User:Kbufdbqjubmjtn/sandbox

From Wikipedia, the free encyclopedia
Mitochondrial outer membrane proteins are identified via proximity labeling.

Enzyme-catalyzed proximity labeling (PL), also known as proximity-based labeling, is a laboratory technique that labels biomolecules, usually proteins or RNA, proximal to a protein of interest.[1] By creating a gene fusion in a living cell between the protein of interest and an engineered labeling enzyme, biomolecules spatially proximal to the protein of interest can then be selectively marked with biotin for pulldown and analysis. Proximity labeling has been used for identifying the components of novel cellular structures and for determining protein-protein interaction partners, among other applications.[2]  

Principles[edit]

Proximity labeling relies on a labeling enzyme that can biotinylate nearby biomolecules promiscuously. Biotin labeling can be achieved through several different methods, depending on the species of labeling enzyme.

  • BioID is a mutant E. coli biotin ligase that catalyzes the activation of biotin by ATP. The activated biotin is short-lived and thus can only diffuse to a region proximal to BioID. Labeling is achieved when the activated biotin reacts with nearby amines, such as the lysine sidechain amines found in proteins.[1] TurboID is a biotin ligase engineered via yeast surface display directed evolution. TurboID enables ~10 minute labeling times instead of the ~18 hour labeling times required by BioID.[3]
  • APEX is an ascorbate peroxidase derivative reliant on hydrogen peroxide for catalyzing the oxidation of biotin-tyramide, also known as biotin-phenol, to a short-lived and reactive biotin-phenol free radical. Labeling is achieved when this intermediate reacts with various functional groups of nearby biomolecules. APEX can also be used for local deposition of diaminobenzidine, a precursor for an electron microscopy stain. APEX2 is a derivative of APEX engineered via yeast surface display directed evolution. APEX2 shows improved labeling efficiency and cellular expression levels.[4]

To label proteins nearby a protein of interest, a typical proximity labeling experiment begins by cellular expression of an APEX2 fusion to the protein of interest, which localizes to the protein of interest's native environment. Cells are next incubated with biotin-phenol, then briefly with hydrogen peroxide, initiating biotin-phenol free radical generation and labeling. To minimize cellular damage, the reaction is then quenched using an antioxidant buffer. Cells are lysed and the labeled proteins are pulled down with streptavidin beads. The proteins are digested with trypsin, and finally the resulting peptidic fragments are analyzed using shotgun proteomics methods such as LC-MS/MS or SPS-MS3.[4]

If instead a protein fusion is not genetically accessible (such as in human tissue samples) but an antibody for the protein of interest is known, proximity labeling can still be enabled by fusing a labeling enzyme with the antibody, then incubating the fusion with the sample.[5][6]

Applications[edit]

Proximity labeling methods have been used to study the proteomes of biological structures that are otherwise difficult to isolate purely and completely, such as cilia,[7] mitochondria,[8] postsynaptic clefts,[2] p-bodies, stress granules,[9] and lipid droplets.[10]

Fusion of APEX2 with G-protein coupled receptors (GPCRs) allows for both tracking GPCR signaling at a 20 second temporal resolution[11] and also identification of unknown GPCR-linked proteins.[12]

Proximity labeling has also been used for transcriptomics and interactomics. Alice Ting and the Ting lab at Stanford University have used APEX to identify RNA localized to specific cellular compartments.[13][14] Proximity labeling has also been used to find interaction partners of heterodimeric protein phosphatases, of the miRISC (microRNA-induced silencing complex) protein Ago2, and of ribonucleoproteins.[15]


