bead" configuration, where the crosslinking reaction takes place between free CC and protein in solution 4 or the presently described "on-bead" configuration (Figure 1B) performs better.
In general, the method should also be compatible with any state-of-the art stable isotope protein or peptide labelling technology, or assessment of the capture sample by 2D gel electrophoresis. In the "off-bead" configuration, it is also possible to capture proteins within whole cells (unpublished results). Furthermore, the drug or cofactor binding site of a protein can be outlined by determining the close-by crosslinking position of the CC within the protein sequence by MS peptide sequencing. The binding mode of a small molecule to a protein can be explored by using different chemical attachment positions at the selectivity function and different linker lengths. As shown in the present study, also protein binding partners of the proteins addressed by the selectivity function (RplK as substrate of PrmA) or unknown small molecule protein interactions (SAH to GlnA) can be identified. Summarized, the additional feature of the CCs, the photo-crosslinking reactivity, allows for the isolation and identification of low abundant proteins or functional protein families from complex protein mixtures with high sensitivity and provides scientists with an additional tool for studying small molecule - protein interactions.

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		<div>
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SAH caproKit&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
<td>1-1010-050 (50 reactions) 1-1010-010 (10 reactions)</td>
<td>Includes the SAH-CC, SAH competitor, streptavidin coated magnetic beads, capture buffer, and wash buffer</td>
</tr>
<tr>
<td>caproBox&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
<td>1-5010-003 (110 V) 1-5010-004 (230 V)</td>
<td>For reproducible photo-activation while cooling the samples</td>
</tr>
<tr>
<td>caproMag&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
<td>1-5100-001</td>
<td>For easy handling of magnetic particles without pipetting</td>
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profiling of methyltransferases and other s-adenosyl-l-homocysteine-binding proteins by capture compound mass spectrometry (ccms)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Watch this Scientific Journal Video about Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS at JoVE.com

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

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