{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,20]],"date-time":"2026-09-20T06:24:17Z","timestamp":1789885457087,"version":"4.0.1"},"reference-count":37,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2016,8,30]],"date-time":"2016-08-30T00:00:00Z","timestamp":1472515200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2016,8,30]],"date-time":"2016-08-30T00:00:00Z","timestamp":1472515200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Analysis of the developing proteome has been complicated by a lack of tools that can be easily employed to label and identify newly synthesized proteins within complex biological mixtures. Here, we demonstrate that the methionine analogs azidohomoalanine and homopropargylglycine can be globally incorporated into the proteome of mice through facile intraperitoneal injections. These analogs contain bio-orthogonal chemical handles to which fluorescent tags can be conjugated to identify newly synthesized proteins. We show these non-canonical amino acids are incorporated into various tissues in juvenile mice and in a concentration dependent manner. Furthermore, administration of these methionine analogs to pregnant dams during a critical stage of murine development, E10.5\u201312.5 when many tissues are assembling, does not overtly disrupt development as assessed by proteomic analysis and normal parturition and growth of pups. This successful demonstration that non-canonical amino acids can be directly administered <jats:italic>in vivo<\/jats:italic> will enable future studies that seek to characterize the murine proteome during growth, disease and repair.<\/jats:p>","DOI":"10.1038\/srep32377","type":"journal-article","created":{"date-parts":[[2016,8,30]],"date-time":"2016-08-30T09:17:02Z","timestamp":1472548622000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":89,"title":["Incorporation of non-canonical amino acids into the developing murine proteome"],"prefix":"10.1038","volume":"6","author":[{"given":"Sarah","family":"Calve","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew J.","family":"Witten","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander R.","family":"Ocken","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tamara L.","family":"Kinzer-Ursem","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2016,8,30]]},"reference":[{"key":"BFsrep32377_CR1","doi-asserted-by":"publisher","first-page":"427","DOI":"10.1038\/nrm2900","volume":"11","author":"C Choudhary","year":"2010","unstructured":"Choudhary, C. & Mann, M. Decoding signalling networks by mass spectrometry-based proteomics. Nat Rev Mol Cell Biol 11, 427\u2013439 (2010).","journal-title":"Nat Rev Mol Cell Biol"},{"key":"BFsrep32377_CR2","doi-asserted-by":"publisher","first-page":"4365","DOI":"10.1038\/srep04365","volume":"4","author":"L Sun","year":"2014","unstructured":"Sun, L. et al. Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development. Sci Rep 4, 4365 (2014).","journal-title":"Sci Rep"},{"key":"BFsrep32377_CR3","doi-asserted-by":"publisher","first-page":"383","DOI":"10.1016\/j.devcel.2015.10.010","volume":"35","author":"L Peshkin","year":"2015","unstructured":"Peshkin, L. et al. On the Relationship of Protein and mRNA Dynamics in Vertebrate Embryonic Development. Dev Cell 35, 383\u2013394 (2015).","journal-title":"Dev Cell"},{"key":"BFsrep32377_CR4","doi-asserted-by":"publisher","first-page":"1257","DOI":"10.1002\/pmic.200700724","volume":"8","author":"D Hartl","year":"2008","unstructured":"Hartl, D. et al. Transcriptome and proteome analysis of early embryonic mouse brain development. Proteomics 8, 1257\u20131265 (2008).","journal-title":"Proteomics"},{"key":"BFsrep32377_CR5","doi-asserted-by":"publisher","first-page":"981","DOI":"10.1074\/mcp.M700382-MCP200","volume":"7","author":"MB Lucitt","year":"2008","unstructured":"Lucitt, M. B. et al. Analysis of the zebrafish proteome during embryonic development. Mol Cell Proteomics 7, 981\u2013994 (2008).","journal-title":"Mol Cell Proteomics"},{"key":"BFsrep32377_CR6","doi-asserted-by":"crossref","unstructured":"Hill, R. C., Calle, E. A., Dzieciatkowska, M., Niklason, L. E. & Hansen, K. C. Quantification of Extracellular Matrix Proteins from a Rat Lung Scaffold to Provide a Molecular Readout for Tissue Engineering. Mol Cell