  1. ^ a b Roux, Kyle J.; Kim, Dae In; Raida, Manfred; Burke, Brian (2012-03-19). "A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells". Journal of Cell Biology. 196 (6): 801–810. doi:10.1083/jcb.201112098. ISSN 0021-9525.
  2. ^ a b Han, Shuo; Li, Jiefu; Ting, Alice Y (2018-06-01). "Proximity labeling: spatially resolved proteomic mapping for neurobiology". Current Opinion in Neurobiology. Neurotechnologies. 50: 17–23. doi:10.1016/j.conb.2017.10.015. ISSN 0959-4388.
  3. ^ Branon, Tess C.; Bosch, Justin A.; Sanchez, Ariana D.; Udeshi, Namrata D.; Svinkina, Tanya; Carr, Steven A.; Feldman, Jessica L.; Perrimon, Norbert; Ting, Alice Y. (2018-10-01). "Efficient proximity labeling in living cells and organisms with TurboID". Nature Biotechnology. 36 (9): 880–887. doi:10.1038/nbt.4201. ISSN 1546-1696.
  4. ^ a b Kalocsay, Marian (2019), Sunbul, Murat; Jäschke, Andres (eds.), "APEX Peroxidase-Catalyzed Proximity Labeling and Multiplexed Quantitative Proteomics", Proximity Labeling: Methods and Protocols, Methods in Molecular Biology, Springer, pp. 41–55, doi:10.1007/978-1-4939-9537-0_4.pdf, ISBN 978-1-4939-9537-0, retrieved 2020-05-04
  5. ^ Rees, Johanna S.; Li, Xue-Wen; Perrett, Sarah; Lilley, Kathryn S.; Jackson, Antony P. (2015-11-01). "Protein Neighbors and Proximity Proteomics". Molecular & Cellular Proteomics. 14 (11): 2848–2856. doi:10.1074/mcp.R115.052902. ISSN 1535-9476. PMID 26355100.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  6. ^ Bar, Daniel Z; Atkatsh, Kathleen; Tavarez, Urraca; Erdos, Michael R; Gruenbaum, Yosef; Collins, Francis S (2018-2). "Biotinylation by antibody recognition - A method for proximity labeling". Nature methods. 15 (2): 127–133. doi:10.1038/nmeth.4533. ISSN 1548-7091. PMC 5790613. PMID 29256494. {{cite journal}}: Check date values in: |date= (help)
  7. ^ Mick, David U.; Rodrigues, Rachel B.; Leib, Ryan D.; Adams, Christopher M.; Chien, Allis S.; Gygi, Steven P.; Nachury, Maxence V. (2015-11-23). "Proteomics of Primary Cilia by Proximity Labeling". Developmental Cell. 35 (4): 497–512. doi:10.1016/j.devcel.2015.10.015. ISSN 1878-1551. PMC 4662609. PMID 26585297.
  8. ^ Rhee, Hyun-Woo; Zou, Peng; Udeshi, Namrata D.; Martell, Jeffrey D.; Mootha, Vamsi K.; Carr, Steven A.; Ting, Alice Y. (2013-03-15). "Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging". Science. 339 (6125): 1328–1331. doi:10.1126/science.1230593. ISSN 0036-8075. PMID 23371551.
  9. ^ Youn, Ji-Young; Dunham, Wade H.; Hong, Seo Jung; Knight, James D.R.; Bashkurov, Mikhail; Chen, Ginny I.; Bagci, Halil; Rathod, Bhavisha; MacLeod, Graham; Eng, Simon W.M.; Angers, Stéphane (2018-02). "High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies". Molecular Cell. 69 (3): 517–532.e11. doi:10.1016/j.molcel.2017.12.020. ISSN 1097-2765. {{cite journal}}: Check date values in: |date= (help)
  10. ^ Bersuker, Kirill; Peterson, Clark W. H.; To, Milton; Sahl, Steffen J.; Savikhin, Victoria; Grossman, Elizabeth A.; Nomura, Daniel K.; Olzmann, James A. (2018-01-08). "A Proximity Labeling Strategy Provides Insights into the Composition and Dynamics of Lipid Droplet Proteomes". Developmental Cell. 44 (1): 97–112.e7. doi:10.1016/j.devcel.2017.11.020. ISSN 1534-5807.
  11. ^ Paek, Jaeho; Kalocsay, Marian; Staus, Dean P.; Wingler, Laura; Pascolutti, Roberta; Paulo, Joao A.; Gygi, Steven P.; Kruse, Andrew C. (04 06, 2017). "Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling". Cell. 169 (2): 338–349.e11. doi:10.1016/j.cell.2017.03.028. ISSN 1097-4172. PMC 5514552. PMID 28388415. {{cite journal}}: Check date values in: |date= (help)
  12. ^ Lobingier, Braden T.; Hüttenhain, Ruth; Eichel, Kelsie; Miller, Kenneth B.; Ting, Alice Y.; von Zastrow, Mark; Krogan, Nevan J. (04 06, 2017). "An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells". Cell. 169 (2): 350–360.e12. doi:10.1016/j.cell.2017.03.022. ISSN 1097-4172. PMC 5616215. PMID 28388416. {{cite journal}}: Check date values in: |date= (help)
  13. ^ Shields, Emily J.; Petracovici, Ana F.; Bonasio, Roberto (2019-04-15). "lncRedibly versatile: biochemical and biological functions of long noncoding RNAs". Biochemical Journal. 476 (7): 1083–1104. doi:10.1042/BCJ20180440. ISSN 0264-6021.
  14. ^ Fazal, Furqan M.; Han, Shuo; Parker, Kevin R.; Kaewsapsak, Pornchai; Xu, Jin; Boettiger, Alistair N.; Chang, Howard Y.; Ting, Alice Y. (2019-07-11). "Atlas of Subcellular RNA Localization Revealed by APEX-Seq". Cell. 178 (2): 473–490.e26. doi:10.1016/j.cell.2019.05.027. ISSN 0092-8674.
  15. ^ Trinkle-Mulcahy, Laura (2019-01-31). "Recent advances in proximity-based labeling methods for interactome mapping". F1000Research. 8: 135. doi:10.12688/f1000research.16903.1. ISSN 2046-1402.{{cite journal}}: CS1 maint: unflagged free DOI (link)
Retrieved from "https://en.wikipedia.org/w/index.php?title=User:Kbufdbqjubmjtn/sandbox&oldid=954937448"