Abstract
The present invention relates to a method for the production of a serine ADP-ribosylated protein or peptide comprising preparing an aqueous buffered solution comprising 5 to 60 mM, preferably 10 to 60 mM of a buffer and having a pH between 5.0 and 9.0, preferably between 5.5 to 8.5, and most preferably between 6.1 to 8.3, said solution further comprising (a) 0.2 to 2.5 mM NAD.sup.+, (b) at least 50 nM, preferably 50 to 3000 nM PARP-1, PARP-2 or the PARP-1 variant E988Q, (c) at least 100 nM, preferably 100 to 5000 nM HPF1, (d) at least 10 μg/mL, preferably 10 μg/mL to 200 μg/mL sonicated DNA, said sonicated DNA preferably comprising DNA fragments of 10 to 330 bp, and (e) up to 600 μM protein or peptide, said protein or peptide comprising at least one serine, thereby generating a reaction mix, in which the protein or peptide becomes serine ADP-ribosylated.
Claims
1. A method for the production of a serine ADP-ribosylated protein or peptide comprising preparing an aqueous buffered solution comprising 5 to 60 mM, preferably 10 to 60 mM of a buffer and having a pH between 5.0 and 9.0, preferably between 5.5 to 8.5, and most preferably between 6.1 to 8.3, said solution further comprising (a) 0.2 to 2.5 mM NAD.sup.+, (b) at least 50 nM, preferably 50 to 3000 nM PARP-1, PARP-2 or the PARP-1 variant E988Q, (c) at least 100 nM, preferably 100 to 5000 nM HPF1, (d) at least 10 μg/mL, preferably 10 μg/mL to 200 μg/mL sonicated DNA, said sonicated DNA preferably comprising DNA fragments of 10 to 330 bp, and (e) up to 600 μM protein or peptide, said protein or peptide comprising at least one serine, thereby generating a reaction mix, in which the protein or peptide becomes serine ADP-ribosylated.
2. The method of claim 1, wherein the solution further comprises at least 1 μM, preferably 1 to 10 μM PARG and/or the ADP-ribosylated protein or peptide is incubated with at least 1 μM, preferably 1 to 10 μM PARG, thereby obtaining a mono-ADP-ribosylated protein or peptide.
3. The method of claim 1, wherein the reaction is carried out at 15-35° C. and preferably at room temperature.
4. The method of claim 1, wherein the reaction is carried out for at least 90 min, preferably at least 120 min and more preferably at least 240 min.
5. The method of claim 1, wherein a fresh pool of 0.2 to 2.5 mM NAD.sup.+ is added to the reaction mix at least every 90 minutes, preferably at least every 60 minutes and more preferably at least every 45 minutes.
6. The method of claim 1, wherein the at least one serine is neighboured by at least one basic amino acid.
7. The method of claim 1, wherein the substrate contains a positively charged tail, a poly-arginine and/or lysine tail.
8. The method of claim 1, wherein the aqueous buffered solution further comprises (f) 10 to 80 mM NaCl or KCl, and/or (g) 0.5 to 2 mM MgCl.sub.2,
9. The method of claim 1, wherein the buffer is a Tris-HCl buffer and is preferably used at a final concentration of 40-60 mM, a Hepes buffer and is preferably used at a final concentration of 40-60 mM, more preferably about 50 mM, or a phosphate buffer and is preferably used at a final concentration of 5-20 mM, more preferably about 10 mM.
10. The method of claim 1, further comprising purifying the serine ADP-ribosylated protein or peptide from the reaction mix.
11. The method of claim 10, wherein the serine ADP-ribosylated protein or peptide is purified from the reaction mix by StageTip fractionation employing C8, C18, SCX, SAX or SDB-RPS chromatography media, cation or anion exchange chromatography, hydrophilic interaction chromatography, phosphopeptide enrichment, enrichment with a ADP-ribose-binding protein domain, boronate affinity chromatography, filtering the reaction with an ultrafiltration device, a spin column or a combination thereof.
12. The method of claim 1, wherein the efficiency of the serine ADP-ribosylation of the protein or peptide is checked by running the reaction product on a polyacrylamide gel with inverted polarity.
13. The method of claim 12, wherein the protein or peptide is stained, preferably with a Coomassie dye, silver staining or a reverse staining technique, such as imidazole reverse staining and zinc reverse staining, and is more preferably stained with Imperial™ protein stain.
