# US 7,407,812 B2 · Method and kit for the isolation of phosphorylated peptides > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,407,812 B2 - Document ID: US7407812B2 - Status: Granted - Kind: B2 - Application number: 11/596,251 - Filed: 2005-04-21 - Published: 2007-10-04 - Granted: 2008-08-05 - Earliest claimed: 2004-05-14 - Assignees: Merck Patent GmbH - Inventors: Jonas Anders; Achim Schwämmle; Sven Andrecht; Robertus Hendriks; Anette Boerner; Cora Rueter - Web page: https://patentgrep.com/patent/US7407812B2 ## CPC - C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes - Y10T 436/25: including sample preparation - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION ## Abstract The invention relates to a method and a kit for the isolation of phosphorylated peptides from complex mixtures using support materials having chelate ligands based on silica and alkaline elution buffers. In preferred embodiments, the method according to the invention enables the isolation of phosphopeptides from complex sample solutions with a reproducibly high yield and at the same time with high purity and it allows efficient ionisation and detection of the isolated phosphopeptides both by MALDI-TOF and also by ESI mass spectrometry without additional requisite method steps, such as, for example, chromatographic desalination of the sample. ## Claims ### Claim 1 Method for the enrichment of phosphopeptides, comprising of the following method steps: a) provision of a support material having chelate ligands based on silica b) activation of the support material from step a) using transition-metal ions, oxides or oxide hydrates of transition-metal ions or trivalent ions of metals from the third main group c) bringing a phosphopeptide-containing sample into contact with the activated support material in the presence of a binding buffer d) removal of the supernatant consisting of the binding buffer and the unbound part of the sample e) optionally washing of the support material f) elution of the phosphopeptides with an elution buffer which has a pH>10 and comprises alkali metal, alkaline earth metal or ammonium salts of thiocyanate, of acids of the complex ligands nitrito, isocyano, nitrile, isocyanato, isothiocyanato, azido, ethylenediamine, isonitrile, fulminato and cyano and/or of the oxygen acids of phosphorus, sulfur, vanadium, ruthenium, niobium, tantalum, tungsten or of molybdenum, and/or chelating agents. ### Claim 2 (depends on 1) Method according to claim 1, wherein the activation in step b) is carried out using iron(III) ions or zirconium(IV) ions. ### Claim 3 (depends on 1) Method according to claim 1 wherein the elution in step f) is carried out with an elution buffer which comprises of an alkali metal, alkaline earth metal or ammonium salts of oxygen acids of phosphorus or of thiocyanate in a concentration of between 0.005 and 2 mol/l. ### Claim 4 (depends on 1) Method according to claim 1, wherein the phosphopeptides eluted in step f) are investigated directly by mass spectrometry, thin-layer chromatography or by sequence analysis. ### Claim 5 (depends on 1) Method according to claim 1, wherein a support material having chelate ligands of the formula Ia and/or Ib is provided in step a) where R=C1 to C6 alkyl or C5 to C18 aryl, optionally mono- or polysubstituted, by hydroxyl, C1-C4-alkoxy, amino, alkylamino, CN or halogen radicals, m=2 to 8, where one or more non-adjacent C atoms may be replaced by O, NH, S or —C═C—. ### Claim 6 (depends on 1) Method according to claim 1, wherein a support material having a chelate ligand which consists of magnetite particles whose surface is at least partly covered by silica is provided in step a). ### Claim 7 Kit for the enrichment of phosphopeptides, at least containing a support material having a chelate ligand based on silica and an elution buffer which has a pH>10 and comprises of an alkali metal, alkaline earth metal or ammonium salts of thiocyanate, of acids of the complex ligands nitrito, isocyano, nitrile, isocyanato, isothiocyanato, azido, ethylenediamine, isonitrile, fulminato or cyano and/or of the oxygen acids of phosphorus, sulfur, vanadium, ruthenium, niobium, tantalum, tungsten and/or molybdenum, and/or chelating agents. ### Claim 8 (depends on 7) Kit according to claim 7, wherein the support material having a chelate ligand has been activated using iron(III) or zirconium(IV) ions. ### Claim 9 (depends on 7) Kit according to claim 7, wherein the kit contains, as support material, magnetite particles which are at least partly coated with silica. ### Claim 10 (depends on 7) Kit according to claim 7, wherein the kit contains a support material having chelate ligands conforming to the formula Ia and/or Ib, where R=methyl and m=2. ### Claim 11 (depends on 7) Kit according to claim 7, wherein the kit contains, as elution buffer, a buffer which comprises alkali metal, alkaline earth metal or ammonium salts of oxygen acids of phosphorus or of thiocyanate in a concentration of between 0.005 and 2 mol/l.