Free Standard AU & NZ Shipping For All Book Orders Over $80!
Register      Login
Australian Journal of Chemistry Australian Journal of Chemistry Society
An international journal for chemical science
Shopping Cart: ( empty )
You are here: Home > Journals > CH > CH13174
RESEARCH FRONT
Contents Vol 66(7)

Molecular Characterization of Collagen Hydroxylysine O-Glycosylation by Mass Spectrometry: Current Status

Irina Perdivara A C , Mitsuo Yamauchi B and Kenneth B. Tomer A
+ Author Affiliations
- Author Affiliations

A Mass Spectrometry Group, National Institutes of Health/National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, NC 27709, USA.

B School of Dentistry, University of North Carolina at Chapel Hill, North Carolina, NC 27599, USA.

C Corresponding author. Email: perdivarai@niehs.nih.gov




Irina Perdivara received her Diploma in Chemistry (2006) and Ph.D. with Honors in Analytical Biochemistry (2009) from the University of Konstanz, Germany. Her research at NIEHS is focussed on structure determination of proteins involved in aging, autoimmune, neurodegenerative and bone diseases by high-performance liquid chromatography – tandem mass spectrometry. Her major area of interest is characterization of protein glycosylation at the glycopeptide level in raw proteolytic mixtures to understand its functional implications in health and disease. Dr Perdivara has set the foundation for quantitative site-specific collagen glycosylation analysis, which has led to significant progress in the research areas of bone physiology and bone-related diseases.


Mitsuo Yamauchi received a DDS (1976) and a Ph.D. in Biochemistry (1983) from the Tokyo Medical and Dental University, Tokyo, Japan. During his residency in Oral Surgery at the same university, he started research on collagen and became a post-doctoral fellow in the Dental Research Center at the University of North Carolina (UNC) at Chapel Hill under the late Professor Gerald L. Mechanic (1978–81). He is currently Sunstar Distinguished Professor of Oral Biology and Professor of Periodontology at UNC at Chapel Hill. His research interests include the biology and chemistry of collagen post-translational modifications, biological functions of matrix proteins in mineralization and aging, and bone-tissue engineering.


Dr. Tomer's research has focussed on characterization of biomolecules involved in immunological and inflammatory responses to environmental exposure. In over 300 research articles, he has reported the development and application of new techniques – nanoflow capillary LC, direct analysis of affinity-bound analytes by MALDI-MS, on-line microdialysis-MS-MS and on-line capillary electrophoresis-MS – and major applications – protein sequences, PTMs, protein structures, epitope mapping, and small-molecule quantitation. Dr Tomer retired at the end of 2011, but remains actively engaged in mass spectrometry at NIEHS, and in the continued mentoring of Fellows as they transition to the next stage in their careers.

Australian Journal of Chemistry 66(7) 760-769 https://doi.org/10.1071/CH13174
Submitted: 11 April 2013  Accepted: 4 May 2013   Published: 18 July 2013

Abstract

The most abundant proteins in vertebrates – the collagen family proteins – play structural and biological roles in the body. The predominant member, type I collagen, provides tissues and organs with structure and connectivity. This protein has several unique post-translational modifications that take place intra- and extra-cellularly. With growing evidence of the relevance of such post-translational modifications in health and disease, the biological significance of O-linked collagen glycosylation has recently drawn increased attention. However, several aspects of this unique modification – the requirement for prior lysyl hydroxylation as a substrate, involvement of at least two distinct glycosyl transferases, its involvement in intermolecular crosslinking – have made its molecular mapping and quantitative characterization challenging. Such characterization is obviously crucial for understanding its biological significance. Recent progress in mass spectrometry has provided an unprecedented opportunity for this type of analysis. This review summarizes recent advances in the area of O-glycosylation of fibrillar collagens and their characterization using state-of-the-art liquid chromatography–mass spectrometry-based methodologies, and perspectives on future research. The analytical characterization of collagen crosslinking and advanced glycation end-products are not addressed here.