14. The method of claim 1, wherein the method is carried out in vitro or ex vivo.
15. A kit for the production of a serine ADP-ribosylated protein or peptide comprising (a) 5 to 60 mM, preferably 10 to 60 mM of a buffer having a pK.sub.a between 5.0 and 9.0, preferably between 5.5 and 8.5, and most preferably between 6.1 to 8.3, (b) 0.2 to 2.5 mM NAD.sup.+, (c) at least 50 nM, preferably 50 to 3000 nM PARP-1, PARP-2 or the PARP-1 variant, (d) at least 100 nM, preferably 100 to 5000 nM HPF1, (e) at least 10 μg/mL, preferably 10 μg/mL to 200 μg/mL sonicated DNA, said sonicated DNA preferably comprising DNA fragments of 10 to 330 bp, (f) optionally at least 1 μM, preferably 1 to 10 μM PARG, (g) optionally 10 to 80 mM NaCl or KCl, and (h) optionally 0.5 to 2 mM MgCl.sub.2, in one or more container(s).
Description
[0088] The figures show.
[0089] FIG. 1—(A) Comparison of the efficiency of the reaction under different conditions (Lane 1) 500 μM Untreated H3 (1-22) peptide, (Lane 2) 500 μM H3 (1-22) peptide reacted under the conditions used in P32-NAD experiments in Bonfiglio et al., 2017, Mol Cell 65, 932-940 e936 (5.5 μM NAD.sup.+, 0.1 μM PARP-1, 2 μM HPF1 for 20′ RT), (Lane 3) 500 μM H3 (1-22) peptide reacted under the conditions used in MSMS experiments Bonfiglio et al., 2017, Mol Cell 65, 932-940 e936 (200 μM NAD.sup.+, 0.1 μM PARP-1, 2 μM HPF1 for 20′ RT), (Lane 4) 500 μM H3 (1-22) peptide reacted under optimized conditions used to illustrate the present invention (2 mM NAD.sup.+, 0.05 μM PARP-1, 500 nM HPF1 for 2 h RT). Note the reduced amount of PARP-1 and HPF1 used in lane 4 compared to lanes 2 and 3. (B) Comparison of the efficiency of the reaction under different conditions (Lane 1) 62 μM Untreated H2B (1-22) peptide, (Lane 2) 62 μM H2B (1-22) peptide reacted under the conditions used in P32-NAD experiments in Bonfiglio et al., 2017, Mol Cell 65, 932-940 e936 (5.5 μM NAD.sup.+, 0.1 μM PARP-1, 2 μM HPF1 for 20′ RT), (Lane 3) 62 μM H2B (1-22) peptide reacted under the conditions used in MSMS experiments Bonfiglio et al., 2017, Mol Cell 65, 932-940 e936 (200 μM NAD.sup.+, 0.1 μM PARP-1, 2 μM HPF1 for 20′ RT), (Lane 4) 62 μM H3 (1-22) peptide reacted under optimized conditions used to illustrate the present invention (0.1 μM PARP-1, 500 nM HPF1, 1 μM PARG, adding 200 μM NAD.sup.+ every 1 hour for up to 6 hours). (C) For specific substrates the addition of a positively charged tail boosts the efficiency of the reaction (Lane 1) 83 μM Untreated H2B (1-22) peptide, (Lane 2) 83 μM H2B (1-22) peptide reacted with 200 μM NAD.sup.+, 0.1 μM PARP-1, 2 μM HPF1 for 30′ RT, (Lane 3) 66 μM Untreated H2B (1-22) peptide C′terminal poly(Arg) tail, (Lane 4) 66 μM H2B (1-22) peptide C′terminal poly(Arg) tail reacted with 200 μM NAD.sup.+, 0.1 μM PARP-1, 2 μM HPF1 for 30′ RT. (D) Comparison of the efficiency of the reaction under different conditions (Lane 1) 64 μM Untreated Histone H4 (1-19)-Biotin peptide, (Lane 2) 64 μM Histone H4 (1-19)-Biotin peptide reacted under the conditions used in P32-NAD experiments in Bonfiglio et al., 2017, Mol Cell 65, 932-940 e936 (5.5 μM NAD.sup.+, 0.1 μM PARP-1, 2 μM HPF1 for 20′ RT), (Lane 3) 64 μM Histone H4 (1-19)-Biotin peptide reacted under the conditions used in MSMS experiments Bonfiglio et al., 2017, Mol Cell 65, 932-940 e936 (200 μM NAD.sup.+, 0.1 μM PARP-1, 2 μM HPF1 for 20′ RT), (Lane 4) 64 μM Histone H4 (1-19)-Biotin peptide reacted under optimized conditions used to illustrate the present invention (0.1 μM PARP-1, 500 nM HPF1, 1 μM PARG, adding 200 μM NAD.sup.+ every 1 hour for up to 6 hours).
