News Column

Patent Issued for Clickable Cross-Linker

July 29, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- According to news reporting originating from Alexandria, Virginia, by NewsRx journalists, a patent by the inventors Sohn, Chang Ho (Pasadena, CA); Beauchamp, Jesse L. (La Canada Flintridge, CA), filed on July 8, 2011, was published online on July 15, 2014 (see also California Institute of Technology).

The assignee for this patent, patent number 8778626, is California Institute of Technology (Pasadena, CA).

Reporters obtained the following quote from the background information supplied by the inventors: "The elucidation of three-dimensional structures of protein complexes and protein protein interactions (PPIs) is one of the central goals in current biological research. Proteins bind to each other to carry out specific biological functions by forming various protein complexes (Hartwell et al., Nature 1999, 402, C47; Pereira-Leal, et al. Philosophical Transactions of the Royal Society B: Biological Sciences 2006, 361, 507). On average, proteins in vivo do not act alone, but rather act as part of a protein complex comprising 10 protein subunits in the cell (Sharan et al., Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 1974. The proteasome is a good example of a functional protein complex (King, R. W.; Deshaies, R. J.; Peters, J.-M.; Kirschner, M. W. Science 1996, 274, 1652). The development of new reagents and methods for identification of binding partners and their interfaces is important for advancement in proteomic science.

"Chemical cross-linkers have been widely employed in analysis of three-dimensional protein structures and protein-protein interactions (PPIs) (Wong, S. S. Chemistry of Protein Conjugation and Cross-Linking; CRC Press, 1991; Phizicky and Fields, Rev. 1995, 59, 94). For identification of cross-linked proteins, traditional experimental methodologies including affinity-based chromatography and Western blot have been performed. However, no detailed structural information relative to the nature of specific protein interfaces is revealed in these experiments. Full atomistic structures of isolated proteins and their complexes can be obtained from NMR spectroscopy and X-ray crystallography but these methodologies usually require large amounts of sample for analysis. Crystallization of diffraction quality protein complexes is often the bottleneck in structure determination by X-ray crystallography.

"Recently, mass spectrometry (MS)-based analysis has allowed detection of binding partners and specific contacting residues in more sensitive ways (Back et al., J. Mol. Biol. 2003, 331, 303; Sinz, A. J. Mass Spectrom. 2003, 38, 1225; Sinz, A. Mass Spectrom. Rev. 2006, 25, 663; Gingras et al., Nat. Rev. Mol. Cell. Biol. 2007, 8, 645; Lee, Y. J. Mol. BioSyst. 2008, 4, 816; Leitner et al., Mol. Cell. Proteomics 2010, 9, 1634; Petrotchenko and Borchers, Mass Spectrom. Rev. 2010, 29, 862; Sinz, A. Anal. Bioanal. Chem. 2010, 397, 3433). In vitro cross-linking and enzymatic digestion produce cross-linked peptides containing spatial information between residues reactive with the cross-linker. This topological information constrains relative distances of amino acid residues, thus aiding in the reconstruction of protein complex subunits.

"For investigation of in vivo PPIs, protein complex immunoprecipitation (i.e., co-IP or 'pull-down') is often performed to recover strongly interacting partners, such as an enzyme bound to its inhibitor. Co-IP requires the use of several antibodies to validate putative binding partners by running successive rounds of experiments. Alternatively, affinity tags can be infused into genes of target proteins to permit efficient purification from cell lysates (Collins and Choudhary, Curr. Opin. Biotechnol. 2008, 19, 324). However, many of the important signaling pathways are believed to be relayed via weak interactions that occur at the outside of strongly bound core protein complexes, and co-IP often fails to identify those weak binding partners. Chemical cross-linking is performed to freeze weak interactions by forming covalent bonds, and then sample analysis is usually combined with other targeted protein purification techniques (Tagwerker et al., Mol. Cell. Proteomics 2006, 5, 737; Guerrero et al., Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 13333.

"For selective and sensitive detection of cross-linked peptides, functionalized chemical cross-linking reagents are used. Various designs of cross-linking reagents have been reported, including biotinylated (Trester-Zedlitz et al.; Tang et al.; Kang et al.), isotope-coded (Chu et al.; Muller et al.; Collins et al.; Petrotchenko et al.), fluorophore labeled (Wine et al.; Sinz et al.; Sinz et al.) mass-tag labeled (Back et al.), amidinating (Lauber et al.), and chromophore labeled (Gardner et al.) cross-linking reagents. However, the addition of functional groups can often cause the cross-linker to become very bulky or less cell-permeable, and thus not very effective for in vivo cross-linking (Zhang et al.). To reduce the total size of the cross-linker, separation of the cross-linking step from conjugation of affinity tags is one effective strategy. (Trester-Zedlitz et al., J. Am. Chem. Soc. 2003, 125, 2416.; Tang et al., Anal. Chem. 2005, 77, 311; Kang et al., Rapid Commun. Mass Spectrom. 2009, 23, 1719; Chu et al., J. Am. Chem. Soc. 2006, 128, 10362; Muller et al., Anal. Chem. 2001, 73, 1927; Collins et al., Bioorg. Med. Chem. Lett. 2003, 13, 4023; Petrotchenko et al., Mol. Cell. Proteomics 2005, 4, 1167; Wine et al., Anal. Chem. 2002, 74, 1939; Sinz et al., Biochemistry 2001, 40, 7903; Sinz and Wang, Anal. Biochem. 2004, 331, 27; Back, J. W.; Hartog, A. F.; Dekker, H. L.; Muijsers, A. O.; de Koning, L. J.; de Jong, L. J. Am. Soc. Mass Spectrom. 2001, 12, 222; Lauber, M. A.; Reilly, J. P. Anal. Chem. 2010, 82, 7736; Gardner et al., Anal. Chem. 2008, 80, 4807; Zhang et al., Mol. Cell. Proteomics 2009, 8, 409.)