References

[1]  E. M. Carter, C. L. Raggio, Curr. Opin. Pediatr. 2009, 21, 46.
         | Crossref | GoogleScholarGoogle Scholar | 19253462PubMed |

[2]  M. K. Gordon, R. A. Hahn, Cell Tissue Res 2010, 339, 247.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhsVymtrjJ&md5=452c0366fce539890f50380c963c9529CAS | 19693541PubMed |

[3]  B. Leitinger, Ann. Rev. Cell Develop. Biol. 2011, 27, 265.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhsFyqtbbJ&md5=196e783e4f2b10cc09d58994414bbd1bCAS |

[4]  K. I. Kivirikko, R. Myllyla, Methods Enzymol. 1982, 82, 245.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL38XktF2msbg%3D&md5=f134cfdc4b03c2105376e6de4bcf0c1fCAS | 6210830PubMed |

[5]  K. I. Kivirikko, R. Myllyla, Methods Enzymol. 1987, 144, 96.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL1cXht1Oitbs%3D&md5=3742ed64ca80ae7375eedc402b818f7eCAS | 3041180PubMed |

[6]  K. Kolble, K. B. Reid, Behring Inst. Mitt. 1993, 93, 81.
         | 1:CAS:528:DyaK2cXis1Oms7w%3D&md5=44cb40abae7dae2fcc9783fbc1a86cb0CAS | 8172588PubMed |

[7]  W. Grassmann, H. Schleich, Biochem. Z. 1935, 277, 320.
         | 1:CAS:528:DyaA2MXjsFGgsA%3D%3D&md5=6159937d820d51e486b881711e32298bCAS |
         | 1:CAS:528:DyaA2MXjsFGgsA%3D%3D&md5=6159937d820d51e486b881711e32298bCAS |

[8]  W. T. Butler, L. W. Cunningham, J. Biol. Chem. 1966, 241, 3882.
         | 1:CAS:528:DyaF28XksV2jtLw%3D&md5=191fdea8cf6eb6190774578863e73d08CAS | 4288358PubMed |

[9]  R. G. Spiro, J. Biol. Chem. 1967, 242, 4813.
         | 1:CAS:528:DyaF2sXltVGjurs%3D&md5=e7ad616f9666a85c63126471ce1b15c7CAS | 6070267PubMed |

[10]  J. M. P. Liefhebber, S. Punt, W. J. M. Spaan, H. C. van Leeuwen, BMC Cell Biol. 2010, 11, 33.

[11]  B. Schegg, A. J. Hulsmeier, C. Rutschmann, C. Maag, T. Hennet, Mol. Cell. Biol. 2009, 29, 943.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXisFCqt7c%3D&md5=b9457fe1818dd421845c9bfda90f716cCAS | 19075007PubMed |

[12]  R. Myllylä, C. G. Wang, J. Heikkinen, A. Juffer, O. Lampela, M. Risteli, H. Ruotsalainen, A. Salo, L. Sipilä, J. Cell. Physiol. 2007, 212, 323.
         | Crossref | GoogleScholarGoogle Scholar | 17516569PubMed |

[13]  C. G. Wang, H. Luosujarvi, J. Heikkinen, M. Risteli, L. Uitto, R. Myllyla, Matrix Biol. 2002, 21, 559.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XptlWltr0%3D&md5=caf0dc3979d8bc994ae4f272eb1bbf55CAS |

[14]  J. Heikkinen, M. Risteli, C. Wang, J. Latvala, M. Rossi, M. Valtavaara, R. Myllylä, J. Biol. Chem. 2000, 275, 36158.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXosVSku7s%3D&md5=de563085acd7e37e1bb4c51e7cc8fcbaCAS | 10934207PubMed |

[15]  K. Mizuno, E. Adachi, Y. Imamura, O. Katsumata, T. Hayashi, Micron 2001, 32, 317.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXos12ju7g%3D&md5=a06697fe5c3774e11f03ff2be2f06627CAS | 11006511PubMed |

[16]  H. Shinkai, K. Yonemasu, Biochem. J. 1979, 177, 847.
         | 1:CAS:528:DyaE1MXkvV2gs7w%3D&md5=ae69cc0bdc08505807d87df8628079b6CAS | 444207PubMed |

[17]  L. Moro, M. Romanello, A. Favia, M. P. Lamanna, E. Lozupone, Calcif. Tissue Int. 2000, 66, 151.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXpslKhug%3D%3D&md5=3d3d5b4f0cb63dbd9ee68685ef89e5b4CAS | 1:CAS:528:DC%2BD3cXpslKhug%3D%3D&md5=3d3d5b4f0cb63dbd9ee68685ef89e5b4CAS | 10652964PubMed |

