This work has been supported by the DFG grant JE 252/7.

1. Preparation of Peptide Arrays
<ol>
	<li>Programming of the Peptide Sequences
	<ol>
		<li>Design peptide sequences for the synthesis using the MultiPep Spotter software. Enter the peptide sequences in single letter codes. 
		Peptide 1: T A R K S T G G K A</li>
		<li>Generate alanine or arginine walk libraries with a specific command (.replace, A) to screen the important amino acids required for the recognition of a defined substrate. For instance in the following example the first line is the wild type sequence and from the second line each amino acid in a peptide is altered to alanine sequentially. 
		replace, A 
		T-A-R-K-S-T-G 
		A-A-R-K-S-T-G 
		T-A-R-K-S-T-G 
		T-A-A-K-S-T-G 
		T-A-R-A-S-T-G 
		T-A-R-K-A-T-G 
		T-A-R-K-S-A-G 
		T-A-R-K-S-T-A</li>
		<li>Use similar commands (.replace R, .replace N etc.) as described in 1.1.2 with all the naturally available amino acid residues (eventually omitting cysteine, methionine and tryptophan, because these residues are prone to oxidation and sometimes lower synthesis yield) to&#160;synthesize a peptide array for determining the consensus substrate sequence motif for an enzyme. 
		NOTE: As shown in Figure 4A, the horizontal axis represents the given peptide sequence and the vertical axis represents the amino acids that are selected for command. Similar to the sequence shown in 1.1.2, A on the vertical axis means that an Ala sequentially replaces&#160;&#160;each position of the given sequence in the first row in Fig. 4A. Similarly arginine sequentially replaces each amino acid in the second&#160;row and so on with the other amino acids. Complete peptide array libraries are synthesized by systematically replacing each residue in the provided peptide substrate sequence with each of the other naturally available amino acid residues, this helps to understand the influence of each residue at each position of the given sequence.</li>
		<li>Alternatively, methylation of known or predicted peptide substrates can be easily verified synthesizing peptide libraries with the target peptides of the PKMT together with positive and negative methylation controls (i.e. peptides known to methylated or known not to be methylated). Synthesize peptides by exchanging the putative target lysine to alanine to confirm methylation of the target lysine as shown below by manual programming. 
		Wild type peptide: S T G G K P R Q F L 
		Mutant peptide : S T G G A P R Q F L 
		NOTE: The software also has inherent scripts to create various libraries, like epitope mapping with a desired frame shift of the sequence to map important amino acids required for an interaction.&#160;</li>
	</ol>
	</li>
	<li>Preparation of Membrane, Amino Acids and Reagents
	<ol>
		<li>Pre-swell the SPOT membrane in DMF (N,N-Dimethylformamide) for 10 min and then place the wet membrane on the SPOT synthesizer frame without air bubbles or wrinkles.</li>
		<li>Wash the membrane three times with 100% ethanol. Dry the membranes completely for at least 10 min before starting the synthesis program.</li>
		<li>In parallel, freshly prepare 0.5 M working concentrations of commercially available Fmoc-protected amino acids by dissolving them in NMP and also prepare the activator and base as described in Table 1.</li>
		<li>Make sure that all the waste solution containers of the SPOT synthesizer were emptied and refill the solvent reservoirs with the DMF, ethanol and piperidine. 
		NOTE: Now the machine is ready for the synthesis.</li>
	</ol>
	</li>
	<li>Spotting of the First Amino Acid
	<ol>
		<li>To generate the amide bond with the free amino group on the membrane, activate the carboxy group of the incoming amino acid by adding the activator (N,N&#8217;-Diisopropylcarbodiimide) and base solution (Ethyl hydroxyimino cyanoacetate) to the amino acid derivative.</li>
		<li>Spot the desired volumes of activated amino acids on the Amino-PEG functionalized membrane using the programmable robot. 
		NOTE: Thereby, the C-terminus of the amino acid is coupled to the amino group of the membrane.</li>
		<li>Repeat this step three times to ensure better coupling of the first activated amino acid. Incubate the membrane for 20 min and then wash with DMF to remove uncoupled amino acids.</li>
	</ol>
	</li>
	<li>Blocking of Free Spaces on Non-spot Areas 
	NOTE: After the coupling of the first C-terminal amino acid of all peptides to the amino group of the membrane, the amino groups between the spots and also some of the amino groups within the spot areas do not form bonds with the amino acids.
	<ol>
		<li>Block these free amino groups by incubating with the capping solution (20% acetic anhydride in DMF) for 5 min. 
		NOTE: This avoids the coupling of amino acids in the further cycles with the membrane instead of the growing peptide chain.</li>
