Optimization of neuropeptide extraction from the mouse hypothalamus

被引:47
作者
Che, Fa-Yun [1 ]
Zhang, Xin [1 ]
Berezniuk, Iryna [1 ]
Callaway, Myrasol [1 ]
Lim, Jihyeon [1 ]
Fricker, Lloyd D. [1 ]
机构
[1] Albert Einstein Coll Med, Dept Mol Pharmacol, Bronx, NY 10461 USA
关键词
peptide processing; carboxypeptidase E; carboxypeptidase D; prohormone convertase;
D O I
10.1021/pr060690r
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Sample preparation for neuropeptidomic studies is a critical issue since protein degradation can produce high levels of peptides that obscure the endogenous neuropeptides. We compared different extraction conditions for the recovery of neuropeptides and the formation of protein breakdown fragments from mouse hypothalami. Sonication and heating in water (70 degrees C for 20 min) followed by cold acid and centrifugation enabled the efficient extraction of many neuropeptides without the formation of protein degradation fragments seen with hot acid extractions. The hot water/cold acid extraction procedure resulted in the reproducible recovery of many hypothalamic peptides, including several novel peptides.
引用
收藏
页码:4667 / 4676
页数:10
相关论文
共 47 条
[11]   Pedtidomics of Cpefat/fat mouse hypothalamus -: Effect of food deprivation and exercise on peptide levels [J].
Che, FY ;
Yuan, Q ;
Kalinina, E ;
Fricker, LD .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2005, 280 (06) :4451-4461
[12]  
Conlon J M, 1997, Methods Mol Biol, V73, P1
[13]   Peptidomics of Cpefat/fat mouse hypothalamus and striatum -: Effect of chronic morphine administration [J].
Decaillot, FM ;
Che, FY ;
Fricker, LD ;
Devi, LA .
JOURNAL OF MOLECULAR NEUROSCIENCE, 2006, 28 (03) :277-284
[14]   Rat neuropeptidomics by LC-MS/MS and MALDI-FTMS: Enhanced dissection and extraction techniques coupled with 2D RP-RP HPLC [J].
Dowell, James A. ;
Vander Heyden, William ;
Li, Lingjun .
JOURNAL OF PROTEOME RESEARCH, 2006, 5 (12) :3368-3375
[15]   PEPTIDES IN THE NERVOUS-SYSTEM [J].
EIPPER, BA ;
MAINS, RE ;
HERBERT, E .
TRENDS IN NEUROSCIENCES, 1986, 9 (10) :463-468
[16]   PEPTIDE ALPHA-AMIDATION [J].
EIPPER, BA ;
MAINS, RE .
ANNUAL REVIEW OF PHYSIOLOGY, 1988, 50 :333-344
[17]   Peptidomics: identification and quantification of endogenous peptides in neuroendocrine tissues [J].
Fricker, LD ;
Lim, JY ;
Pan, H ;
Che, FY .
MASS SPECTROMETRY REVIEWS, 2006, 25 (02) :327-344
[18]   Carboxypeptidase E activity is deficient in mice with the fat mutation - Effect on peptide processing [J].
Fricker, LD ;
Berman, YL ;
Leiter, EH ;
Devi, LA .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (48) :30619-30624
[19]  
FRICKER LD, 2004, HDB PROTEOLYTIC ENZY, P840
[20]   De novo sequencing of neuropeptides using reductive isotopic methylation and investigation of ESI QTOF MS/MS fragmentation pattern of neuropeptides with N-terminal dimethylation [J].
Fu, Q ;
Li, LJ .
ANALYTICAL CHEMISTRY, 2005, 77 (23) :7783-7795