The Structural Genomics Consortium is a registered charity (no: 1097737) that receives funds from AbbVie, Bayer AG, Boehringer Ingelheim, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in Canada and US), Pfizer, Takeda and the Wellcome Trust [106169/ZZ14/Z].

1. Cell culturing and plating
NOTE: Culture cells with recommended media and test routinely for mycoplasma contamination. HEK293T, MCF7, and C2C12 cells were chosen as examples since these cell lines were successfully used in PRMT assays.
<ol>
	<li>Culture HEK293T, MCF7, and C2C12 in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 U mL&#8722;1), and streptomycin (100 &#181;g mL&#8722;1) in 10 cm tissue-culture treated (TC) dishes.</li>
	<li>For the PRMT8 assay, grow PRMT1 inducible knockdown HEK293T cells in media containing doxycycline (2 &#181;g/mL) for 3 days before assay start.</li>
	<li>To plate the cells, remove and discard media from the plate.</li>
	<li>Add 10 mL of PBS (without Ca+2 and Mg+2 ions) to wash cells and discard the solution.</li>
	<li>Add 1 mL of Trypsin-EDTA (0.25%), incubate for 1 min at room temperature (RT), and then discard the solution. Incubate until cells become round and detach from the plate. Tap the plate to help detach cells, if needed. For hard-to-trypsinize cells, such as C2C12, incubate the plate for 1-2 min at 37 &#176;C. 
	NOTE: Avoid cell exposure to trypsin solution for longer periods (&#62;10 min) as it will reduce cell viability.</li>
	<li>Add 1 mL of prewarmed media to the plate, and gently pipette cells up and down to break-up cell clumps. Transfer cells to a 15 mL tube, and add 3-5 mL of media.</li>
	<li>To measure cell number, mix 10 &#181;L of cells with 10 &#181;L of Trypan blue and transfer 10 &#181;L to hemocytometer or use any other cell counting method.</li>
	<li>Dilute cells to recommended cell density and put 500 &#181;L/well into 24-well TC plates (Table 1). For endogenous assays (PRMT1, PRMT4, PRMT5, PRMT7, and PRMT9), move to Step 3.1.</li>
</ol>
2. Cell transfection
<ol>
	<li>For exogenous assays (PRMT3, PRMT6, PRMT8) transfect HEK293T cells with the recommended amount of DNA (Table 2). HEK293T cells are easy to transfect so any transfection reagent can be used, following the manufacturer&#39;s instruction.</li>
</ol>
3. Compound treatment
NOTE: Do not exceed 0.1% final dimethyl sulfoxide (DMSO) concentration in culture media. Keep the same DMSO concentration in each well. The selective PRMT inhibitors (chemical probes) and their closely related inactive analogs can be found in Table 3.
<ol>
	<li>For endogenous assays (PRMT1, PRMT4, PRMT5, PRMT7, and PRMT9), remove media from cells and replace with 500 &#181;L of media with compound or DMSO alone (control). 
	NOTE: It usually takes 2 days to observe over 80% decrease in R methylation levels.</li>
	<li>For exogenous assays (PRMT3, PRMT6, PRMT8), remove media 4 h after transfection, add 500 &#181;L of media with compound or DMSO alone (control), and incubate for 20-24 h.</li>
</ol>
4. Cell lysate preparation
<ol>
	<li>Remove all media from wells, wash with 100 &#181;L of PBS to remove residual media, and add 60 &#181;L of lysis buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 10 mM MgCl2, 0.5% Triton X-100, 12.5 U mL&#8722;1 benzonase, complete EDTA-free protease inhibitor cocktail) to each well.
	<ol>
		<li>Incubate for less than 1 min at RT, rocking the plate to distribute the lysis buffer over the cells. Then add 3 &#181;L of 20% w/v sodium dodecyl sulfate (SDS), to a final 1 % concentration, and mix by gently shaking. Transfer lysate into microcentrifuge tubes and keep it on ice. 
		NOTE: Add benzonase and protein inhibitor cocktail fresh before use. The addition of benzonase rapidly hydrolyzes nucleic acids which reduces cell lysate viscosity.</li>
	</ol>
	</li>
	<li>Determine protein concentration of the samples using BCA Protein Assay Kit or use any other method which tolerates 1% SDS in solution.
	<ol>
		<li>Add 2 &#181;L of lysate and protein standards (0, 1, 2, 4, and 8 &#181;g/mL of BSA in lysis buffer) into the wells of the 96-well clear plate.</li>
