A new strategy for studying proteome of velvet antler cartilage — ASN Events
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  3. A new strategy for studying proteome of velvet antler cartilage

A new strategy for studying proteome of velvet antler cartilage (#301)

zhigang sui 1 , qun zhao 1 , nan deng 1 , hao jiang 1 , zhen liang 1 , lihua zhang 1 , yukui zhang 1
  1. National Chromatographic R. & A. Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, dalian/116023, China

Velvet antlers are the unique organs that display an annual cycle of full regeneration in mammals [1]. Surprisingly, within the cartilage of velvet antlers, numerous blood vessels were evenly distributed. The proteomic analysis of differences in normal and this vascularized cartilage samples will be viable to elucidate the mechanisms of vasculogenesis. However, in cartilage, there are high content of proteoglycan (PGs) and collagens [2], which could impair the proteomic analysis of cellular component. Recently, cetylpyridinium chloride (CPC) precipitation was developed for PGs removal [3]. However, it suffered from complicated operation procedure and potential protein loss. Herein we proposed an ionic liquid based strategy for the selective protein extraction from cartilage, allowing rapid and efficient profiling of proteins in antler cartilage.

Cartilage was first washed, cut into 10 μm slices and suspended in 4% (w/v) 1-dodecyl-3-methylimidazolium chloride ([Domim]Cl). Then samples were agitated at room temperature for 15 min and the tissue slices were picked out. The extracted solutionwas further sonicated for 120 s. After the removal of insoluble fraction by centrifugation, protein concentrations were estimated by BCA assay. After the lysates were supplemented with DTT (final concentration at 0.1 M) and incubated at 95 ºC for 5 min, filter assistant sample preparation (FASP) was applied to treat the sample using filtration device (MWCO 10kD) [4]. Finally, peptides were collected by centrifugation for further nanoRPLC-ESI-MS/MS analysis.

Compared with previous method [3], the developed method can obviously enhance the numbers of identified peptides (3290 vs 1609), protein groups (893 vs 509) and membrane proteins (103 vs 41). It is mainly because that methanol/chloroform precipitation for removal of ionic detergent was unneeded and the sonication was introduced to improve the efficiency of protein extraction in the developed method. In summary, our proposed method, followed by analysis with nanoRPLC-ESI-MS/MS, provides a rapid and efficient approach for proteomic characterization of cartilage tissue of velvet antler.

  1. C.Y. Li, Birth Defects Res. C Embryo Today 96 (2012) 51-62.
  2. J.B. Vincourt, F. Lionneton, G. Kratassiouk, F. Guillemin, P. Netter, D. Mainard, J. Magdalou, Mol. Cell. Proteomics 5 (2006) 1984-1995.
  3. R. Wilson, A.F. Diseberg, L. Gordon, S. Zivkovic, L. Tatarczuch, E.J. Mackie, J.J. Gorman, J.F. Bateman, Mol. Cell. Proteomics 9 (2010) 1296-1313.
  4. J.R. Wisniewski, A. Zougman, N. Nagaraj, M. Mann, Nat. Methods 6 (2009) 359-360.