[0090] FIG. 2—(A) TBE sequencing gel run with inverted polarity and stained with Imperial™ Protein Stain (Thermo). Contrary to what occurs when using radioactivity with standard SDS-PAGE, both unmodified and Ser-ADPr histone H3 (1-21) peptides can be resolved and visualized by this novel strategy. By this method, the inventors were able to test different conditions to optimize the efficiency of the reaction (NAD.sup.+, substrate, PARP-1 and HPF1 concentrations, buffer compositions, incubation times). As shown with this particular example, after testing different concentrations of NAD.sup.+ in the presence or absence of 1 μM HPF1, a condition was found in which after a 90 minutes reaction, ˜100% of the peptide (110 μM) is ADP-ribosylated on serine. (B) SDS-PAGE Tricine gel run with normal polarity and stained by InstantBlue™ Ultrafast Protein Stain (SIGMA). Although this commonly-used methodology does not permit discrimination between Ser-ADPr and unmodified peptides, it is useful to determine the purity of the sample. As depicted in the figure the modified Ser-ADPr peptides can be completely separated from the other components that were present in the in vitro ADPr reaction (PARP-1 and HPF1). Importantly, although not shown, NAD.sup.+ is also removed by this separation strategy. (C) TBE sequencing gel run with inverted polarity and stained with Imperial™ Protein Stain (Thermo). Comparable to what is shown in panel A, Ser-ADPr PARP-1 (494-524) peptides can be resolved by this novel visualization strategy (TBE sequencing gel run with inverted polarity). With this example, the inventors demonstrate that the optimized conditions used to illustrate the present invention require adding HPF1 to the reaction mix (2 mM NAD.sup.+, 0.05 μM PARP-1) and incubating for 90 minutes to obtain ˜100% Ser-ADP-ribosylation of 110 μM of a PARP-1 (494-524) peptide. (D) TBE sequencing gel run with inverted polarity and stained with Imperial™ Protein Stain (Thermo). Comparable to what is shown in panel A, inventors performed in vitro ADP-ribosylation assays under different experimental conditions in order to optimize the yield and efficiency of the reaction. (Lane 1) 200 μM Untreated H3 (1-22) peptide, (Lane 2) 200 μM H3 (1-22) peptide reacted with 2 mM NAD.sup.+, 0.1 μM PARP-1, 2 μM HPF1 in the presence of activated DNA for 2 hours RT, (Lane 3) 200 μM H3 (1-22) peptide reacted with 2 mM NAD.sup.+, 0.1 μM PARP-1, 2 μM HPF1 in the absence of activated DNA for 2 hours RT, (Lane 4) 82 μM Untreated H3 (1-22) peptide, (Lane 5) 82 μM H3 (1-22) peptide reacted with 2 mM NAD.sup.+, 0.1 μM PARP-1, 500 nM HPF1 in 50 mM Tris pH: 7.5, 50 mM NaCl, 1 mM MgCl.sub.2, for 2 hours RT, (Lane 6) 82 μM H3 (1-22) peptide reacted with 2 mM NAD.sup.+, 0.1 μM PARP-1, 500 nM HPF1 in 40 mM Tris pH: 7.5, 50 mM NaCl, 1 mM MgCl.sub.2, for 2 hours RT, (Lane 7) 82 μM H3 (1-22) peptide reacted with 2 mM NAD.sup.+, 0.1 μM PARP-1, 500 nM HPF1 in 60 mM Tris pH: 7.5, 50 mM NaCl, 1 mM MgCl.sub.2, for 2 hours RT, (Lane 8) 82 μM H3 (1-22) peptide reacted with 2 mM NAD.sup.+, 0.1 μM PARP-1, 500 nM HPF1 in 50 mM Tris pH: 7.5, 10 mM NaCl, 1 mM MgCl.sub.2, for 2 hours RT, (Lane 9) 82 μM H3 (1-22) peptide reacted with 2 mM NAD.sup.+, 0.1 μM PARP-1, 500 nM HPF1 in 50 mM Tris pH: 7.5, 100 mM NaCl, 1 mM MgCl.sub.2, for 2 hours RT. (E) The serine specificity of the ADPr reaction can be checked by treating a portion of the serine ADP-ribosylated protein or peptide with an enzyme that specifically removes serine ADP-ribosylation from the peptide or protein. Such enzyme is preferably ARH3. (Lane 1) 74 μM Untreated Histone H3 (1-21) peptide, (Lane 2) 74 μM Histone H3 (1-21) peptide reacted with 2 mM NAD.sup.+, 0.1 μM PARP-1 and 2 μM HPF1, for 2 hours RT (Lane 3) Half of the reaction from Lane 2 was incubated with 0.5 μM ARH3 for 30 minutes at RT.