Proteomics (2015).","DOI":"10.1074\/mcp.M114.045260"},{"key":"BFsrep32377_CR7","doi-asserted-by":"publisher","first-page":"1741","DOI":"10.1074\/mcp.M113.037267","volume":"13","author":"ML Decaris","year":"2014","unstructured":"Decaris, M. L. et al. Proteomic analysis of altered extracellular matrix turnover in bleomycin-induced pulmonary fibrosis. Mol Cell Proteomics 13, 1741\u20131752 (2014).","journal-title":"Mol Cell Proteomics"},{"key":"BFsrep32377_CR8","doi-asserted-by":"publisher","first-page":"1709","DOI":"10.1074\/mcp.M112.024919","volume":"12","author":"T Geiger","year":"2013","unstructured":"Geiger, T. et al. Initial quantitative proteomic map of 28 mouse tissues using the SILAC mouse. Mol Cell Proteomics 12, 1709\u20131722 (2013).","journal-title":"Mol Cell Proteomics"},{"key":"BFsrep32377_CR9","doi-asserted-by":"publisher","first-page":"1352","DOI":"10.1074\/mcp.M113.031914","volume":"13","author":"JD Bagert","year":"2014","unstructured":"Bagert, J. D. et al. Quantitative, time-resolved proteomic analysis by combining bioorthogonal noncanonical amino acid tagging and pulsed stable isotope labeling by amino acids in cell culture. Mol Cell Proteomics 13, 1352\u20131358 (2014).","journal-title":"Mol Cell Proteomics"},{"key":"BFsrep32377_CR10","doi-asserted-by":"publisher","first-page":"9482","DOI":"10.1073\/pnas.0601637103","volume":"103","author":"DC Dieterich","year":"2006","unstructured":"Dieterich, D. C., Link, A. J., Graumann, J., Tirrell, D. A. & Schuman, E. M. Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc Natl Acad Sci USA 103, 9482\u20139487 (2006).","journal-title":"Proc Natl Acad Sci USA"},{"key":"BFsrep32377_CR11","doi-asserted-by":"publisher","first-page":"25","DOI":"10.1016\/S0014-5793(01)02657-6","volume":"502","author":"KL Kiick","year":"2001","unstructured":"Kiick, K. L., Weberskirch, R. & Tirrell, D. A. Identification of an expanded set of translationally active methionine analogues in Escherichia coli. FEBS Lett 502, 25\u201330 (2001).","journal-title":"FEBS Lett"},{"key":"BFsrep32377_CR12","doi-asserted-by":"publisher","first-page":"2596","DOI":"10.1002\/1521-3773(20020715)41:14<2596::AID-ANIE2596>3.0.CO;2-4","volume":"41","author":"VV Rostovtsev","year":"2002","unstructured":"Rostovtsev, V. V., Green, L. G., Fokin, V. V. & Sharpless, K. B. A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective \u201cligation\u201d of azides and terminal alkynes. Angew Chem Int Ed Engl 41, 2596\u20132599 (2002).","journal-title":"Angew Chem Int Ed Engl"},{"key":"BFsrep32377_CR13","doi-asserted-by":"publisher","first-page":"40","DOI":"10.1021\/cn2000876","volume":"3","author":"FI Hinz","year":"2012","unstructured":"Hinz, F. I., Dieterich, D. C., Tirrell, D. A. & Schuman, E. M. Non-canonical amino acid labeling in vivo to visualize and affinity purify newly synthesized proteins in larval zebrafish. ACS Chem Neurosci 3, 40\u201349 (2012).","journal-title":"ACS Chem Neurosci"},{"key":"BFsrep32377_CR14","doi-asserted-by":"publisher","first-page":"15046","DOI":"10.1021\/ja044996f","volume":"126","author":"NJ Agard","year":"2004","unstructured":"Agard, N. J., Prescher, J. A. & Bertozzi, C. R. A strain-promoted [3\u2009+\u20092] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. J Am Chem Soc 126, 15046\u201315047 (2004).","journal-title":"J Am Chem Soc"},{"key":"BFsrep32377_CR15","doi-asserted-by":"publisher","first-page":"2004","DOI":"10.1002\/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5","volume":"40","author":"HC Kolb","year":"2001","unstructured":"Kolb, H. C., Finn, M. G. & Sharpless, K. B. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew Chem Int Ed Engl 40, 2004\u20132021 (2001).","journal-title":"Angew Chem Int Ed Engl"},{"key":"BFsrep32377_CR16","doi-asserted-by":"publisher","first-page":"897","DOI":"10.1038\/nn.2580","volume":"13","author":"DC Dieterich","year":"2010","unstructured":"Dieterich, D. C. et al. In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nat Neurosci 13, 897\u2013905 (2010).","journal-title":"Nat Neurosci"},{"key":"BFsrep32377_CR17","doi-asserted-by":"publisher","first-page":"737","DOI":"10.1016\/j.celrep.2014.01.024","volume":"6","author":"W Shen","year":"2014","unstructured":"Shen, W. et al. Acute synthesis of CPEB is required for plasticity