		<li>Mix reagent A with reagent B at 50:1 ratio and add 200 &#181;L per well. Incubate for 20 min at 37 &#176;C and read the absorbance.</li>
	</ol>
	</li>
	<li>Adjust the protein concentration with lysis buffer to be equal across the samples.</li>
	<li>Add 20 &#181;L of 4x Loading Buffer to 60 &#181;L of cell lysate and heat at 95 &#176;C for 5 min. After heat denaturation, the lysates can be stored at -20 &#176;C.</li>
</ol>
5. Western blot analysis
<ol>
	<li>Load 5-20 &#181;g of total cell lysate for analysis of histone proteins and 20-100 &#181;g for other proteins into a 4-12% Bis-Tris protein gel.</li>
	<li>Run the gel in MOPS SDS running buffer (50 mM MOPS, 50 mM Tris Base, 0.1% SDS w/v, 1 mM EDTA, pH 7.7) for about 2 h at 100 V or until the dye front reaches the bottom of the gel.</li>
	<li>If performing a wet transfer, assemble the transfer sandwich in ice-cold Tris-Glycine transfer buffer (25 mM Tris, 192 mM Glycine, 20% v/v methanol, and 0.05% w/v SDS).
	<ol>
		<li>Place sponges, filter paper, PVDF membrane, and gel according to manufacturer&#39;s instructions. Activate PVDF membrane by soaking in methanol and equilibrate gel in transfer buffer for 30 s before assembly. 
		NOTE: Use recommended PVDF western blotting membrane since it has low autofluorescence and suitability for low molecular weight proteins, such as histones (Table of Materials).</li>
	</ol>
	</li>
	<li>Transfer proteins from the gel to PVDF membrane in Tris-Glycine transfer buffer at 70 V for 1.5 h on ice.</li>
	<li>Block membrane for 30 min in blocking buffer (5% w/v milk in phosphate-buffered saline, PBS). Rinse with wash buffer (PBST: 0.1% v/v Tween-20 in PBS), and incubate with primary antibodies in bovine serum albumin (BSA) solution&#160;(5% BSA in PBST) overnight at 4 &#176;C (Table 3). 
	NOTE: For longer storage, filter-sterilize BSA solution, add 0.02% w/v sodium azide, and keep at 4 &#176;C.</li>
	<li>Wash membrane 3 x 5 min with PBST. Then incubate with goat-anti-rabbit (IR800) and donkey anti-mouse (IR680) antibodies in recommended blocking buffer (Table of Materials, Table 3) for 30 min at RT and wash 3 x 5 min with PBST.</li>
	<li>Read the signal on a fluorescent western blot imager at 800 and 700 nm. Preferably use the instrument which allows imaging strong and faint bands clearly in a single image with high sensitivity and dynamic range, high signal-to-noise ratio, a warning when an image saturation is reached as well as multiplexing of two fluorescent colors in the same sample band.</li>
	<li>Determine band intensities for western blot analysis using appropriate software for fluorescent western imaging.</li>
</ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+modern+quantitative+fluorescent+western+blotting+with+troubleshooting+strategies.">A guide to modern quantitative fluorescent western blotting with troubleshooting strategies.</a> The Journal of Visualized Experiments. (93), e52099 (2014).</li><li>Thandapani, P., O'Connor, T. R., Bailey, T. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRMT8,+a+new+membrane-bound+tissue-specific+member+of+the+protein+arginine+methyltransferase+family.">PRMT8, a new membrane-bound tissue-specific member of the protein arginine methyltransferase family.</a> Journal of Biological Chemistry. 280 (38), 32890-32896 (2005).</li><li>Kim, J. D., Kako, K., Kakiuchi, M., Park, G. G., Fukamizu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EWS+is+a+substrate+of+type+I+protein+arginine+methyltransferase,+PRMT8.">EWS is a substrate of type I protein arginine methyltransferase, PRMT8.</a> International Journal of Molecular Medicine. 22 (3), 309-315 (2008).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRMT9+is+a+type+II+methyltransferase+that+methylates+the+splicing+factor+SAP145.">PRMT9 is a type II methyltransferase that methylates the splicing factor SAP145.</a> Nature Communications. 6, 6428 (2015).</li><li>Rakow, S., Pullamsetti, S. S., Bauer, U. 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The authors do not have any competing financial interests or other conflicting interests to declare.