[0091] FIG. 3—Autoradiogram showing ADP-ribosylation of two synthetic peptide variants corresponding to amino acids 1-21 of human H3. To generate these modified peptides, we performed an in vitro ADPr reaction using PARP-1, HPF1, and radioactive (32P) NAD.sup.+. As depicted in the figure, only species with 32P radioactivity (ADPr species) are detected. Unmodified species cannot be detected as they are not radioactively labelled, which prevents any estimation of the efficiency of the reaction and, in the same line, the purity of the species present (FIG. 3 is taken from in Bonfiglio et al., 2017, loc. lit.).
[0092] FIG. 4—TBE sequencing gels run with inverted polarity and stained with Imperial™ Protein Stain (Thermo). To further demonstrate that the claimed 100% serine ADP-ribosylation method is applicable to virtually any peptide or protein with an amino acid sequence containing or comprising at least one serine, the inventors have applied the present invention on different substrates. (A) Histone H4 (1-19)-Biotin peptide (Lane 1) 170 μM Untreated Histone H4 (1-19)-Biotin peptide, (Lane 2) 170 μM Histone H4 (1-19)-Biotin peptide reacted with 2 mM NAD.sup.+, 0.12 μM PARP-1, 1.5 μM HPF1 and 1 μM PARG in the presence of activated DNA for 6 hours RT. (B) Histone H3 (21-44)-Biotin peptide (Lane 1) 195 μM Untreated Histone H3 (21-44)-Biotin peptide, (Lanes 2 to 8) Different replicates for 195 μM Histone H3 (21-44)-Biotin peptide reacted with 2 mM NAD.sup.+, 0.12 μM PARP-1 and 1 μM HPF1 in the presence of activated DNA for 5 hours RT. (C) Histone H3 (1-21)-K9Me Biotin peptide (Lane 1) 95 μM Untreated Histone H3 (1-21)-K9Me Biotin peptide, (Lane 2) 95 μM Histone H3 (1-21)-K9Me Biotin peptide reacted with 2 mM NAD.sup.+ every 2 h, 0.1 μM PARP-1 and 1 μM HPF1, in the presence of activated DNA for 6 hours RT. (D) Histone H3 (1-21)-K9Me2 Biotin peptide (Lane 1) 95 μM Untreated Histone H3 (1-21)-K9Me2 Biotin peptide, (Lane 2) 95 μM Histone H3 (1-21)-K9Me2 Biotin peptide reacted with 2 mM NAD.sup.+ every 2 h, 0.1 μM PARP-1 and 1 μM HPF1, in the presence of activated DNA for 6 hours RT. (E) Histone H1.0 (94-112) peptide (Lane 1) 90 μM Histone H1.0 (94-112) peptide, (Lane 2) 90 μM Histone H1.0 (94-112) peptide reacted with 2 mM NAD.sup.+, 0.1 μM PARP-1 and 0.5 μM HPF1 in the presence of activated DNA for 2 hours RT, (Lane 3) 90 μM Histone H1.0 (94-112) S103A peptide, (Lane 4) 90 μM Histone H1.0 (94-112) S103A peptide reacted with 2 mM NAD.sup.+, 0.1 μM PARP-1 and 0.5 μM HPF1 in the presence of activated DNA for 2 hours RT. (F) Histone H1.2 (178-195) peptide (Lane 1) 100 μM Histone H1.2 (178-195) peptide, (Lane 2) 100 μM Histone H1.2 (178-195) peptide reacted with 2 mM NAD.sup.+, 0.1 μM PARP-1 and 0.5 μM HPF1 in the presence of activated DNA for 2 hours RT, (Lane 3) 100 μM Histone H1.2 (178-195) S187A peptide, (Lane 4) 100 μM Histone H1.2 (178-195) S187A peptide reacted with 2 mM NAD.sup.+, 0.1 μM PARP-1 and 0.5 μM HPF1 in the presence of activated DNA for 2 hours RT. (G) TMA16 (2-22) peptide (Lane 1) 115 μM Untreated TMA16 (2-22) peptide, (Lane 2) 115 μM Untreated TMA16 (2-22) S9A peptide, (Lane 3) 115 μM TMA16 (2-22) peptide reacted with 2 mM NAD.sup.+ and 0.1 μM PARP-1 in the presence of activated DNA for 6 hours RT, (Lane 4) 115 μM TMA16 (2-22) S9A peptide reacted with 2 mM NAD.sup.+ and 0.1 μM PARP-1 in the presence of activated DNA for 6 hours RT, (Lane 5) 115 μM TMA16 (2-22) peptide reacted with 2 mM NAD.sup.+, 0.1 μM PARP-1 and 1.5 μM HPF1 in the presence of activated DNA for 6 hours RT, (Lane 6) 115 μM TMA16 (2-22) S9A peptide reacted with 2 mM NAD.sup.+, 0.1 μM PARP-1 and 1.5 μM HPF1 in the presence of activated DNA for 6 hours RT.
