Optimization of Neuropeptide Extraction from the Mouse Hypothalamus

Fei, Yun; Zhang, Xin; Berezniuk, Iryna; Callaway, Myrasol; J, Lim; Fricker, Lloyd D.

doi:10.1021/pr060690r

Cited by 61 publications

(83 citation statements)

References 42 publications

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“…2B. These two cerebellin forms were previously identified from mouse hypothalamus studies (8); however, References found in SCN studies were for the prohormones, which were previously reported in the studies. there was no report localizing these peptides to the SCN.…”

Section: Resultsmentioning

confidence: 79%

“…Mass spectrometry, a rapid and sensitive method that has been used for the analysis of complex biological samples, can detect and identify the precise forms of neuropeptides without prior knowledge of peptide identity, with these approaches making up the field of peptidomics (7)(8)(9)(10)(11)(12). The direct tissue and single neuron analysis by MALDI MS has enabled the discovery of hundreds of neuropeptides in the last decade, and the neuronal homogenate analysis by fractionation and subsequent ESI or MALDI MS has yielded an equivalent number of new brain peptides (5).…”

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confidence: 99%

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Endogenous Peptide Discovery of the Rat Circadian Clock

Lee

Atkins

Hatcher

et al. 2010

Molecular & Cellular Proteomics

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Understanding how a small brain region, the suprachiasmatic nucleus (SCN), can synchronize the body's circadian rhythms is an ongoing research area. This important time-keeping system requires a complex suite of peptide hormones and transmitters that remain incompletely characterized. Here, capillary liquid chromatography and FTMS have been coupled with tailored software for the analysis of endogenous peptides present in the SCN of the rat brain. After ex vivo processing of brain slices, peptide extraction, identification, and characterization from tandem FTMS data with <5-ppm mass accuracy produced a hyperconfident list of 102 endogenous peptides, including 33 previously unidentified peptides, and 12 peptides that were post-translationally modified with amidation, phosphorylation, pyroglutamylation, or acetylation. This characterization of endogenous peptides from the SCN will aid in understanding the molecular mechanisms that mediate rhythmic behaviors in mammals. Molecular & Cellular Proteomics 9:285-297, 2010.

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Section: Resultsmentioning

confidence: 79%

mentioning

confidence: 99%

Endogenous Peptide Discovery of the Rat Circadian Clock

Lee

Atkins

Hatcher

et al. 2010

Molecular & Cellular Proteomics

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“…Peptidomics-Peptides were extracted from whole mouse brain (minus the olfactory bulb and cerebellum) as described (19,20). In brief, four mice (C57B6/J, 2-3 months old) were sacrificed by decapitation, and the head was immediately irradiated in a conventional microwave oven for 8 s to raise the brain temperature to 80°C and inactivate proteases that can rapidly degrade cellular proteins during postmortem processing of the tissue (21).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Substrate Specificity of Human Carboxypeptidase A4 and Implications for a Role in Extracellular Peptide Processing

Tanco

Zhang

Morano

et al. 2010

Journal of Biological Chemistry

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CPA4 (carboxypeptidase A4) is a member of the metallocarboxypeptidase family. CPA4 was originally found in a screen of mRNAs up-regulated by sodium butyrate-induced differentiation of cancer cells. Further studies suggested a relation between CPA4 and prostate cancer aggressiveness. In the present study, we determined that CPA4 is secreted from cells as a soluble proenzyme (pro-CPA4) that can be activated by endoproteases, such as trypsin. Three complementary approaches were used to study the substrate specificity of CPA4; kinetic analysis was performed using a new series of chromogenic substrates and some biologically relevant peptides, the cleavage of synthetic peptides was tested individually, and the cleavage of a mixture of >100 mouse brain peptides was examined using a quantitative peptidomics mass spectrometry-based approach. CPA4 was able to cleave hydrophobic C-terminal residues with a preference for Phe, Leu, Ile, Met, Tyr, and Val. However, not all peptides with C-terminal hydrophobic residues were cleaved, indicating the importance of additional residues within the peptide. Aliphatic, aromatic, and basic residues in the P1 position have a positive influence on the cleavage specificity. In contrast, acidic residues, Pro, and Gly have a negative influence in the P1 position. Some of the peptides identified as CPA4 substrates (such as neurotensin, granins, and opioid peptides) have been previously shown to function in cell proliferation and differentiation, potentially explaining the link between CPA4 and cancer aggressiveness. Taken together, these studies suggest that CPA4 functions in neuropeptide processing and regulation in the extracellular environment. Carboxypeptidases (CPs)3 hydrolyze a single amino acid from the C terminus of peptides and proteins. Metallo-CPs use a catalytic mechanism in which nucleophilic attack on a peptide bond is mediated by a Zn 2ϩ -activated water molecule (1). Most metallo-CPs are classified based on amino acid sequence, structure, and function into subfamilies within clan MC, family M14 (2). These subfamilies include M14A, also known as the CPA/ CPB subfamily; M14B, also known as the CPN/CPE subfamily; M14C, which includes ␥-D-glutamyl-(L)-meso-diaminopimelate peptidase I from Bacillus sphaericus; and a proposed fourth subfamily, including cytosolic CPs (3, 4). Metallo-CPs have also been divided into subgroups based on substrate specificity: A-like enzymes with preference for hydrophobic C-terminal amino acids; B-like enzymes, which cleave C-terminal basic residues; CPs with preference for glutamate in the C-terminal position; and CPs with a broad substrate specificity.CPA4 (carboxypeptidase A4) belongs to the M14A subfamily of carboxypeptidases. The pancreatic members of this subfamily (CPA1, CPA2, and CPB) act in the degradation of dietary proteins in the digestive tract. Other members of this subfamily display a wide spectrum of physiological roles. Mast cell carboxypeptidase (recently renamed CPA3) is found in the secretory granules of mast cells and plays a role ...

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“…The labeling procedure has been previously described in detail (35,36). In brief, each group of cells within an experiment was labeled with one of the isotopic TMAB-Nhydroxysuccinimide labels.…”

Section: Isotopic Labeling and Mass Spectrometry For Peptide Identifimentioning

confidence: 99%

A Novel Intracellular Peptide Derived from G1/S Cyclin D2 Induces Cell Death

Araujo

Russo

Castro

et al. 2014

Journal of Biological Chemistry

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Background: Intracellular peptides probably regulate several biological processes. Results: pep5 derived from G 1 /S cyclin D2 specifically increases during the S phase of the cell cycle and, reintroduced into the cell, induces apoptosis and necrosis. Conclusion: pep5 has potential therapeutic applications and could have biological functions. Significance: pep5 discovery advances our understanding of limited proteolysis.

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Optimization of Neuropeptide Extraction from the Mouse Hypothalamus

Cited by 61 publications

References 42 publications

Endogenous Peptide Discovery of the Rat Circadian Clock

Endogenous Peptide Discovery of the Rat Circadian Clock

Characterization of the Substrate Specificity of Human Carboxypeptidase A4 and Implications for a Role in Extracellular Peptide Processing

A Novel Intracellular Peptide Derived from G1/S Cyclin D2 Induces Cell Death

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