{"search_session":{},"preferences":{"l":"en","queryLanguage":"en"},"patentId":"US_8367366_B2","frontPageModel":{"patentViewModel":{"ref":{"entityRefId":"127-726-288-575-880","entityRefType":"PATENT"},"entityMetadata":{"linkedIds":{"empty":true},"tags":[],"collections":[{"id":12138,"type":"PATENT","title":"University of Arkansas at Fayetteville - Patent Portfolio","description":"","access":"OPEN_ACCESS","displayAvatar":true,"attested":false,"itemCount":3026,"tags":[],"user":{"id":91044780,"username":"Cambialens","firstName":"","lastName":"","created":"2015-05-04T00:55:26.000Z","displayName":"Cambialens","preferences":"{\"usage\":\"public\",\"beta\":false}","accountType":"PERSONAL","isOauthOnly":false},"notes":[{"id":8643,"type":"COLLECTION","user":{"id":91044780,"username":"Cambialens","firstName":"","lastName":"","created":"2015-05-04T00:55:26.000Z","displayName":"Cambialens","preferences":"{\"usage\":\"public\",\"beta\":false}","accountType":"PERSONAL","isOauthOnly":false},"text":"
Search Applicants and Owners separately:Univ* Arkansas. Select more for logical variants. Add to collection. Select all patents in the collection and expand by simple families. Add to collection. Total patents: 2874
Search Applicants and Owners separately:Univ* Arkansas. Select more for logical variants. Add to collection. Select all patents in the collection and expand by simple families. Add to collection. Total patents: 2874
(a) incubating the enzyme or enzyme mixture in vitro with a polypeptide substrate under conditions that allow methylation and/or demethylation of the polypeptide substrate to add methyl groups to or remove methyl groups from one or more lysine residues of the polypeptide substrate to generate an enzymatically methylated and/or demethylated polypeptide, wherein the polypeptide substrate is a peptide fragment of a full-length protein;\n
(b) reacting the enzymatically methylated and/or demethylated polypeptide with one or more substances to non-enzymatically react with the polypeptide to add one or more methyl groups to the polypeptide to generate a fully dimethylated or trimethylated polypeptide that is fully dimethylated or trimethylated at least one lysine residue; wherein the methyl groups non-enzymatically added to the polypeptide differ in molecular weight from the added or removed methyl groups of the enzymatically methylated and/or demethylated protein or peptide; and\n
(c) subjecting the fully methylated polypeptide to mass spectrometry to quantitatively determine a ratio of enzymatically produced nonmethyl, monomethyl, and dimethyl and optionally trimethyl lysine species at least one lysine residue."],"number":1,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 1 further comprising determining amino acid positions of enzymatically added or removed methyl groups on the polypeptide."],"number":2,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 2 wherein the method comprises quantitatively determining a ratio of enzymatically produced nonmethyl, monomethyl, and dimethyl and optionally trimethyl lysine species at a first lysine residue identified by position and quantitatively determining a ratio of enzymatically produced nonmethyl, monomethyl, and dimethyl and optionally trimethyl lysine species at a second lysine residue identified by position."],"number":3,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 2 wherein the mass spectrometry is tandem mass spectrometry."],"number":4,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 1 wherein the mass spectrometry comprises MALDI mass spectrometry."],"number":5,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 1 wherein the polypeptide substrate is methylated."],"number":6,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 1 wherein the polypeptide substrate is acetylated."],"number":7,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 1 wherein the enzyme or enzyme mixture comprises lysine specific demethylase 1 (LSD1)."],"number":8,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 1 wherein the polypeptide substrate is a polypeptide of histone H3."],"number":9,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 9 wherein the polypeptide substrate comprises H3K4 or H3K9."],"number":10,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 1 wherein step (b) comprises reacting the enzymatically methylated and/or demethylated polypeptide with formaldehyde non-enzymatically to generate a formaldehyde-reacted polypeptide and reducing the formaldehyde reacted polypeptide to generate a fully dimethylated polypeptide."],"number":11,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 1 wherein step (a) comprises incubating the enzyme or enzyme mixture in vitro with two or more polypeptide substrates differing in post-translational modifications but identical in their amino acid sequence, and the method comprises comparing activity of the enzyme or enzyme mixture on the two or more polypeptide substrates."],"number":12,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 12 wherein the two or more polypeptide substrates differ in methylation at