[0093] The examples illustrate the invention.
EXAMPLE 1—COMPARISON OF THE EFFICIENCY OF THE REACTION UNDER DIFFERENT CONDITIONS
[0094] The present invention provides means and methods for the efficient and scalable production of essentially pure Ser-ADPr proteins or peptides. This scalable system for high-yield and efficient generation of the pure Ser-ADP-ribosylated version of a given protein or peptide represents a significant advance in the field. As revealed in FIGS. 1A, B and D, the in vitro modification of different synthetic peptides performed under the experimental conditions published in the state of the art (Bonfiglio et al., 2017, Mol Cell 65, 932-940 e936) does not result in the complete Ser-ADPr of the peptide, in particular no pure Ser-ADP-ribosylated version of a protein or a peptide can be obtained. In contrast, by reacting the same synthetic peptides under the optimized conditions used to illustrate the present invention, a ˜100% pure Ser-ADPr protein or peptide can be obtained. A complementary advantage of the present invention is that by evaluating the efficiency of the reaction (see Example 4), the inventors were able to determine and set forth the experimental conditions to obtain the highest amount of pure Ser-ADPr peptide using the smallest amount of expensive reagents, in particular PARP-1, HPF1 and PARG, if present.
EXAMPLE 2—TESTED CONDITIONS UNDER WHICH AN ABOUT 100% EFFICIENCY WAS OBTAINED
[0095] Among the tested conditions the following conditions resulted in about 100% efficiency:
Solvent:
Water
Buffer:
[0096] (i) 40-60 mM Tris-HCl, pH=7.5; (ii) 50 mM Hepes pH=7.5; or (iii) 10 mM phosphate buffer (pHs=6.1, 6.6, 7.2, 7.7, 8.3);
Salt:
[0097] (i) No salts, or (ii) 10-80 mM NaCl and/or 50 mM KCl (iii) and/or 0.5-2 mM MgCl.sub.2;
NAD.SUP.+.: 1.8 to 2.2 mM;
[0098] PARP-1: at least 50 nM PARP-1;
HPF1: at least 100 nM;
Substrate peptide: 100 to 600 μM (e.g. histone H3 (1-21) peptides);
Incubation time: at least 90 min;
PARG: if present, at least 1 μM
[0099] The required elements for producing Ser-ADPr peptides are PARP-1, HPF1, NAD.sup.+, activated DNA and the substrate itself, all of them contained in a reactor (e.g. tube) in which the modification of the substrate occurs. As the in vitro modification of different synthetic peptides performed under the experimental conditions published in the state of the art does not result in the complete Ser-ADPr of the peptide (FIGS. 1A, B and D), different variables needed to be adjusted in order to optimize the efficiency of the modification, such as the incubation time, the amount of substrate, the amount of PARP-1, HPF1, PARG (if present), and/or NAD.sup.+. Using the visualization methodology described in Example 4, the production of the ADPr version for various substrate peptides was optimized and ˜100% of the modification was reached under the above shown conditions. After reaching ˜100% of the modification (FIGS. 2A, 2C and 2D), a cleaning step can be carried out to eliminate all the reacting elements except the Ser-ADPr peptide. For the cleaning step, for example, a stage tip with a C8 resin that enables fast and efficient purification of the peptide can be used (FIG. 2B). To note, all of the materials required are also available in a much larger scale to make much higher levels of production possible (see Example 3).