	</ol>
	</li>
	<li>Removal of Fmoc Group
	<ol>
		<li>After the blocking, wash the membrane with DMF 4 times and then incubate with 20% piperidine in DMF for 20 min to remove the Fmoc protection group. 
		NOTE: This step leads to removal of the amino-protecting Fmoc groups and it is called deprotection.</li>
		<li>Afterwards, wash the membrane twice with DMF and twice with ethanol and allow it to dry. 
		NOTE: Now the membrane is ready for the coupling of the next amino acids.&#160;</li>
	</ol>
	</li>
	<li>Chain Elongation 
	NOTE: Addition of each amino acid is called a cycle. Apart from the coupling of first amino acid, the cycle begins with the deprotection of Fmoc group of the coupled amino acid and it is followed by the coupling of the activated carboxyl group of an incoming amino acid to the amino group of the growing peptide chain.
	<ol>
		<li>Repeat steps 1.3 and 1.5 until the desired peptide length is achieved.</li>
	</ol>
	</li>
	<li>Staining 
	NOTE: After the final cycle, the peptide arrays are stained with bromophenol blue to confirm the successful synthesis of peptides. Bromophenol blue binds with the free amino groups of the peptides. Peptide spots show light blue color after the staining but the intensity of the spots varies depending on the peptide sequence. This staining allows confirmation of the complete removal of piperidine, because in presence of piperidine bromophenol blue does not stain the peptides. In addition, this also helps to mark the peptide arrays for further usage.
	<ol>
		<li>After the Fmoc group deprotection in the last synthesis cycle, wash the membrane with DMF and 100% ethanol. Then, treat the membrane with bromophenol blue (0.02% bromophenol blue in DMF) for a minimum of 5 min until it completely turns blue.</li>
		<li>Afterwards, wash the membrane with DMF and ethanol followed by drying for 10 min. Now take out the membrane from the synthesizer and perform side chain deprotection (section 1.8) manually. If needed, photograph the membrane to document the results of the peptide synthesis (Fig. 2).&#160;</li>
	</ol>
	</li>
	<li>Side Chain Deprotection
	<ol>
		<li>Treat the membranes with 20 to 25 ml of side chain deprotection mixture consisting of 95% Trifluoroacetic acid (TFA) to cleave the side chain protection groups and scavenger reagents (2.5% water and 2.5% triisopropylsilane) to protect the side chains of amino acids from modification during this step. Take a sufficient volume of deprotection mixture to ensure that it covers the membrane completely in a tightly closed chemical resistant box and incubate it for 1 to 2 hr while shaking gently.</li>
		<li>Wash the membrane six times with 20-25 ml of DCM (Dichloromethane) for 2 min each and finally twice with 100% ethanol and dry it in a desiccator overnight. 
		NOTE: Now the membrane can be used for experiments. A scheme of the peptide array synthesis is provided in Figure 1.&#160;</li>
	</ol>
	</li>
</ol>
2. Protein Expression and Purification
<ol>
	<li>Protein Expression of PKMTs as GST Fusions
	<ol>
		<li>Using standard techniques, clone the gene encoding the full length PKMT or its catalytic domain into a bacterial expression vector (like pGEX-6p2) to express it as GST-fusion protein.</li>
		<li>Transfer the vector with the desired PKMT insert into BL21 or BL21 codon plus E. coli cells by the heat shock method or any other method.</li>
		<li>Prepare a pre-culture with 30 ml of Luria-Bertani (LB) media on the day of expression and incubate at 37 &#176;C for 7 to 8 hr with continuous shaking.</li>
		<li>Next, transfer 10 ml of the pre-culture into a 2 L big baffled flasks containing 1 L of LB media and incubate at 37 &#176;C in the incubator with continuous shaking until the culture reaches a defined optical density at 600 nm. 
		NOTE: While this can be optimized for each protein, we routinely induce at an OD600nm of about 0.8. The induction temperature has to be optimized for each protein individually, some proteins show good expression at 22 &#176;C and some at higher temperatures.</li>
		<li>Shift the cells to the induction temperature for 15 min, then induce with 1 mM of isopropyl-beta-D-thiogalactopyranoside and allow the culture to grow for 10-12 hr at induction temperature.</li>
		<li>Afterwards harvest the cells by centrifugation at 5,000 x g for 15 min and finally wash the pellet with 30 ml of STE buffer (10 mM Tris pH 8.0, 100 mM NaCl and 0.1 mM EDTA) and store at -20 &#176;C for further usage.</li>
	</ol>
	</li>
	<li>Protein Purification&#160;
	<ol>
		<li>Thaw the cell pellet and re-suspend in 30 ml of sonication buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT and 5% glycerol) and disrupt by sonication. 
		NOTE: This buffer needs to be optimized for each protein eventually.</li>