one or more amino acid residues."],"number":13,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 1 wherein the polypeptide substrate comprises a synthetic ligand and the method further comprises before step (c) purifying the enzymatically acetylated and/or deacetylated polypeptide by contacting the polypeptide with a solid substrate coupled to a receptor for the ligand."],"number":14,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 1 wherein the enzyme or enzyme mixture comprises a methyltransferase, and step (a) comprises incubating the methyltransferase in vitro with S-adenosyl methionine and the polypeptide substrate to generate an enzymatically methylated polypeptide."],"number":15,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 1 wherein the enzyme or enzyme mixture comprises a demethylase, and step (a) comprises incubating the demethylase in vitro with a partially or fully methylated polypeptide substrate to generate an enzymatically demethylated polypeptide."],"number":16,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 1 wherein the enzymatically added or removed methyl groups are of natural isotopic composition and the non-enzymatically added methyl groups contain at least one deuterium."],"number":17,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 1 wherein the polypeptide substrate is a peptide fragment of a histone."],"number":18,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 1 wherein the polypeptide substrate is enzymatically methylated and/or demethylated on two or more amino acid residues."],"number":19,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 2 wherein the polypeptide substrate is enzymatically methylated and/or demethylated on two or more amino acid residues."],"number":20,"annotation":false,"claim":true,"title":false},{"lines":["A kit for characterizing the activity of a methyltransferase or demethylase on a substrate, the kit comprising:\n
(a) (i) a polypeptide substrate for a methyltransferase and/or demethylase, wherein the polypeptide substrate is a peptide fragment of a full-length protein or (ii) a methyltransferase or demethylase; and\n
(b) a methylation reagent for nonenzymatic methylation selected from the group consisting of formaldehyde, iodomethane, and a reductant capable of reducing a Schiff base in vitro; and\n
optionally (c) S-adenosyl methionine;\n
wherein the methylation reagent or the polypeptide substrate have a non-natural isotopic composition, or if neither has a non-natural isotopic composition then S-adenosyl methionine is a component of the kit and has a non-natural isotopic composition."],"number":21,"annotation":false,"claim":true,"title":false},{"lines":["The kit of claim 21 wherein the polypeptide substrate is a fragment of a full-length histone."],"number":22,"annotation":false,"claim":true,"title":false},{"lines":["The kit of claim 21 wherein the kit comprises d2-formaldehyde or 13C-formaldehyde."],"number":23,"annotation":false,"claim":true,"title":false},{"lines":["The kit of claim 21 wherein the polypeptide substrate comprises a synthetic ligand and the kit further comprises a solid substrate coupled to a receptor for the ligand."],"number":24,"annotation":false,"claim":true,"title":false},{"lines":["The kit of claim 21 wherein the polypeptide substrate is methylated."],"number":25,"annotation":false,"claim":true,"title":false},{"lines":["The kit of claim 21 wherein the polypeptide substrate is acetylated."],"number":26,"annotation":false,"claim":true,"title":false},{"lines":["The kit of claim 21 wherein the kit contains two or more polypeptide substrates that differ in post-translational modifications but are identical in their amino acid sequence."],"number":27,"annotation":false,"claim":true,"title":false},{"lines":["The kit of claim 21 wherein the kit comprises S-adenosyl methionine."],"number":28,"annotation":false,"claim":true,"title":false},{"lines":["The kit of claim 21 wherein the polypeptide substrate is a substrate for methylation and/or demethylation by the methyltransferase and/or demethylase at two or more amino acid residues."],"number":29,"annotation":false,"claim":true,"title":false},{"lines":["The kit of claim 22 wherein the polypeptide substrate is a peptide fragment of histone H3 and comprises H3K4 or H3K9."],"number":30,"annotation":false,"claim":true,"title":false},{"lines":["The kit of claim 21 wherein the kit comprises:\n
(a) (i) a polypeptide substrate for a methyltransferase and/or demethylase, wherein the polypeptide substrate is a peptide fragment of a full-length protein and (ii) a methyltransferase or demethylase."],"number":31,"annotation":false,"claim":true,"title":false}]}},"filters":{"npl":[],"notNpl":[],"applicant":[],"notApplicant":[],"inventor":[],"notInventor":[],"owner":[],"notOwner":[],"tags":[],"dates":[],"types":[],"notTypes":[],"j":[],"notJ":[],"fj":[],"notFj":[],"classIpcr":[],"notClassIpcr":[],"classNat":[],"notClassNat":[],"classCpc":[],"notClassCpc":[],"so":[],"notSo":[],"sat":[]},"sequenceFilters":{"s":"SEQIDNO","d":"ASCENDING","p":0,"n":10,"sp":[],"si":[],"len":[],"t":[],"loc":[]}}