Here, the detailed cellular assay protocols for members of the PRMT family are described that use fluorescent western blotting methods. Unique substrates for which the changes in arginine methylation can be easily detected upon individual PRMT loss or catalytic inhibition and cannot be compensated by other family members were selected. Some proteins are methylated by multiple PRMTs21,23, suggesting an overlap in substrate specificity where some PRMTs contribute only a small amount of cellular mark in a given protein substrate24,25,26,27, for example, both PRMT8 and PRMT1 contribute to methylation of EWS. Therefore, each assay required thorough validation of substrates and antibodies with knockdown and/or overexpression experiments and further validation with well-characterized selective inhibitors. PRMT specific substrates were identified for which methylation mark changes could be detected within 2-3 days post-PRMT loss/inhibition to avoid compounding effects of reduced cell viability and proliferation that may indirectly affect the methyl-arginine mark levels. Although it was possible to find unique substrates for PRMT1, 4, 5, 7, and 9; for PRMT3, 6, and 8 the gain of function approach had to be employed. Several arginine methyl-specific antibodies were tested for various cellular targets, but none were able to detect significant changes within 3 days of PRMT3 and PRMT6 knockdown; therefore, biomarker assays were developed using ectopically expressed enzymes together with catalytically inactive mutants, which served as a control for the baseline substrate methylation. PRMT8 is a close PRMT1 homolog and shares similar substrate preferences. As a PRMT8 selective biomarker could not be identified, an assay in PRMT1 knockdown cells was developed, where PRMT8 was co-expressed together with EWS. PRMT1 is also a major enzyme responsible for H4R3 asymmetric methylation, therefore, to use H4R3me2a as a biomarker for PRMT3 and PRMT6 cellular assays, cells with low basal H4R3me2a levels were chosen as well as catalytically inactive mutants were used as a background control. Although endogenous assays are preferred, exogenous assays prove invaluable for testing the cellular potency of several selective PRMT inhibitors7,8,9. With growing knowledge of PRMT biology, we expect to improve the assays by finding more specific biomarker proteins for PRMT3, PRMT6, and PRMT8.
The use of validated antibodies and appropriate controls are critical for the PRMT assay performance. All antibodies recommended here have been thoroughly validated by knockdown and overexpression experiments, however, batch-to-batch differences, especially in the case of polyclonal antibodies, may still influence their performance. Therefore, it is crucial to use genetic methods and chemical probes together with their closely related negative controls to confirm assay reliability. Additionally, for PRMT assays that require protein overexpression, it is crucial to use catalytically inactive mutants along with wild-type protein to determine the basal methylation levels.
This collection of quantitative assays for profiling the activity of PRMTs in cells can be broadly useful for the scientific community since it can be rapidly and easily implemented with minimal equipment and limited technical expertise, involving only basic cell culturing and fluorescent western blotting techniques. The recommended antibodies and chemical probes for PRMTs can also be utilized for activity-based protein profiling (ABPP) assays to establish the suitability of a given ABPP probe, monitor target engagement, and assess off-target effects by using the competitive ABPP format. The assay development approaches discussed here can also be extrapolated for other enzyme families such as protein lysine-methyltransferases and acetyltransferases.