EXAMPLE 3—EXPERIMENTAL CONDITIONS UNDER WHICH LARGE AMOUNTS OF PURE SERINE MONO-ADP-RIBOSYLATED PEPTIDE WERE OBTAINED
[0100] Important applications, including the generation of antibodies and structural studies, require large amounts (several milligrams) of serine ADP-ribosylated peptides. In addition, the possibility of scaling up the reaction significantly facilitates the commercialisation of peptides, as a bulk of a serine ADP-ribosylated peptide can be aliquoted in tens or hundreds of vials that can be then sold separately. Under the following tested conditions, the inventors were able to produce ˜5 mg of pure serine mono-ADP-ribosylated peptide:
Solvent: Water
[0101] Buffer: 50 mM Tris-HCl, pH=7.5;
Salts: 50 mM NaCl and 1 mM MgCl.SUB.2.;
NAD.SUP.+.: 2 mM;
PARP-1: 100 nM;
HPF1: 1.5 μM;
[0102] Substrate peptide: 450 μM (Histone H3 (1-15) peptide)
Incubation time: 320 min
[0103] The reaction mix was incubated for 320 minutes at RT and stopped by adding 1 μM Olaparib. Afterwards, 1 μM PARG was added and the reaction mix and it was incubated for 60 minutes at RT. After checking the efficiency, yield and specificity of the reaction by using the novel strategy described in this application (see Example 4), the serine mono-ADP-ribosylated peptides were separated from the other constituents of the reaction mix by using reverse chromatography (C18 cartridge). Pure mono-Ser ADP-ribosylated H3 (1-15) were eluted with 30% Acetonitrile.
EXAMPLE 4—REACTION ASSAY—A METHOD FOR CHECKING THE PURITY, YIELD AND SPECIFICITY OF SERINE ADP-RIBOSYLATION
[0104] In order to produce pure ADPr peptides a visualization methodology is necessary that allows not only the optimization of the reaction but also the assessment of the specificity, purity and yield of the modified peptide. The use of radioactive NAD.sup.+ as a substrate coupled with standard SDS-PAGE electrophoresis was the first and is still the primary means of visualizing in vitro ADP-ribosylated peptides (or proteins) in the art. In fact, it was the gel electrophoresis method that the inventors initially used to visualize Ser-ADPr peptides (and recombinant proteins) that was generated by reacting PARP-1, HPF1, and NAD.sup.+ in vitro (see FIG. 3 and Bonfiglio et al., 2017, loc. lit.).
[0105] However, with this radioactive NAD.sup.+ approach it is impossible to monitor the efficiency and the yield of the reaction generating Ser-ADPr peptides or proteins, since the unmodified substrate peptides or proteins are completely “invisible” with this visualization strategy. In addition standard gel electrophoresis conditions, such as those employed with the radioactive NAD.sup.+ approach, do not allow the spatial separation of ADPr peptides from the unmodified counterpart. Therefore, in order to be able to optimize the ADPr reaction, an unbiased and straightforward method for visualizing both the ADPr-modified and unmodified species was needed.
[0106] The novel visualization method is presented herein in FIGS. 1, 2A, C, D and E. The novelty is that a TBE-polyacrylamide gel, intended for electrophoresis of short nucleic acids, is adapted for the separation of peptides by switching the polarity of the electrophoresis runs. SDS-PAGE, which is the standard gel electrophoresis system for the separation of ADP-ribosylated species (FIG. 2B), does not allow the separation of an ADP-ribosylated peptide from its unmodified counterpart, as both peptides have similarly strong negative charges in the presence of SDS. Considering that ADP-ribose is a nucleotide (more precisely a dinucleotide) the inventors reasoned that an electrophoresis system, such as the TBE-polyacrylamide gel, that is capable of resolving one nucleotide difference in length of nucleic acid fragments would allow a clear separation between an ADP-ribosylated peptide and its unmodified counterpart. However, in a TBE gel the negatively-charged nucleic acids are separated by migrating toward the positively charged anode. The serine ADP-ribosylation substrate peptides, in contrast, have a net positive charge in the absence of SDS, even when modified by ADP-ribose, and, therefore, cannot be run on a TBE gel in its standard configuration. A simple solution to this practical problem is changing the polarity of the electrodes by reversing the jacks when connecting to the power supply. By this means, the positively-charged peptides can run into the commercial off-the-shelf TBE gel and be separated according to their charge. ADP-ribosylated peptides are less positively charged and have a higher mass compared to their unmodified counterparts and therefore migrate significantly more slowly, which allows a clear spatial separation between the bands of the modified and unmodified peptides. After the run, any simple peptide/protein staining strategy (e.g. Coomassie) can be used, and both species (unmodified and modified) are detected, as depicted in FIGS. 1, 2A, C, D and E.
