Rat neuropeptidomics by LC-MS/MS and MALDI-FTMS: Enhanced dissection and extraction techniques coupled with 2D RP-RP HPLC

J Proteome Res. 2006 Dec;5(12):3368-75. doi: 10.1021/pr0603452.


Recently developed sample preparation techniques employing microwave irradiation have enabled the comprehensive study of endogenous mammalian neuropeptides. These methods reduce interference from post-mortem protein degradation by deactivating proteases via heat denaturation. Alternatively, we have developed a protocol using cryostat dissection and a boiling extraction buffer to achieve a similar effect. This novel methodology greatly reduces post-mortem protein contamination and increases neuropeptide identification without the use of specialized equipment. In addition, a 2D HPLC scheme employing differential pH selectivity in the first and second dimensions has been used to enhance neuropeptidome coverage. By using our novel dissection protocol in tandem with 2D RP-RP HPLC, we were able to identify a total of 56 peptides from known neuropeptide precursors, including 17 previously unidentified peptides. The use of cryostat dissection and two-dimensional RP-RP HPLC enhances the detection of novel neuropeptides by deactivating proteases and reducing sample complexity.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Chromatography, High Pressure Liquid
  • Cryoultramicrotomy / methods
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data
  • Neuropeptides / genetics*
  • Proteomics / methods*
  • Rats / genetics*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods


  • Neuropeptides