Arginine methylation is an important post-translational modification that regulates protein-protein and protein-RNA interactions, thus playing an important role in various cellular processes such as pre-mRNA splicing, DNA damage, transcription response, and growth factor-mediated transduction1,2. Arginine is methylated by protein arginine methyltransferases (PRMTs) resulting in monomethyl arginine (Rme1), asymmetrical dimethylarginine (Rme2a), or symmetrical dimethylarginine (Rme2s)3. Based on the methylation type, PRMTs are classified into three groups: Type I (PRMT1, 2, 3, 4, 6, and 8), which catalyze mono- and asymmetric dimethylation; Type II (PRMT5 and PRMT9), which catalyze mono- and symmetric dimethylation; and Type III (PRMT7), which can only monomethylate arginine3.
Due to a growing number of commercially available arginine methylation-specific antibodies, PRMT activity can be measured using western blotting. Fluorescent-based western blot is the preferred technique over chemiluminescent detection due to a greater dynamic range and linearity, higher sensitivity, and allowing for multiplexing4. To quantify the protein methylation levels, normalization of the methylation signal to total protein levels is required. By choosing the antibodies for total and methylated protein raised in different host species (e.g., mouse and rabbit), secondary antibodies labeled with different fluorophores can be used and the signal for both antibodies can be determined in the same sample band. Methyl-arginine antibodies were developed to identify and characterize monomethylated, asymmetrically, or symmetrically dimethylated proteins where methyl-arginine is found in a specific context. Since the majority of PRMTs methylate glycine- and arginine-rich motifs within their substrates5, several antibodies were raised for the peptides containing monomethyl or asymmetric, symmetric dimethyl-arginine-glycine repeats such as D5A12, ASYM24, or ASYM25, and SYM11, respectively. Other methyl-arginine antibodies were generated against a peptide library containing asymmetric, symmetric dimethyl- and monomethyl arginine in a repeat context facilitating the detection of methyl-arginine in these particular contexts6. There is also an increasing number of antibodies that recognize specific arginine mark on a single protein which enable selective detection of methylation such as histone H4R3me2a or BAF155-R1064me2a.
There are several commercially available PRMT inhibitors, which can be used as tools for PRMT cellular assays. However, not all of them are thoroughly characterized for selectivity and off-target effects and some should be used with caution. The Structural Genomic Consortium, in collaboration with academic labs and pharma partners, has developed well-characterized potent, selective, and cell-permeable PRMT inhibitors (chemical probes) that can be used with no restrictions by the scientific community. Information on these inhibitors can be found on https://www.thesgc.org/chemical-probes/epigenetics and https://www.chemicalprobes.org/.&#160;Chemical probes are small-molecule inhibitors with&#160;in vitro IC50 or Kd &#60; 100 nM, over 30-fold selectivity over proteins in the same family, and significant cellular activity at 1 &#181;M. Additionally, each chemical probe has a close chemical analog that is inactive against the intended target7,8,9,10,11,12.
The goal of this protocol is to measure the cellular activity of individual PRMT family members using the fluorescent western blot method. Here detailed information on validated assay biomarkers, antibodies, and potent cell-active inhibitors as well as valuable strategies for successful assay implementation are provided.

