Journal of Molecular Neuroscience

, Volume 28, Issue 3, pp 265–275 | Cite as

Quantitative peptidomics in mice

Effect of cocaine treatment
Original Article


We recently developed a quantitative peptidomics method using stable isotopic labels and mass spectrometry to both quantify and identify a large number of peptides. To test this approach and screen for peptides regulated by cocaine administration, 32 Cpe fat/fat mice and 16 wild-type mice were treated twice daily for 5 d either with saline or 10 mg/kg cocaine. Peptides were extracted from striatum, hypothalamus, hippocampus, and trimethylammonium butyrate containing either nine deuterium or nine hydrogen atoms. Pools of heavy- and light-labeled peptides were combined, purified on an anhydrotrypsin affinity column, and analyzed on a reverse-phase column coupled to an electrospray ionization quadrapole time-of-flight mass spectrometer. Changes in peptide levels upon cocaine treatment were determined from the relative peak intensities of the cocaine versus saline peaks, and peptides were identified from collision-induced dissociation spectra. Ten peptides were found to increase or decrease in each of two separate analyses from distinct groups of mice. Peptides found to increase corresponded to fragments of proenkephalin, prothyrotropin-releasing hormone, provasopressin, proSAAS, secretogranin II, chromogranin B, and peptidyl-glycine-α-amidating mono-oxygenase in the hypothalamus. The same peptidyl-glycine-α-amidating mono-oxygenase peptide decreased in the prefrontal cortex, along with striatal neurokinin B and two unidentified peptides. Thirty other peptides were not substantially affected by cocaine treatment in both replicates. Taken together, the quantitative peptidomics approach provides an efficient method to screen for changes in a large number of peptides.