of visual avoidance behavior in Xenopus. Cell Rep 6, 737\u2013747 (2014).","journal-title":"Cell Rep"},{"key":"BFsrep32377_CR18","doi-asserted-by":"publisher","first-page":"2237","DOI":"10.1038\/nprot.2014.150","volume":"9","author":"M Ullrich","year":"2014","unstructured":"Ullrich, M. et al. Bio-orthogonal labeling as a tool to visualize and identify newly synthesized proteins in Caenorhabditis elegans. Nat Protoc 9, 2237\u20132255 (2014).","journal-title":"Nat Protoc"},{"key":"BFsrep32377_CR19","doi-asserted-by":"publisher","first-page":"413","DOI":"10.1073\/pnas.1111561108","volume":"109","author":"J Liu","year":"2012","unstructured":"Liu, J., Xu, Y., Stoleru, D. & Salic, A. Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin. Proc Natl Acad Sci U S A 109, 413\u2013418 (2012).","journal-title":"Proc Natl Acad Sci U S A"},{"key":"BFsrep32377_CR20","doi-asserted-by":"publisher","first-page":"7364","DOI":"10.1002\/anie.200602114","volume":"45","author":"KE Beatty","year":"2006","unstructured":"Beatty, K. E. et al. Fluorescence visualization of newly synthesized proteins in mammalian cells. Angew Chem Int Ed Engl 45, 7364\u20137367 (2006).","journal-title":"Angew Chem Int Ed Engl"},{"key":"BFsrep32377_CR21","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1073\/pnas.012583299","volume":"99","author":"KL Kiick","year":"2002","unstructured":"Kiick, K. L., Saxon, E., Tirrell, D. A. & Bertozzi, C. R. Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc Natl Acad Sci USA 99, 19\u201324 (2002).","journal-title":"Proc Natl Acad Sci USA"},{"key":"BFsrep32377_CR22","doi-asserted-by":"publisher","first-page":"4815","DOI":"10.1021\/acs.jproteome.5b00653","volume":"14","author":"DB McClatchy","year":"2015","unstructured":"McClatchy, D. B. et al. Pulsed Azidohomoalanine Labeling in Mammals (PALM) Detects Changes in Liver-Specific LKB1 Knockout Mice. J Proteome Res 14, 4815\u20134822 (2015).","journal-title":"J Proteome Res"},{"key":"BFsrep32377_CR23","doi-asserted-by":"publisher","first-page":"3966","DOI":"10.1021\/pr5002862","volume":"13","author":"LM Schiapparelli","year":"2014","unstructured":"Schiapparelli, L. M. et al. Direct detection of biotinylated proteins by mass spectrometry. J Proteome Res 13, 3966\u20133978 (2014).","journal-title":"J Proteome Res"},{"key":"BFsrep32377_CR24","doi-asserted-by":"publisher","first-page":"1203","DOI":"10.1161\/hh1101.092180","volume":"88","author":"S Dayal","year":"2001","unstructured":"Dayal, S. et al. Endothelial dysfunction and elevation of S-adenosylhomocysteine in cystathionine beta-synthase-deficient mice. Circ Res 88, 1203\u20131209 (2001).","journal-title":"Circ Res"},{"key":"BFsrep32377_CR25","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1016\/j.ymgme.2007.02.001","volume":"91","author":"CL Elmore","year":"2007","unstructured":"Elmore, C. L. et al. Metabolic derangement of methionine and folate metabolism in mice deficient in methionine synthase reductase. Mol Genet Metab 91, 85\u201397 (2007).","journal-title":"Mol Genet Metab"},{"key":"BFsrep32377_CR26","doi-asserted-by":"publisher","first-page":"F280","DOI":"10.1152\/ajprenal.00113.2010","volume":"299","author":"ME Chapman","year":"2010","unstructured":"Chapman, M. E., Hu, L., Plato, C. F. & Kohan, D. E. Bioimpedance spectroscopy for the estimation of body fluid volumes in mice. Am J Physiol Renal Physiol 299, F280\u2013283 (2010).","journal-title":"Am J Physiol Renal Physiol"},{"key":"BFsrep32377_CR27","doi-asserted-by":"publisher","first-page":"15089","DOI":"10.1073\/pnas.2436385100","volume":"100","author":"AM Troen","year":"2003","unstructured":"Troen, A. M., Lutgens, E., Smith, D. E., Rosenberg, I. H. & Selhub, J. The atherogenic effect of excess methionine intake. Proc Natl Acad Sci USA 100, 15089\u201315094 (2003).","journal-title":"Proc Natl Acad Sci USA"},{"key":"BFsrep32377_CR28","doi-asserted-by":"publisher","first-page":"68","DOI":"10.1016\/S0014-5793(98)00489-X","volume":"428","author":"JC van Hest","year":"1998","unstructured":"van Hest, J. C. & Tirrell, D. A. Efficient introduction of alkene functionality into proteins in vivo . FEBS Lett 428, 68\u201370 (1998).","journal-title":"FEBS Lett"},{"key":"BFsrep32377_CR29","doi-asserted-by":"publisher","first-page":"259","DOI":"10.1016\/j.yjmcc.2012.05.005","volume":"53","author":"JA