"More recent cross-linking and enrichment strategies for separation of the cross-linking reaction from enrichment steps have recently been developed based on bio-orthogonal chemistries such as the azide-alkyne 'click' cycloaddition (Rostovtsev et al.; Tornoe et al.; Baskin et al.) and Staudinger ligation (Saxon et al.) using alkyne (Chowdhury et al.; Trnka et al.) or azide (Nessen et al.; Vellucci et al.) tagged cross-linkers. Azides and alkynes are not naturally found in proteins, peptides, nucleic acids, or glycans. The orthogonality of azides and alkynes to biological processes (i.e., competing reactions) is a significant advantage of this approach. Moreover, the 'click' cycloaddition can be performed under aqueous conditions, allowing the enrichment of cross-linked products by conjugation of an appropriate affinity or labeling tag. However, existing clickable cross-linkers still require screening and analysis of all cross-linked products. This analysis can require time consuming confirmation to eliminate false positives. Accordingly, an efficient and effective clickable cross-linker is desired. (Rostovtsev et al., Angew. Chem.-Int. Edit. 2002, 41, 2596; Tornoe et al., J. Org. Chem. 2002, 67, 3057; Baskin et al., Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16793; Saxon et al., Science 2000, 287, 2007; Chowdhury et al., Anal. Chem. 2009, 81, 5524; Trnka et al., Mol. Cell. Proteomics 2010, 9, 2306; Nessen et al., J. Proteome Res. 2009, 8, 3702; Vellucci et al., J. Am. Soc. Mass Spectrom. 2010, 21, 1432.)"

In addition to obtaining background information on this patent, NewsRx editors also obtained the inventors' summary information for this patent: "In some embodiments of the present invention, a composition including a cross-linker compound of Formula I

"##STR00001## is provided, where each of M.sub.1 and M.sub.2 is a target conjugation group capable of conjugating to a protein, peptide or nucleic acid; each of L.sub.1 and L.sub.2 is independently a linkage having 1 to 20 carbon atoms or 1 to 6 polyethylene glycol groups; and R is a reporter group selected from hexynyl groups, pentynyl groups, heptynyl groups, azido-propyl groups, azido-butyl groups, or azido-pentyl groups.

"In some embodiments, the composition also includes a labeling tag conjugated to the cross-linker compound, the labeling tag having a reactive group selected from hexynyl groups, pentynyl groups, heptynyl groups, azido-propyl groups, azido-butyl groups, azido-pentyl groups, cyclooctynyl groups, or difluorinated cyclooctynyl groups.

"In other embodiments, a method of cross-linking at least one protein or peptide includes mixing a sample containing the at least one protein or peptide with the cross-linker compound of Formula I; conjugating a tag to the cross-linker compound in the cross-linked sample to form a tagged sample; isolating the tagged sample to form an enriched sample; ionizing the enriched sample to form an ionized sample; fragmenting the ionized sample to form fragment ions; and detecting a mass-to-charge ratio of the fragment ions.

"In some embodiments of the method of cross-linking, R is selected from azido-propyl groups, azido-butyl groups and azido-pentyl groups, and the labeling tag has a reactive group selected from hexynyl groups, pentynyl groups, heptynyl groups, cyclooctynyl groups, and difluorinated cyclooctynyl groups.

"In some embodiments of the method of cross-linking, R is selected from hexynyl groups, pentynyl groups, and heptynyl groups, and the labeling tag has an azide group.

"In some embodiments, the method of cross-linking also includes digesting the cross-linked sample using a protease prior to conjugating a labeling tag to the cross-linker compound.

"In some embodiments of the method of cross-linking, the sample containing at least one protein or peptide also includes a nucleic acid.

"In some embodiments, a method of identifying intermolecular and intramolecular protein interactions in a sample includes mixing a sample containing at least one protein or peptide with the cross-linker compound of Formula Ito form a cross-linked sample; conjugating a labeling tag to the cross-linker compound in the cross-linked sample to form a tagged sample; isolating the tagged sample to form an enriched sample; ionizing the enriched sample to form an ionized sample; fragmenting the ionized sample to form fragment ions; detecting a mass-to-charge ratio of the fragment ions; and based on the mass-to-charge ratio of the fragment ions, determining a peptide sequence associated with the fragment ions.

"In some embodiments of the method of identifying intermolecular and intramolecular protein interactions in a sample, the sample containing at least one protein or peptide also contains a nucleic acid.

"In some embodiments of the method of identifying intermolecular and intramolecular protein interactions in a sample, R is selected from azido-propyl, azido-butyl, and azido-pentyl groups, and the labeling tag has an alkyne group selected from hexynyl groups, pentynyl groups, heptynyl groups, cyclooctynyl groups, and difluorinated cyclooctynyl groups.

"In some embodiments of the method of identifying intermolecular and intramolecular protein interactions in a sample, R is selected from hexynyl groups, pentynyl groups and heptynyl groups, and the labeling tag has an azide group."

For more information, see this patent: Sohn, Chang Ho; Beauchamp, Jesse L.. Clickable Cross-Linker. U.S. Patent Number 8778626, filed July 8, 2011, and published online on July 15, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=8778626.PN.&OS=PN/8778626RS=PN/8778626

Keywords for this news article include: California Institute of Technology, Genetic Phenomena, Genetic Structures, Genome Components, Peptides, Proteins, Proteomics, Technology, Viral Genes, rev Genes.

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