Index Entries

Peptide processing carboxypeptidase proSAAS proenkephalin secretogranin II preprotachykinin B 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Adams D. H., Hanson G. R., and Keefe K. A. (2001) Differential effects of cocaine and methamphetamine on neurotensin/neuromedin N and preprotachykinin messenger RNA expression in unique regions of the striatum. Neuroscience 102, 843–851.PubMedCrossRefGoogle Scholar
  2. Adams D. H., Hanson G. R., and Keefe K. A. (2003) Distinct effects of methamphetamine and cocaine on preprodynorphin messenger RNA in rat striatal patch and matrix. J. Neurochem. 84, 87–93.PubMedCrossRefGoogle Scholar
  3. Aebersold R. and Mann M. (2003) Mass spectrometry-based proteomics. Nature 422, 198–207.PubMedCrossRefGoogle Scholar
  4. Alburges M. E., Ramos B. P., Bush L., and Hanson G. R. (2000) Responses of the extrapyramidal and limbic substance P systems to ibogaine and cocaine treatments. Eur. J. Pharmacol. 390, 119–126.PubMedCrossRefGoogle Scholar
  5. Arroyo M., Baker W. A., and Everitt B. J. (2000) Cocaine self-administration in rats differentially alters mRNA levels of the monoamine transporters and striatal neuropeptides. Mol. Brain Res. 83, 107–120.PubMedCrossRefGoogle Scholar
  6. Baggerman G., Verleyen P., Clynen E., Huybrechts J., De Loof A., and Schoofs L. (2004) Peptidomics. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 803, 3–16.PubMedCrossRefGoogle Scholar
  7. Blouin R. A., Kolpek J. H., and Mann H. J. (1987) Influence of obesity on drug disposition. Clin. Pharmacol. 6, 706–714.Google Scholar
  8. Branch A. D., Unterwald E. M., Lee S. E., and Kreek M. J. (1992) Quantitation of preproenkephalin mRNA levels in brain regions from male Fischer rats following chronic cocaine treatment using a recently developed solution hybridization assay. Brain Res. Mol. Brain Res. 14, 231–238.PubMedCrossRefGoogle Scholar
  9. Cain B. M., Wang W., and Beinfeld, M. C. (1997) Cholecystokinin (CCK) levels are greatly reduced in the brains but not the duodenums of Cpe fat/Cpefat mice: a regional difference in the involvement of carboxypeptidase E (Cpe) in pro-CCK processing. Endocrinology 138, 4034–4037.PubMedCrossRefGoogle Scholar
  10. Chard T. (1987) An Introduction to Radioimmunoassay and Related Techniques. Elsevier, Amsterdam.Google Scholar
  11. Che F.-Y. and Fricker L. D. (2002) Quantitation of neuropeptides in Cpe fat/Cpefat mice using differential isotopic tags and mass spectrometry. Anal. Chem. 74, 3190–3198.PubMedCrossRefGoogle Scholar
  12. Che F.-Y. and Fricker L. D. (2005) Quantitative peptidomics of mouse pituitary: Comparison of different stable isotopic tags. J. Mass Spectrom. 40, 238–249.PubMedCrossRefGoogle Scholar
  13. Che F.-Y., Biswas R., and Fricker L. D. (2005a) Relative quantitation of peptides in wild type and Cpe fat/fat mouse pituitary using stable isotopic tags and mass spectrometry. J. Mass Spectrom. 40, 227–237.PubMedCrossRefGoogle Scholar
  14. Che F.-Y., Eipper B. A., Mains R. E., and Fricker L. D. (2003) Quantitative peptidomics of pituitary glands from mice deficient in copper transport. Cell. Mol. Biol. 49, 713–722.Google Scholar
  15. Che F.-Y., Lim J., Biswas R., Pan H., and Fricker L. D. (2005b) Quantitative neuropeptidomics of microwave-irradiated mouse brain and pituitary. Mol. Cell. Proteomics, 4, 1391–1405.PubMedCrossRefGoogle Scholar
  16. Che F.-Y., Yan L., Li H., Mzhavia N., Devi L., and Fricker L. D. (2001) Identification of peptides from brain and pituitary of Cpe fat/Cpefat mice. Proc. Natl. Acad. Sci. U. S. A. 98, 9971–9976.PubMedCrossRefGoogle Scholar
  17. Che F.-Y., Yuan Q., Kalinina E., and Fricker L. D. (2005c) Peptidomics of Cpe fat/fat mouse hypothalamus: effect of food deprivation and exercise on peptide levels. J. Biol. Chem. 280, 4451–4461.PubMedCrossRefGoogle Scholar
  18. Cheymol G. (2000) Effects of obesity on pharmacokinetics implications for drug therapy. Clin. Pharmacokinet. 39, 215–231.PubMedCrossRefGoogle Scholar
  19. Douglass J., McKinzie A. A., and Couceyro P. (1995) PCR differential display identifies a rat brain mRNA that is transcriptionally regulated by cocaine and amphetamine. J. Neurosci. 15, 2471–2481.PubMedGoogle Scholar
  20. Erstad B. L. (2004) Dosing of medications in morbidly obese patients in the intensive care unit setting. Intensive Care Med. 30, 18–32.PubMedCrossRefGoogle Scholar
  21. Eugene P. A., Senanayake S., and Sattin A. (2002) Cocaine regulates TRH-related peptides in rat brain. Neurochem. Int. 41, 415–428.CrossRefGoogle Scholar
  22. Fricker L. D., Berman Y. L., Leiter E. H., and Devi L. A. (1996) Carboxypeptidase E activity is deficient in mice with the fat mutation: effect on peptide processing. J. Biol. Chem. 271, 30,619–30,624.Google Scholar
  23. Fricker L. D., McKinzie A. A., Sun J., Curran E., Qian Y., Yan L., et al. (2000) Identification and characterization of proSAAS, a granin-like neuroendocrine peptide precursor that inhibits prohormone processing. J. Neurosci. 20, 639–648.PubMedGoogle Scholar
  24. Gygi S. P. and Aebersold R. (2000) Mass spectrometry and proteomics. Curr. Opin. Chem. Biol. 4, 489–494.PubMedCrossRefGoogle Scholar
  25. Hanson G. R., Midgley L. P., Bush L. G., Johnson M., and Gibb J. W. (1989) Comparison of responses by neuropeptide systems in rat to the psychotropic drugs, methamphetamine, cocaine and PCP. NIDA Res. Monogr. 95, 348.PubMedGoogle Scholar