EXAMPLE 5—ADDITIONAL EXAMPLES FOR OBTAINING PURE SER-ADPR SUBSTRATES BY USING THE PRESENT INVENTION
[0107] To further demonstrate that the claimed ˜100% serine ADP-ribosylation method is applicable to virtually any peptide or protein with an amino acid sequence containing or comprising at least one serine, the inventors have applied the method of the present invention to different substrates as follows. [0108] Substrate: 170 μM Histone H4 (1-19)-Biotin peptide [0109] Solvent: Water [0110] Buffer: 50 mM Tris-HCl, pH=7.5; [0111] Salts: 50 mM NaCl and 1 mM MgCl.sub.2; [0112] NAD.sup.+: 2 mM (added every 120 min); [0113] PARP-1: 120 nM; [0114] HPF1: 1.5 μM; [0115] PARG: 1 μM [0116] Incubation time: 360 min [0117] Substrate: 195 μM Histone H3 (21-44)-Biotin peptide [0118] Solvent: Water [0119] Buffer: 50 mM Tris-HCl, pH=7.5; [0120] Salts: 50 mM NaCl and 1 mM MgCl.sub.2; [0121] NAD.sup.+: 2 mM; [0122] PARP-1: 120 nM; [0123] HPF1: 1 μM; [0124] Incubation time: 300 min [0125] Substrate: 95 μM Histone H3 (1-21)-K9Me Biotin peptide [0126] Solvent: Water [0127] Buffer: 50 mM Tris-HCl, pH=7.5; [0128] Salts: 50 mM NaCl and 1 mM MgCl.sub.2; [0129] NAD.sup.+: 2 mM (added every 120 min); [0130] PARP-1: 100 nM; [0131] HPF1: 1 μM; [0132] Incubation time: 360 min [0133] Substrate: 95 μM Histone H3 (1-21)-K9Me2 Biotin peptide [0134] Solvent: Water [0135] Buffer: 50 mM Tris-HCl, pH=7.5; [0136] Salts: 50 mM NaCl and 1 mM MgCl.sub.2; [0137] NAD.sup.+: 2 mM (added every 120 min); [0138] PARP-1: 100 nM; [0139] HPF1: 1 μM; [0140] Incubation time: 360 min [0141] Substrate: 90 μM Histone H1.0 (94-112) peptide [0142] Solvent: Water [0143] Buffer: 50 mM Tris-HCl, pH=7.5; [0144] Salts: 50 mM NaCl and 1 mM MgCl.sub.2; [0145] NAD.sup.+: 2 mM; [0146] PARP-1: 100 nM; [0147] HPF1: 0.5 μM; [0148] Incubation time: 120 min [0149] Substrate: 100 μM Histone H1.2 (178-195) peptide [0150] Solvent: Water [0151] Buffer: 50 mM Tris-HCl, pH=7.5; [0152] Salts: 50 mM NaCl and 1 mM MgCl.sub.2; [0153] NAD.sup.+: 2 mM; [0154] PARP-1: 100 nM; [0155] HPF1: 0.5 μM; [0156] Incubation time: 120 min [0157] Substrate: 115 μM TMA16 (2-22) peptide [0158] Solvent: Water [0159] Buffer: 50 mM Tris-HCl, pH=7.5; [0160] Salts: 50 mM NaCl and 1 mM MgCl.sub.2; [0161] NAD.sup.+: 2 mM; [0162] PARP-1: 100 nM; [0163] HPF1: 1.5 μM; [0164] Incubation time: 240 min
[0165] The results are shown in FIG. 4 and it is evident that ˜100% pure Ser-ADPr protein or peptide was obtained.