[18]  B. P. Toole, A. H. Kang, R. L. Trelstad, J. Gross, Biochem. J. 1972, 127, 715.
         | 1:CAS:528:DyaE38XksF2jtbc%3D&md5=79029c7c6956a9fa78822f7f0848420bCAS |
         | 1:CAS:528:DyaE38XksF2jtbc%3D&md5=79029c7c6956a9fa78822f7f0848420bCAS | 4651137PubMed |

[19]  M. Risteli, H. Ruotsalainen, A. M. Salo, R. Sormunen, L. Sipila, N. L. Baker, S. R. Lamandé, L. Vimpari-Kauppinen, R. Myllylä, J. Biol. Chem. 2009, 284, 28204.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXht1SqsbrP&md5=ae03a8081676a59cbfd402e450ba1d85CAS | 19696018PubMed |

[20]  A. M. Salo, H. Cox, P. Farndon, C. Moss, H. Grindulis, M. Risteli, S. P. Robins, R. Myllylä, Am. J. Hum. Genet. 2008, 83, 495.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXht1Omtb3L&md5=baa0a8c8522d0eaa2bd028c1baca29ecCAS | 1:CAS:528:DC%2BD1cXht1Omtb3L&md5=baa0a8c8522d0eaa2bd028c1baca29ecCAS | 18834968PubMed |

[21]  J. F. Bateman, T. Mascara, D. Chan, W. G. Cole, Biochem. J. 1984, 217, 103.
         | 1:CAS:528:DyaL2cXotlOjtg%3D%3D&md5=9811d6d4c9df0f4047c92a12c0dc6dcbCAS | 6421277PubMed |

[22]  R. E. Brenner, U. Vetter, A. Nerlich, O. Worsdorfer, W. M. Teller, P. K. Muller, Eur. J. Clin. Invest. 1990, 20, 8.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3cXitFClurk%3D&md5=175fc2bda59464c3027f791bb3d0cae6CAS | 2108039PubMed |

[23]  J. Brinckmann, H. Notbohm, M. Tronnier, Y. Acil, P. P. Fietzek, W. Schmeller, P. K. Müller, B. Bätge, J. Invest. Dermatol. 1999, 113, 617.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXmslamtrs%3D&md5=ee9b84d5b15393bc1e5c056230ac9e9bCAS | 10504450PubMed |

[24]  G. Cetta, G. De Luca, R. Tenni, G. Zanaboni, L. Lenzi, A. A. Castellani, Connect. Tissue Res. 1983, 11, 103.
         | Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaL3s3os1Gktw%3D%3D&md5=f1cf3353f66e84a157c4b3b33888aa93CAS | 6224635PubMed |

[25]  H. W. Lehmann, E. Wolf, K. Roser, M. Bodo, G. Delling, P. K. Muller, J. Cancer Res. Clin. Oncol. 1995, 121, 413.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXosVChsb0%3D&md5=08e2c6a287ab95e101c8b4cc1944cabaCAS | 7635871PubMed |

[26]  M. Michalsky, K. Norris-Suarez, P. Bettica, A. Pecile, L. Moro, Biochem. Biophys. Res. Commun. 1993, 192, 1281.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3sXktVWhtr8%3D&md5=67e24d5052872ebaea2f39fbd2891ab4CAS | 8507198PubMed |

[27]  R. Tenni, M. Valli, A. Rossi, G. Cetta, Am. J. Med. Genet. 1993, 45, 252.
         | Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK3s3gs12hsA%3D%3D&md5=49e3709e8cd92e4d930e9baa9a421ff7CAS | 8456811PubMed |

[28]  M. Yamauchi, M. Sricholpech, Essays Biochem. 2012, 52, 113.
         | 1:CAS:528:DC%2BC38Xht1ShurrK&md5=0dcf0d23c554a4a0269a1099e5493fbfCAS |
         | 1:CAS:528:DC%2BC38Xht1ShurrK&md5=0dcf0d23c554a4a0269a1099e5493fbfCAS | 22708567PubMed |

[29]  K. Bhadriraju, K. H. Chung, T. A. Spurlin, R. J. Haynes, J. T. Elliott, A. L. Plant, Biomaterials 2009, 30, 6687.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXht1Oiu77M&md5=d40193b7a36b159c5df305fc41a54ce0CAS | 1:CAS:528:DC%2BD1MXht1Oiu77M&md5=d40193b7a36b159c5df305fc41a54ce0CAS | 19762078PubMed |