		<li>Centrifuge the cell lysate at 22,000 x g for 1 hr and, later collect the supernatant and pass through a column containing 600 &#181;l of glutathione Sepharose 4B resin.</li>
		<li>Wash the column with 50 ml of sonication buffer and next with 100 ml of high salt buffer (50 mM Tris pH 7.5, 500 mM NaCl, 1 mM DTT and 5% glycerol) to remove unspecifically bound proteins. Elute the bound protein with 5 ml of high salt buffer containing 40 mM glutathione.</li>
		<li>Dialyze the eluted protein in 2 L of dialysis buffer with low glycerol (20 mM Tris pH 7.4, 100 mM KCl, 0.5 mM DTT and 10% glycerol) for 2 hr and later in 20 mM Tris pH 7.4, 100 mM KCl, 0.5 mM DTT and 70% glycerol for 8 hr or overnight. Make sure all the purification buffers are at 4 &#176;C and perform the protein purification in the cold room to avoid protein denaturation.</li>
	</ol>
	</li>
</ol>
3. Peptide Array Methylation
<ol>
	<li>Pre-incubation of Peptide Arrays
	<ol>
		<li>Perform peptide array methylation either in a box of appropriate size or in a plastic bag to avoid the wastage of enzyme and expensive radioactively labelled AdoMet. Pre-incubate the peptide array membrane in sealed plastic bag containing the respective methylation buffer without enzyme and labeled [methyl-3H]-AdoMet for 10 min. The volume of buffer depends upon the size of the array, for example use 8 ml of methylation buffer for a full specificity profile peptide array. Optimize the amount and concentration of the AdoMet for each enzyme.</li>
	</ol>
	</li>
	<li>Methylation of Peptide Arrays
	<ol>
		<li>Discard the pre-incubation buffer and incubate the membrane in 8 ml of methylation buffer containing the respective PKMT and labeled [methyl-3H]-AdoMet for 1 to 2 hr. 
		NOTE: The amount of enzyme required for a methylation reaction depends upon the activity of specific PKMT, we initiate our screening experiments typically with 50 nM enzyme final concentration.</li>
	</ol>
	</li>
	<li>Washing of Peptide Arrays
	<ol>
		<li>Afterwards discard the methylation buffer in a radioactive waste container and wash the peptide arrays for 5 times with 20 ml of buffer containing 100 mM ammonium bicarbonate and 1% SDS for 5 min to remove the bound protein. Discard the washing buffer in a radioactive waste container.</li>
	</ol>
	</li>
	<li>Detection of the Radioactive Signal
	<ol>
		<li>After the washes incubate the membrane for 5 min&#160;in 10 ml of amplify solution (NAMP100V).</li>
		<li>Then, discard the amplify solution into the radioactive waste container for organic solvents, seal the peptide array in a plastic bag and put it in an autoradiography cassette.</li>
		<li>Place the autoradiography film on the peptide array in the dark room. Close the cassette carefully and expose it at -80 &#176;C for the necessary time. Then, develop the film to analyze the results. Capture the image couple of times with different exposition times to avoid saturation of strongly methylated peptide substrates.</li>
	</ol>
	</li>
</ol>
4. Data Processing and Analysis
<ol>
	<li>Densitometric Analysis of Spot Intensities
	<ol>
		<li>Scan the film in a conventional scanner and analyze the spot intensities by densitometry. Do this using ImageJ (which is freeware) or a commercial program. 
		NOTE: We use Phoretix software for this task.</li>
	</ol>
	</li>
	<li>Normalization and Averaging of Results from Different Membranes
	<ol>
		<li>Conduct the peptide array methylation experiments at least in triplicate. Normalize the scanned intensities for each experiment by background subtraction and setting the activity at a reference substrate as 1.0. Average the data for the individual spots and report them as mean values and standard deviations.</li>
	</ol>
	</li>
	<li>Data Analysis and Display
	<ol>
		<li>Plot the data in 2D using a grayscale or color scheme to indicate the relative activity. Do this with Excel or Sigmaplot as shown here. Also, prepare a plot of the distribution of the standard deviations of the repeated experiments to analyze the quality of the data.</li>
		<li>To compare and display the accuracy of the recognition of each residue in the substrate peptide quantitatively, calculate the relative preference of the PKMT for each amino acid i at position x by a discrimination factor D 16,17: 
		DX;i = &#60; Vj&#8800;i&#62;/Vi - 1 
		Where vi is the rate of methylation of the peptide variant carrying amino acid i at position x and &#60;vj&#8800;i&#62; is the average rate of methylation of all 19 peptides carrying a different amino acid j&#8800;i at position x (including the wild type sequence). 
		NOTE: The discrimination factor defined by this depends on the background level. If only one residue is accepted at a certain position with a background of 3%, a discrimination factor of 32.3 is obtained. No preference at a position results in a discrimination factor of 0.</li>
	</ol>
	</li>
</ol>