<table><tbody><tr><td>10 cm TC dishes</td><td>Greiner bio-one</td><td>664160</td><td></td></tr><tr><td>24-well TC plates</td><td>Greiner bio-one</td><td>662160</td><td></td></tr><tr><td>4&amp;ndash;12% Bis-Tris Protein Gels</td><td>ThermoFisher Scientiffic</td><td>NP0323BOX, NP0322BOX,NP0321BOX</td><td></td></tr><tr><td>Amersham Hybond P PVDF membrane</td><td>Millipore-Sigma</td><td>10600021</td><td></td></tr><tr><td>anti-Asym 24</td><td>Millipore-Sigma</td><td>07-414</td><td></td></tr><tr><td>anti-Asym 25</td><td>Millipore-Sigma</td><td>09-814</td><td></td></tr><tr><td>anti-B-actin</td><td>Santa Cruz Biotechnologies</td><td>sc-47778</td><td></td></tr><tr><td>anti-BAF155</td><td>Santa Cruz Biotechnologies</td><td>sc-32763</td><td></td></tr><tr><td>anti-BAF155-R1064me2a</td><td>Millipore-Sigma</td><td>ABE1339</td><td></td></tr><tr><td>anti-FLAG (#, 1:5000)</td><td>Millipore-Sigma</td><td>F4799</td><td></td></tr><tr><td>anti-GFP</td><td>Clontech</td><td>632381</td><td></td></tr><tr><td>anti-H3</td><td>Abcam</td><td>ab10799</td><td></td></tr><tr><td>anti-H3R2me2a</td><td>Millipore-Sigma</td><td>04-848</td><td></td></tr><tr><td>anti-H3R8me2a</td><td>Rockland</td><td>600-401-I67</td><td></td></tr><tr><td>anti-H4</td><td>Abcam</td><td>ab174628</td><td></td></tr><tr><td>anti-H4R3me2a</td><td>Active Motif</td><td>39705</td><td></td></tr><tr><td>anti-Hsp/Hsc70</td><td>Enzo</td><td>ADI-SPA-820</td><td></td></tr><tr><td>anti-PRMT1</td><td>Millipore-Sigma</td><td>07-404</td><td></td></tr><tr><td>anti-PRMT3</td><td>Abcam</td><td>ab191562</td><td></td></tr><tr><td>anti-PRMT4</td><td>Bethyl</td><td>#A300-421A</td><td></td></tr><tr><td>anti-PRMT5</td><td>Abcam</td><td>ab109451</td><td></td></tr><tr><td>anti-PRMT6</td><td>Abcam</td><td>ab47244</td><td></td></tr><tr><td>anti-PRMT7</td><td>Abcam</td><td>ab179822</td><td></td></tr><tr><td>anti-Rme1</td><td>CST</td><td>8015</td><td></td></tr><tr><td>anti-Rme2a</td><td>CST</td><td>13522</td><td></td></tr><tr><td>anti-Rme2s</td><td>CST</td><td>13222</td><td></td></tr><tr><td>anti-Rme2s (ASYM25), Millipore, , 1:2000)</td><td></td><td>09-814</td><td></td></tr><tr><td>anti-SAP145 (Abcam, #, 1:1000)</td><td>Abcam</td><td>ab56800</td><td></td></tr><tr><td>anti-SAP145-R508me2s</td><td></td><td></td><td>kind gift from Dr. Yanzhong Yang, Beckman Research Institute of City of Hope</td></tr><tr><td>anti-SmBB&amp;rsquo;</td><td>Santa Cruz Biotechnologies</td><td>sc-130670</td><td></td></tr><tr><td>benzonase</td><td></td><td></td><td>PRODUCED IN-HOUSE</td></tr><tr><td>BSA</td><td>Millipore-Sigma</td><td>A7906</td><td></td></tr><tr><td>C2C12</td><td></td><td></td><td>gift from Dr. Stephane Richard, McGill University</td></tr><tr><td>cOmplete, EDTA-free Protease Inhibitor Cocktail</td><td>Millipore-Sigma</td><td>11873580001</td><td></td></tr><tr><td>DMEM</td><td>Wisent</td><td>319-005-CL</td><td></td></tr><tr><td>DMSO</td><td>Bioshop</td><td>DMS666.100</td><td></td></tr><tr><td>donkey anti-mouse IgG-IR680</td><td>Licor</td><td>926-68072</td><td></td></tr><tr><td>doxycycline</td><td>Millipore-Sigma</td><td>D9891</td><td></td></tr><tr><td>EDTA</td><td>Bioshop</td><td>EDT111.500</td><td></td></tr><tr><td>FBS</td><td>Wisent</td><td>80150</td><td></td></tr><tr><td>glycine</td><td>Bioshop</td><td>GLN002.5</td><td></td></tr><tr><td>goat-anti-rabbit IgG-IR800</td><td>Licor</td><td>926-32211</td><td></td></tr><tr><td>HEK293T</td><td></td><td></td><td>gift from Dr. Sam Benchimol, York University</td></tr><tr><td>Image Studio Software ver 5.2</td><td>Licor</td><td></td><td></td></tr><tr><td>Loading buffer: NuPAGE LDS Sample Buffer (4x)</td><td>ThermoFisher Scientiffic</td><td>NP0007</td><td></td></tr><tr><td>MCF7</td><td></td><td>ATCC&amp;reg; HTB-22&amp;trade;</td><td></td></tr><tr><td>NaCl</td><td>Bioshop</td><td>SOD001.1</td><td></td></tr><tr><td>NuPAGE MOPS SDS Running Buffer</td><td>ThermoFisher Scientiffic</td><td>NP0001</td><td></td></tr><tr><td>Odyssey Blocking Buffer (dilute 4 x with PBST)</td><td>Licor</td><td>927-40000</td><td>Intercept (PBS) Blocking Buffer can also be used # 927-70001</td></tr><tr><td>Odyssey CLX Imaging System</td><td>Licor</td><td>model number 9140</td><td></td></tr><tr><td>PBS (tissue culture)</td><td>Wisent</td><td>311-010-CL</td><td></td></tr><tr><td>PBS (western blot)</td><td>Bioshop</td><td>PBS405.4</td><td></td></tr><tr><td>penstrep</td><td>Wisent</td><td>450-201-EL</td><td></td></tr><tr><td>Pierce&amp;trade; BCA Protein Assay Kit</td><td>ThermoFisher Scientiffic</td><td>23225</td><td></td></tr><tr><td>SDS</td><td>Bioshop</td><td>SDS001.1</td><td></td></tr><tr><td>skim milk powder</td><td>Bioshop</td><td>SKI400.500</td><td></td></tr><tr><td>TC20 automated cell counter</td><td>Biorad</td><td>1450102</td><td></td></tr><tr><td>Tripsin-EDTA (0.25%)</td><td>Wisent</td><td>325-043-EL</td><td></td></tr><tr><td>Tris</td><td>Bioshop</td><td>TRS003.5</td><td></td></tr><tr><td>Tritton X-100</td><td>Bioshop</td><td>TRX506</td><td></td></tr><tr><td>trypan blue</td><td>GIBCO</td><td>15250-061</td><td></td></tr><tr><td>Tween-20</td><td>Bioshop</td><td>TWN510.500</td><td></td></tr></tbody></table>