[30]  H. J. Jurgensen, D. H. Madsen, S. Ingvarsen, M. C. Melander, H. Gardsvoll, L. Patthy, L. H. Engelholm, N. Behrendt, J. Biol. Chem. 2011, 286, 32736.
         | Crossref | GoogleScholarGoogle Scholar | 21768090PubMed |

[31]  W. Vogel, G. D. Gish, F. Alves, T. Pawson, Mol. Cell 1997, 1, 13.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXlt1KjsA%3D%3D&md5=ee7487e1145742212999bf23b249eb02CAS | 9659899PubMed |

[32]  H. F. Xu, N. Raynal, S. Stathopoulos, J. Myllyharju, R. W. Farndale, B. Leitinger, Matrix Biol. 2011, 30, 16.
         | Crossref | GoogleScholarGoogle Scholar |

[33]  J. L. Lauer-Fields, N. B. Malkar, G. Richet, K. Drauz, G. B. Fields, J. Biol. Chem. 2003, 278, 14321.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXjtVShsL8%3D&md5=b1a0bbe06f309c1b34fe438c68a736f5CAS | 12574156PubMed |

[34]  P. E. Van den Steen, P. Proost, D. D. Brand, A. H. Kang, J. Van Damme, G. Opdenakker, Biochemistry 2004, 43, 10809.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXmtVKksbw%3D&md5=96009f3df4667feea9f1e9c992f6e0a5CAS | 15311942PubMed |

[35]  M. Yamauchi, M. Shiiba, Methods Mol. Biol. 2008, 446, 95.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXls1Khuro%3D&md5=a259feed3dcb1508431859070b2b764fCAS | 18373252PubMed |

[36]  M. Sricholpech, I. Perdivara, H. Nagaoka, M. Yokoyama, K. B. Tomer, M. Yamauchi, J. Biol. Chem. 2011, 286, 8846.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXjtFOjurY%3D&md5=8617c26bf01a95dbd15eaad0f7f6027fCAS | 1:CAS:528:DC%2BC3MXjtFOjurY%3D&md5=8617c26bf01a95dbd15eaad0f7f6027fCAS | 21220425PubMed |

[37]  M. Sricholpech, I. Perdivara, M. Yokoyama, H. Nagaoka, M. Terajima, K. B. Tomer, M. Yamauchi, J. Biol. Chem. 2012, 287, 22998.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XptlWmtrY%3D&md5=c9ff9547cda7684288d46321693be4fdCAS | 1:CAS:528:DC%2BC38XptlWmtrY%3D&md5=c9ff9547cda7684288d46321693be4fdCAS | 22573318PubMed |

[38]  K. J. Bos, G. J. Rucklidge, B. Dunbar, S. P. Robins, Matrix Biol. 1999, 18, 149.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXjsVOrs7w%3D&md5=7d8f84ae9513a0250716ff01187fe23fCAS | 1:CAS:528:DyaK1MXjsVOrs7w%3D&md5=7d8f84ae9513a0250716ff01187fe23fCAS | 10372555PubMed |

[39]  K. Mann, D. E. Mechling, H. P. Bachinger, C. Eckerskorn, F. Gaill, R. Timpl, J. Mol. Biol. 1996, 261, 255.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK28XltFKrtrg%3D&md5=2c7e432a2ce872782c0c749c22e12dceCAS | 8757292PubMed |

[40]  P. E. Van Den Steen, P. Proost, B. Grillet, D. D. Brand, A. H. Kang, J. Van Damme, G. Opdenakker, FASEB J. 2002, 16, 379.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XhvVCmurY%3D&md5=2de325613104ae4a239e7a48d400c602CAS | 11874987PubMed |

[41]  W. Henkel, K. Dreisewerd, J. Proteome Res. 2007, 6, 4269.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtFKksr3K&md5=b7783495b63b2122c97738d1f7a775e8CAS | 17939700PubMed |

[42]  J. Rauterberg, K. Kuhn, Eur. J. Biochem. 1971, 19, 398.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE3MXktVSgt7c%3D&md5=40dfe46236f6671fdbfa98e617666103CAS | 1:CAS:528:DyaE3MXktVSgt7c%3D&md5=40dfe46236f6671fdbfa98e617666103CAS | 5102923PubMed |

[43]  R. K. Rhodes, K. D. Gibson, E. J. Miller, Biochemistry 1981, 20, 3117.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL3MXktF2qsbY%3D&md5=b6208d0f25c0948abe12d9ce22f2bc76CAS | 7248273PubMed |