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No conflicts of interest declared.

SPOT synthesis as described here is a powerful method to map protein-protein interaction sites and investigate the substrate recognition of peptide modifying enzymes. However, SPOT synthesis still has certain drawbacks, because although the peptides synthesized by the SPOT method are reported to have more than 90% purity 21 this is difficult to confirm in each instance. Therefore, results have to be reproduced by other methods for instance using protein domains or with purified peptides synthesized by standard...

In the last two decades, several reports demonstrated the importance of post-translational modifications (PTM) in cellular development and several diseases like cancer, but recently protein lysine methylation has emerged as an another vital PTM. While initially histone lysine methylation was found to be an essential chromatin mark, later work also showed lysine methylation of several non-histone proteins 1-4. The sequential transfer of methyl groups from S-adenosyl-L-methionine to the &#949;-amino group of lysine residues is catalyzed by a family of enzymes called Protein Lysine Methyltransferases (PKMTs) that contains over 60 proteins in the human genome. PKMTs were initially discovered as histone modifying enzymes that methylate specific lysine residue but later reports demonstrated that they could also methylate non-histone proteins 5. Up to now, approximately 5,000 lysine methylation sites were identified on different proteins 6, but the enzymes responsible for these modifications are often not identified. One reason for this is that the specificity of most PKMTs has not been studied extensively. Therefore, it recurrently occurs that novel substrates of PKMTs are discovered. The lack of a detailed knowledge of the substrate specificity of PKMTs hinders understanding of their biological function and role. To study the specificity of a PKMT in detail, the methylation rates of many peptide substrates that differ in one or few amino acids must be measured and compared, which is ideally done using peptide arrays. Based on the resulting specificity profiles, potential substrates of PKMTs can be identified that can be studied further.
Peptide arrays are widely used tools for the biochemical analysis of antibodies, peptide modifying enzymes and mapping of protein-protein interaction sites (antibody-antigen, receptor-ligand) 7-9. Several hundreds of peptides are needed for such applications. Different methods are available for peptide synthesis, among them peptide synthesis on resin is very commonly used, but it has limitations in throughput and it is relatively expensive. These issues were resolved with the introduction of the SPOT synthesis method by Frank and colleagues 10. The SPOT synthesis method allows synthesis of several hundred peptides in parallel and on average it is inexpensive compared to resin synthesis. The peptides synthesized on cellulose membrane can be used either directly for various applications or peptides can be cleaved from the membrane and used as free peptides for in solution assays or to prepare peptide microarrays 10-13.
SPOT synthesis is a variant of the solid phase peptide synthesis, which uses a cellulose membrane as a solid support and employs the standard Fmoc-chemistry 10-13. Hence, the synthesis of peptide chains starts at the C-terminal end and proceeds towards the N-terminal end in contrast to the biological synthesis in ribosomes. Cellulose membranes are functionalized for the attachment of the first activated amino acids (Fig. 1). The SPOT method is based on the sequential delivery of activated amino acids in a droplet of solvent to defined spots on the membrane using an automated pipetting system. The droplet of liquid is dispensed on the porous membrane where it forms a circular wet spot, which later acts as an open reactor for the chemical reactions in peptide synthesis. The spot size is determined by the volume dispensed and the absorptive capacity of the membrane, multiples of such spots are arranged as arrays. The scale of synthesis correlates with the spot size and the loading capacity of the membrane. The distance between the spots and the density of arrays are managed by varying the spot sizes. Cellulose membrane has several advantages as solid phase in peptide synthesis, it is inexpensive, tolerant to the chemicals used in peptide synthesis, stable in aqueous solutions and easy to handle. In addition, its hydrophilic nature makes it suitable for several biological assay systems. SPOT synthesis can be carried out manually or automated (for 1000s of peptides) depending on the required number of peptides. A fully automated SPOT synthesizer from Intavis (K&#246;ln, Germany) is used for our applications. It permits synthesis of peptides in different amounts and of different length. Linear peptides are regularly synthesized with 15 to 20 amino acid