Rat neuropeptidomics by LC-MS/MS and MALDI-FTMS: Enhanced dissection and extraction techniques coupled with 2D RP-RP HPLC.
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.
Dowell
James A
JA
School of Pharmacy and Department of Chemistry, University of Wisconsin-Madison, 777 Highland Avenue, Madison, Wisconsin 53705-2222, USA.
Heyden
William Vander
WV
Li
Lingjun
L
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D016616:Cryoultramicrotomy / Q000379:methods; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000235:genetics*; D040901:Proteomics / Q000379:methods*; D051381:Rats / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D009479:Neuropeptides