  26. Julka S. and Regnier F. E. (2004) Quantification in proteomics through stable isotope coding: a review. J. Proteome Res. 3, 350–363.PubMedCrossRefGoogle Scholar
  27. Kuzmin A. and Johansson B. (1999) Expression of c-fos, NGFI-A and secretogranin II mRNA in brain regions during initiation of cocaine self-administration in mice. Eur. J. Neurosci. 11, 3694–3700.PubMedCrossRefGoogle Scholar
  28. Maj M., Turchan J., Smialowska M., and Przewlocka B. (2003) Morphine and cocaine influence on CRF biosynthesis in the rat central nucleus of amygdala. Neuropeptides 37, 105–110.PubMedCrossRefGoogle Scholar
  29. Mathieu-Kia A. M. and Besson M. J. (1998) Repeated administration of cocaine, nicotine and ethanol: effects on preprodynorphin, preprotachykinin A and preproenkephalin mRNA expression in the dorsal and the ventral striatum of the rat. Brain Res. Mol. Brain Res. 54, 141–151.PubMedCrossRefGoogle Scholar
  30. Naggert J. K., Fricker L. D., Varlamov O., Nishina P. M., Rouille Y., Steiner D. F., et al. (1995) Hyperproinsulinemia in obese fat/fat mice associated with a point mutation in the carboxypeptidase E gene and reduced carboxypeptidase E activity in the pancreatic islets. Nat. Genet. 10, 135–142.PubMedCrossRefGoogle Scholar
  31. Rovere C., Viale A., Nahon J., and Kitabgi P. (1996) Impaired processing of brain proneurotensin and promelanin-concentrating hormone in obese fat/fat mice. Endocrinology 137, 2954–2958.PubMedCrossRefGoogle Scholar
  32. Schrader M. and Schulz-Knappe P. (2001) Peptidomics technologies for human body fluids. Trends Biotechnol. 19, S55-S60.PubMedCrossRefGoogle Scholar
  33. Skold K., Svensson M., Kaplan A., Bjorkesten L., Astrom J., and Andren P.E. (2002) A neuroproteomic approach to targeting neuropeptides in the brain. Proteomics 2, 447–454.PubMedCrossRefGoogle Scholar
  34. Smiley P. L., Johnson M., Bush L., Gibb J. W., and Hanson G. R. (1990) Effects of cocaine on extrapyramidal and limbic dynorphin systems. J. Pharmacol. Exp. Ther. 253, 938–943.PubMedGoogle Scholar
  35. Spangler R., Unterwald E. M., and Kreek M. J. (1993) ‘Binge’ cocaine administration induces a sustained increase of prodynorphin mRNA in rat caudateputamen. Brain Res. Mol. Brain Res. 19, 323–327.PubMedCrossRefGoogle Scholar
  36. Svensson M., Skold K., Svenningsson P., and Andren P. F. (2003) Peptidomics-based discovery of novel neuropeptides. J. Proteome Res. 2, 213–219.PubMedCrossRefGoogle Scholar
  37. Turchan J., Maj M., Przewlocka B., and Przewlocki R. (2002) Effect of cocaine and amphetamine on biosynthesis of proenkephalin and prodynorphin in some regions of the rat limbic system. Pol. J. Pharmacol. 54, 367–372.PubMedGoogle Scholar
  38. Varlamov O. and Fricker L. D. (1998) Intracellular trafficking of metallocarboxypeptidase D in AtT-20 cells: localization to the trans-Golgi network and recycling from the cell surface. J. Cell Sci. 111, 877–885.PubMedGoogle Scholar
  39. Varlamov O., Eng F. J., Novikova E. G., and Fricker L. D. (1999a) Localization of metallocarboxypeptidase D in AtT-20 cells: Potential role in prohormone processing. J. Biol. Chem. 274, 14,759–14,767.Google Scholar
  40. Varlamov O., Leiter E. H., and Fricker L. D. (1996) Induced and spontaneous mutations at Ser202 of carboxypeptidase E: Effect on enzyme expression, activity, and intracellular routing. J. Biol. Chem. 271, 13,981–13,986.Google Scholar
  41. Varlamov O., Wu F., Shields D., and Fricker L. D. (1999b) Biosynthesis and packaging of carboxypeptidase D into nascent secretory vesicles in pituitary cell lines. J. Biol. Chem. 274, 14,040–14,045.Google Scholar
  42. Wahlestedt C., Karoum F., Jaskiw G., Wyatt R. J., Larhammar D., Ekman R., and Reis D. J. (1991) Cocaine-induced reduction of brain neuropeptide Y synthesis dependent on medial prefrontal cortex. Proc. Natl. Acad. Sci. U. S. A. 88, 2078–2082.PubMedCrossRefGoogle Scholar
  43. Westwood S. C. and Hanson G. R. (1999) Effects of stimulants of abuse on extrapyramidal and limbic neuropeptide Y systems. J. Pharmacol. Exp. Ther. 288, 1160–1166.PubMedGoogle Scholar
  44. Yuferov V., Nielsen D., Butelman E., and Kreek M. J. (2005) Microarray studies of psychostimulant-induced changes in gene expression. Addict. Biol. 10, 101–118.PubMedCrossRefGoogle Scholar
  45. Zhang R., Sioma C. S., Thompson R. A., Xiong L., and Regnier F. E. (2002) Controlling deuterium isotope effects in comparative proteomics. Anal. Chem. 74, 3662–3669.PubMedCrossRefGoogle Scholar
  46. Zhou Y., Spangler R., Schlussman S. D., Yuferov V. P., Sora I., Ho A., et al. (2002) Effects of acute “binge” cocaine on preprodynorphin, preproenkephalin, proopiomelanocortin, and corticotropin-releasing hormone receptor mRNA levels in the striatum and hypothalamic-pituitary-adrenal axis of mu-opioid receptor knockout mice. Synapse 45, 220–229.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2006

Authors and Affiliations

  1. 1.Department of Molecular PharmacologyAlbert Einstein College of MedicineBronx
  2. 2.Department of Psychiatry and Behavioral SciencesAlbert Einstein College of MedicineBronx
  3. 3.Department of NeuroscienceAlbert Einstein College of MedicineBronx

Personalised recommendations