length, in addition peptides of up to 42 amino acid can also be prepared by step-wise synthesis 14,15. However, increasing the number of amino acids leads to reductions in the overall coupling yields, which affects the quality of the peptides. Because of the low amount of peptides per spot, the products are often difficult to purify and the quality of individual peptides cannot be easily assessed. Therefore, the results obtained from SPOT peptide arrays must be confirmed either with peptides synthesized by standard methods in larger scale, which can be purified and analyzed according to standards in peptide synthesis or by synthesizing the proteins containing the desired peptide sequences. Still, we found the SPOT synthesis to be highly reliable and results generally to be reproducible. SPOT synthesis is not restricted to proteinogenic amino acids, several commercially available modified amino acids also can be used for synthesis, allowing peptides to be modified before and after the final cleavage of the side-chain protection group and, furthermore, it also allows incorporation of phosphorylated, methylated or acetylated amino acids 11.
Immobilized peptide libraries synthesized by the SPOT method can be directly used for many biological and biochemical assays. We employed peptide arrays comprising 300-400 peptides to investigate the substrate specificity of PKMTs. For enzymatic modification, the peptide arrays are incubated with the respective PKMT and labeled [methyl-3H]-AdoMet in an appropriate buffer. The methylation of the respective substrate is analyzed by following the enzymatic transfer of the radioactively labelled methyl groups from AdoMet to the peptide substrate via autoradiography (Fig. 3). By this procedure, the peptide arrays allow the study of methylation of different peptide substrates at the same time. One important advantage of this method is that all the peptides are methylated in competition, such that during the linear phase of the methylation kinetics, the relative methylation of each peptide is proportional to the catalytic rate constant divided by the dissociation constant (kcat/KD) of the enzyme for the respective peptide substrate. Therefore, the amount of radioactivity incorporated into each spot is directly correlated with the enzymatic activity towards the particular peptide. Using the results of a peptide array methylation experiment, the specificity profile of the PKMT can be defined and based on this novel substrates can be predicated. Peptide arrays allow the rapid and cost efficient validation of the methylation of novel substrates at the peptide level. For this, arrays are prepared that contain the predicted novel substrates together with modified peptides containing an Ala instead of Lys at the target sites as well as positive and negative control peptides. Finally, the novel substrates can be prepared as proteins together with mutants, in which the target Lys is altered to Ala and the methylation can be confirmed at the protein level. Depending on the results, this is then followed by biological studies addressing potential roles of the methylation of the newly described protein substrates.

<table><tbody><tr><td>Ethanol abs Gradient Grade HPLC</td><td>Honeywell</td><td>10299901</td><td>Flammable</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide Peptide Synthesis</td><td>Biosolve</td><td>4193302</td><td>Flammable, Toxic</td></tr><tr><td>Piperidine &amp;ge;99% for Peptide Synthesis</td><td>Roth</td><td>A122.1</td><td>Flammable, Toxic, corrosive</td></tr><tr><td>Oxyma Pure (Ethyl (hydroxyimino)cyanoacetate )</td><td>Novabiochem</td><td>8510860100</td><td></td></tr><tr><td>&lt;em&gt;N,N&amp;rsquo;&lt;/em&gt;-Diisopropylcarbodiimide purum &amp;ge;98% GC</td><td>Fluka</td><td>38370</td><td>Flammable, Toxic, corrosive</td></tr><tr><td>Acetic anhydride</td><td>Roth</td><td>CP28.1</td><td>Flammable, Toxic, corrosive</td></tr><tr><td>Triisopropylsilane 99%</td><td>Aldrich</td><td>233781</td><td>Flammable, Toxic</td></tr><tr><td>Dichlormethane &amp;ge;99.9%</td><td>Roth</td><td>P089.1</td><td>Carcinogenic</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-Methyl-2-Pyrrolidone</td><td>Roth</td><td>4306.2</td><td>Toxic</td></tr><tr><td>Derivatized cellulose Membrane</td><td>Intavis AG K&amp;ouml;ln</td><td>32.1</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Roth</td><td>P088.2</td><td>Toxic, corrosive</td></tr><tr><td>Bromphenolblue</td><td>AppliChem</td><td>A3640.0010</td><td></td></tr><tr><td>Ammonium Hydrogen Carbonate</td><td>Roth</td><td>T871.2</td><td>Toxic</td></tr><tr><td>Sodium Dodecyl Sulfate Pellets</td><td>Roth</td><td>CN30.3</td><td>Toxic, Flammable</td></tr><tr><td>MultiPep Synthesizer</td><td>Intavis AG K&amp;ouml;ln</td><td>n.a.</td><td></td></tr><tr><td>HyperfilmTM high performance film</td><td>GE Healthcare</td><td>28906837</td><td></td></tr><tr><td>Phoretix software</td><td>TotalLab</td><td>n.a.</td><td></td></tr></tbody></table>