quantitative methods to study protein arginine methyltransferase 1-9 activity in cells

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine members that can monomethylate or symmetrically/asymmetrically dimethylate arginine residues. Several antibodies recognizing different types of arginine methylation of various proteins are available; thus, providing tools for the development of PRMT activity biomarker assays. PRMT antibody-based assays are challenging due to overlapping substrates and motif-based antibody specificities. These issues and the experimental setup to investigate the arginine methylation contributed by individual PRMTs are discussed. Through the careful selection of the representative substrates that are biomarkers for eight out of nine PRMTs, a panel of PRMT activity assays were designed. Here, the protocols for cellular assays quantitatively measuring the enzymatic activity of individual members of the PRMT family in cells are reported. The advantage of the described methods is their straightforward performance in any lab with cell culture and fluorescent western blot capabilities. The substrate specificity and chosen antibody reliability were fully validated with knockdown and overexpression approaches. In addition to detailed guidelines of the assay biomarkers and antibodies, information on the use of an inhibitor tool compound collection for PRMTs is also provided.

Watch this Scientific Journal Video about Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells at JoVE.com

Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells

These protocols provide the methodology used to assess the enzymatic activity of individual members of the protein arginine methyltransferase (PRMT) family in cells. Detailed guidelines on assessing PRMT activity using endogenous and exogenous biomarkers, methyl-arginine recognizing antibodies, and inhibitor tool compounds are described.

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine ...

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Research

JoVE Journal

Biology

4.1K Views.  University of Toronto. Het doel van deze procedure is om de enzymatische activiteit van individuele leden van de protein arginine methyltransferase familie in cellen te meten. De activiteit wordt gemeten door het bepalen van hun arginine methylatie niveaus en totale niveaus van bio-marker eiwit met behulp van kwantitatieve fluorescerende westerse blotting. Het voordeel van de beschreven methode is de eenvoudige prestaties in elk lab met celcultuur en fluorescerende westerse vlekmogelijkheden.De gedetailleerd...

Video: Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

Genetics

A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known small molecule-protein interactions and to find potential protein targets for natural products. Compared with other methods, DARTS uses native, unmodified, small molecules and is simple and easy to operate. In this study, we further enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The protein-ligand interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used the mTOR-rapamycin interaction as an exemplary case for establishment of our protocol. From the proteolytic curve we saw that the proteolysis of mTOR by pronase was inhibited by the presence of rapamycin. The dose-dependency curve allowed us to estimate the binding affinity of rapamycin and mTOR. This method is likely to be a powerful and simple method for accurately identifying novel target proteins and for the optimization of drug target engagement.

A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction

In this study, we enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used mTOR-rapamycin interaction as an exemplary case.

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known ...

Biochemistry

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

Cancer Research

Proximity Ligation Assay Allows the Detection, Localization, and Quantification of Protein Arginine Methylation in Fixed Tissue

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often limited by the lack of a specific antibody recognizing the target arginine residue. Proximity ligation assay (PLA) was originally developed to study protein/protein interactions. Here, we describe in detail a PLA protocol dedicated to the detection of arginine methylation that we applied to the glucocorticoid receptor (GR). Having previously shown that PRMT5 dimethylates GRs in cells, we used PLA with a pan symmetrical dimethyl antibody and an anti-GR antibody to measure GR methylation in breast tumors. We demonstrate that PLA offers a unique approach to measure arginine methylation of a target protein, even when the site of methylation has not been identified. This technique could be extended to other post-translational modifications where effective pan antibodies are available. Hence, we detail the PLA technology used to detect arginine methylation in fixed tissue using GR as an example.

Proximity ligation assay is a very useful technique to localize and quantify arginine methylation of a given protein when the modified arginine residue is unknown and/or if no specific antibody is available.

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.

The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells

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