Vekich","year":"2012","unstructured":"Vekich, J. A., Belmont, P. J., Thuerauf, D. J. & Glembotski, C. C. Protein disulfide isomerase-associated 6 is an ATF6-inducible ER stress response protein that protects cardiac myocytes from ischemia\/reperfusion-mediated cell death. J Mol Cell Cardiol 53, 259\u2013267 (2012).","journal-title":"J Mol Cell Cardiol"},{"key":"BFsrep32377_CR30","doi-asserted-by":"publisher","first-page":"562","DOI":"10.1016\/j.molcel.2014.01.004","volume":"53","author":"D Eletto","year":"2014","unstructured":"Eletto, D., Eletto, D., Dersh, D., Gidalevitz, T. & Argon, Y. Protein disulfide isomerase A6 controls the decay of IRE1alpha signaling via disulfide-dependent association. Mol Cell 53, 562\u2013576 (2014).","journal-title":"Mol Cell"},{"key":"BFsrep32377_CR31","doi-asserted-by":"publisher","first-page":"229","DOI":"10.1016\/S1567-133X(02)00057-1","volume":"2","author":"Y Liu","year":"2002","unstructured":"Liu, Y. & Hogan, B. L. Differential gene expression in the distal tip endoderm of the embryonic mouse lung. Gene Expr Patterns 2, 229\u2013233 (2002).","journal-title":"Gene Expr Patterns"},{"key":"BFsrep32377_CR32","doi-asserted-by":"publisher","first-page":"1028","DOI":"10.1096\/fj.10-168799","volume":"25","author":"CA Goodman","year":"2011","unstructured":"Goodman, C. A. et al. Novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique. FASEB J 25, 1028\u20131039 (2011).","journal-title":"FASEB J"},{"key":"BFsrep32377_CR33","doi-asserted-by":"publisher","first-page":"376","DOI":"10.1074\/mcp.M200025-MCP200","volume":"1","author":"SE Ong","year":"2002","unstructured":"Ong, S. E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1, 376\u2013386 (2002).","journal-title":"Mol Cell Proteomics"},{"key":"BFsrep32377_CR34","doi-asserted-by":"publisher","first-page":"5707","DOI":"10.1021\/pr5006982","volume":"13","author":"G Zhang","year":"2014","unstructured":"Zhang, G. et al. In-depth quantitative proteomic analysis of de novo protein synthesis induced by brain-derived neurotrophic factor. J Proteome Res 13, 5707\u20135714 (2014).","journal-title":"J Proteome Res"},{"key":"BFsrep32377_CR35","doi-asserted-by":"publisher","first-page":"392","DOI":"10.1021\/bc200365k","volume":"23","author":"R van Geel","year":"2012","unstructured":"van Geel, R., Pruijn, G. J., van Delft, F. L. & Boelens, W. C. Preventing thiol-yne addition improves the specificity of strain-promoted azide-alkyne cycloaddition. Bioconjug Chem 23, 392\u2013398 (2012).","journal-title":"Bioconjug Chem"},{"key":"BFsrep32377_CR36","doi-asserted-by":"publisher","first-page":"3057","DOI":"10.1021\/jo011148j","volume":"67","author":"CW Tornoe","year":"2002","unstructured":"Tornoe, C. W., Christensen, C. & Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J Org Chem 67, 3057\u20133064 (2002).","journal-title":"J Org Chem"},{"key":"BFsrep32377_CR37","doi-asserted-by":"publisher","first-page":"9879","DOI":"10.1002\/anie.200905087","volume":"48","author":"V Hong","year":"2009","unstructured":"Hong, V., Presolski, S. I., Ma, C. & Finn, M. G. Analysis and optimization of copper-catalyzed azide-alkyne cycloaddition for bioconjugation. Angew Chem Int Ed Engl 48, 9879\u20139883 (2009).","journal-title":"Angew Chem Int Ed Engl"}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/srep32377.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/srep32377","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/srep32377.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,1,5]],"date-time":"2023-01-05T05:53:19Z","timestamp":1672897999000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/srep32377"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2016,8,30]]},"references-count":37,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2016,9,30]]}},"alternative-id":["BFsrep32377"],"URL":"https:\/\/doi.org\/10.1038\/srep32377","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2016,8,30]]},"assertion":[{"value":"21 March 2016","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"9 August 2016","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"30 August 2016","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing financial interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"32377"}}