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 were successfully used to biochemically characterize the specificity of PKMTs, and several novel histone and non-histone substrates of PKMT were identified by this approach 5,16-19. Defining the correct substrate spectrum of a PKMT (or any enzyme) is an essential step towards understanding of its molecular mechanism and cellular functions.
As an example of the application of the peptide SPOT array methylation method, we describe the results of the specificity analysis...

Watch this Scientific Journal Video about Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays at JoVE.com

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.9K Views.  Stuttgart University. 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.

Video: Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Biochemistry

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important for revealing protease function. The method proposed in this study determines protease specificity by measuring the molecular weight of unique substrates using Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The substrates contain iminobiotin, while the cleaved site consists of amino acids, and the spacer consists of polyethylene glycol. The cleaved substrate will generate a unique molecular weight using a cleaved amino acid. One of the merits of this method is that it may be carried out in one pot using crude samples, and it is also suitable for assessing multiple samples. In this article, we describe a simple experimental method optimized with samples extracted from mouse lung tissue, including tissue extraction, placement of digestive substrates into samples, purification of digestive substrates under different pH conditions, and measurement of the substrates&#39; molecular weight using MALDI-TOF mass spectrometry. In summary, this technique allows for the identification of protease specificity in crude samples derived from tissue extracts using MALDI-TOF mass spectrometry, which may easily be scaled up for multiple sample processing.

Here, we present a protocol to determine protease specificity in crude mouse tissue extracts using MALDI-TOF spectrometry.

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Immunology and Infection

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, ...

Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized. Peptidomic studies frequently analyze peptides that are naturally generated in cells. Here, a sample preparation protocol based on heat-inactivation is described, which eliminates protease activity, and extraction with mild conditions, so there is no peptide bonds cleavage. In addition, the relative quantitation of peptides using stable isotope labeling by reductive methylation of amines is also shown. This labeling method has some advantages as the reagents are commercially available, inexpensive compared to others, chemically stable, and allows the analysis of up to five samples in a single LC-MS run.

This article describes a sample preparation method based on heat-inactivation to preserve endogenous peptides avoiding degradation post-mortem, followed by relative quantitation using isotopic labeling plus LC-MS.

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to human brain function, as neurons are postmitotic and rely heavily on the autophagy-lysosome pathway to maintain cellular homeostasis. Despite advancements in the understanding of various lysosomal functions, capturing the highly dynamic communications between lysosomes and other cellular components is technically challenging, particularly in a high-throughput fashion. Here, a detailed protocol is provided for the recently published endogenous (knock-in) lysosome proximity labeling proteomic method in human induced pluripotent stem cell (hiPSC)-derived neurons. 
Both lysosomal membrane proteins and proteins surrounding lysosomes within a 10-20 nm radius can be confidently identified and accurately quantified in live human neurons. Each step of the protocol is described in detail, i.e., hiPSC-neuron culture, proximity labeling, neuron harvest, fluorescence microscopy, biotinylated protein enrichment, protein digestion, LC-MS analysis, and data analysis. In summary, this unique endogenous lysosomal proximity labeling proteomics method provides a high-throughput and robust analytical tool to study the highly dynamic lysosomal activities in live human neurons.

A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to ...

Neuroscience

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The &#34;one-pot&#34; affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into ...