[44]  R. K. Rhodes, E. J. Miller, J. Biol. Chem. 1979, 254, 12084.
         | 1:CAS:528:DyaL3cXhvFSh&md5=0623c15917fa43cfb5968c87e618ed2bCAS | 500696PubMed |

[45]  G. Zhang, A. Sun, W. Li, T. Liu, Z. Su, J. Chromatogr. A 2006, 1114, 274.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XktVyis7Y%3D&md5=ab1bc368e6b30ae45ac5971f19888d20CAS | 16600269PubMed |

[46]  Y. Taga, M. Kusubata, K. Ogawa-Goto, S. Hattori, Mol. Cell. Proteomics 2012, 11, M111.010397.
         | Crossref | GoogleScholarGoogle Scholar | 22247541PubMed |

[47]  C. X. Yang, A. C. Park, N. A. Davis, J. D. Russell, B. Kim, D. D. Brand, M. J. Lawrence, Y. Ge, M. S. Westphall, J. J. Coon, D. S. Greenspan, J. Biol. Chem. 2012, 287, 40598.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XhslansbfE&md5=18c827621e0f01388518bc4ed4890ffeCAS |

[48]  I. Perdivara, L. Perera, M. Sricholpech, M. Terajima, N. Pleshko, M. Yamauchi, K. B. Tomer, J. Am. Soc. Mass Spectrom. 2013,
         | Crossref | GoogleScholarGoogle Scholar | 23633013PubMed |

[49]  J. Peter-Katalinic, Method Enzymol. 2005, 405, 139.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXmt1Snt74%3D&md5=81453ddfef32d067be79bbcdc8370429CAS | 1:CAS:528:DC%2BD2sXmt1Snt74%3D&md5=81453ddfef32d067be79bbcdc8370429CAS |

[50]  J. E. P. Syka, J. J. Coon, M. J. Schroeder, J. Shabanowitz, D. F. Hunt, Proc. Natl. Acad. Sci. USA 2004, 101, 9528.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXlvVahs70%3D&md5=abf0e5ee5d43d0eb0e8068663451ed7cCAS | 1:CAS:528:DC%2BD2cXlvVahs70%3D&md5=abf0e5ee5d43d0eb0e8068663451ed7cCAS |

[51]  S. R. Pinnell, R. Fox, S. M. Krane, Biochim. Biophys. Acta 1971, 229, 119.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE3MXntFOjug%3D%3D&md5=21ab88dcb2038318b5ad988aa4b0bff1CAS | 1:CAS:528:DyaE3MXntFOjug%3D%3D&md5=21ab88dcb2038318b5ad988aa4b0bff1CAS | 5101159PubMed |

[52]  J. D. Schofiel, I. L. Freeman, D. S. Jackson, Biochem. J. 1971, 124, 467.

[53]  J. P. Segrest, L. W. Cunningh, J. Clin. Invest. 1970, 49, 1497.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE3cXkslCms7c%3D&md5=07c5e8a996bc87ef444f17e14d3f2bbeCAS | 1:CAS:528:DyaE3cXkslCms7c%3D&md5=07c5e8a996bc87ef444f17e14d3f2bbeCAS | 5431661PubMed |

[54]  L. J. Dominguez, M. Barbagallo, L. Moro, Biochem. Biophys. Res. Commun. 2005, 330, 1.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXisVCnu7w%3D&md5=bc4bc4fff92b91743fbba09a6d65a1ddCAS | 1:CAS:528:DC%2BD2MXisVCnu7w%3D&md5=bc4bc4fff92b91743fbba09a6d65a1ddCAS | 15781223PubMed |

[55]  M. Gilar, P. Olivova, A. E. Daly, J. C. Gebler, J. Sep. Sci. 2005, 28, 1694.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtVyhur%2FM&md5=b21663e6093376e5ff302222bdc986c3CAS | 1:CAS:528:DC%2BD2MXhtVyhur%2FM&md5=b21663e6093376e5ff302222bdc986c3CAS | 16224963PubMed |

[56]  M. Gilar, P. Olivova, A. E. Daly, J. C. Gebler, Anal. Chem. 2005, 77, 6426.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXovFWhtr0%3D&md5=0c17a67ec0411b85daa7957f8a7148ffCAS | 1:CAS:528:DC%2BD2MXovFWhtr0%3D&md5=0c17a67ec0411b85daa7957f8a7148ffCAS | 16194109PubMed |