Relative quantitation of peptides in wild‐type and Cpefat/fat mouse pituitary using stable isotopic tags and mass spectrometry

Che, Fa-Yun; Biswas, Reeta; Fricker, Lloyd D.

doi:10.1002/jms.742

Cited by 80 publications

(90 citation statements)

References 66 publications

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“…For most secreted analytes, the amounts of analyte collected from releasates were insufficient for MS/MS. To obtain sufficient amounts of these compounds, SCN tissue punches were subjected to peptide extraction protocols commonly used for MS-based peptidomic studies (19)(20)(21). Following separation of these extracts by liquid chromatography, analytes from the tissue-extracted samples were matched by mass to corresponding releasate analytes for MS/MS by MALDI TOF MS/MS and, in some instances, were matched separately by linear ion trap quadrupole Fourier transform mass spectrometry for sequence confirmation.…”

Section: Resultsmentioning

confidence: 99%

Mass spectrometry-based discovery of circadian peptides

Hatcher

Atkins

Annangudi

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

151

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A significant challenge to understanding dynamic and heterogeneous brain systems lies in the chemical complexity of secreted intercellular messengers that change rapidly with space and time. Two solid-phase extraction collection strategies are presented that relate time and location of peptide release with mass spectrometric characterization. Here, complex suites of peptide-based cell-to-cell signaling molecules are characterized from the mammalian suprachiasmatic nucleus (SCN), site of the master circadian clock. Observed SCN releasates are peptide rich and demonstrate the corelease of established circadian neuropeptides and peptides with unknown roles in circadian rhythms. Additionally, the content of SCN releasate is stimulation specific. Stimulation paradigms reported to alter clock timing, including electrical stimulation of the retinohypothalamic tract, produce releasate mass spectra that are notably different from the spectra of compounds secreted endogenously over the course of the 24-h cycle. In addition to established SCN peptides, we report the presence of proSAAS peptides in releasates. One of these peptides, little SAAS, exhibits robust retinohypothalamic tract-stimulated release from the SCN, and exogenous application of little SAAS induces a phase delay consistent with light-mediated cues regulating circadian timing. These mass spectrometry-based analyses provide a new perspective on peptidergic signaling within the SCN and demonstrate that the integration of secreted compounds with information relating time and location of release generates new insights into intercellular signaling in the brain.little SAAS ͉ neuropeptides ͉ solid-phase extraction ͉ peptidomics ͉ suprachiasmatic nucleus A fundamental component of cell-to-cell signaling in the brain is the release of endogenously derived neuropeptidebased transmitters and modulators within dynamic neural networks. Neuropeptides include a broad set of structurally diverse molecules that are physiologically active at low concentrations and localize across heterogeneous brain regions, particularly throughout neuroendocrine systems. These properties contribute marked chemical complexity to neurotransmission within heterogeneous and dynamic brain systems. Directly acquiring releasate information about chemical content, release site distribution, and stimulation dependence is a significant challenge to the study of neuronal networks incorporating neuropeptide intercellular signaling.This article describes the use of several unique peptide sampling approaches to characterize chemically complex releasates from the rat suprachiasmatic nucleus (SCN), the site of the master circadian clock (1, 2). The SCN is highly innervated with peptidergic efferents, afferents, and interneurons (3). Moreover, SCN humoral signals are critical elements for the coordination of biological rhythms because SCN transplants can restore aspects of circadian rhythms in SCN-ablated animals (4). In the present work, SCN releasates were collected and concentrated directly from brain sl...

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Section: Resultsmentioning

confidence: 99%

Mass spectrometry-based discovery of circadian peptides

Hatcher

Atkins

Annangudi

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

151

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“…For all three approaches, the following criteria were used to consider a peptide as identified by MS/MS: (i) the peptide mass had to be within 40 ppm (preferably 20 ppm) of the theoretical mass, (ii) 80% or more of the CID major fragments observed in MS/MS matched to predicted fragmentation ions, and (iii) there were at least five matches to either b-and/or y-series ions. The relative levels of peptides in treated versus control mice were calculated by the ratio of peak intensity of the H 9 -TMAB-and d 9 -TMAB-labeled peptide pairs as described previously (5,12).…”

Section: Methodsmentioning

confidence: 99%

Quantitative Neuropeptidomics of Microwave-irradiated Mouse Brain and Pituitary

Fei

Pan

et al. 2005

Molecular & Cellular Proteomics

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166

304

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In neuropeptidomics, the degradation of a small fraction of abundant proteins overwhelms the low signals from neuropeptides, and many neuropeptides cannot be detected by mass spectrometry without extensive purification. Protein degradation was prevented when mice were sacrificed with focused microwave irradiation, permitting the detection of hypothalamic neuropeptides by mass spectrometry. Here we report an alternative and very simple method utilizing an ordinary microwave oven to inhibit enzymatic degradation. We used this technique to identify brain and pituitary neuropeptides. Quantitative analysis using mass spectrometry in combination with stable isotopic labeling was performed to determine the effect of microwave irradiation on relative levels of neuropeptides and protein degradation fragments. Microwave irradiation greatly reduced the levels of degradation fragments of proteins. In contrast, neuropeptide levels were increased about 2-3 times in hypothalamus by the microwave irradiation but not increased in pituitary. In a second experiment, three brain regions (hypothalamus, hippocampus, and striatum) from microwave-irradiated mice were analyzed. Altogether 41 neuropeptides or fragments of secretory pathway proteins were identified after microwave treatment; some of these are novel. These peptides were derived from 15 proteins: proopiomelanocortin, proSAAS, proenkephalin, preprotachykinins A and B, provasopressin, prooxytocin, melanin-concentrating hormone, proneurotensin, chromogranins A and B, secretogranin II, prohormone convertases 1 and 2, and peptidyl amidating monooxygenase. Although some protein degradation fragments were still found after microwave irradiation, these appear to result from protein breakdown during the extraction and not to an enzymatic reaction during the postmortem period. Two of the protein fragments corresponded to novel protein forms: VAP-33 with a 7-residue N-terminal extension and ␤ tubulin with a glutathione on the Cys near the N terminus. In conclusion, microwave irradiation with an ordinary microwave oven effectively inhibits enzymatic postmortem protein degradation, increases the recovery of neuropeptides, and makes it possible to conduct neuropeptidomic studies with mouse brain tissues. Peptides perform many important functions throughout the body as hormones and neurotransmitters. Neuropeptides are involved in a wide variety of systems, including reward mechanisms, pain, memory, food intake and body weight regulation, circadian rhythms, and many others (1, 2). A large number of studies have examined the changes in levels of various peptides upon different treatments or in different physiological states. These studies typically measured peptide levels using radioimmunoassays (RIAs).1 Although this approach is sensitive, most antisera are not specific for a single peptide and are able to cross-react with N-and/or C-terminally extended peptides and with peptides modified by post-translational modifications such as acetylation, phosphorylation, or sulfation (3). Also RIAs ...

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“…Sodium phosphate, dibasic anhydrous (Na 2 HPO 4 ), and sodium phosphate monobasic anhydrous (NaH 2 PO 4 ) were from Amresco. The 4-trimethylammoniumbutyryl (TMAB) stable isotopic labeling reagents containing either three, six, and nine atoms of deuterium (D3, D6, and D9-TMAB) or no deuterium (D0-TMAB) were synthesized as described (29)(30)(31) Mice. TAP1/β2m double double-knockout mice were originally provided by Luc Van Kaer from Vanderbilt University School of Medicine Nashville, USA.…”

Section: Methodsmentioning

confidence: 99%

Similar Intracellular Peptide Profile of TAP1/β2 Microglobulin Double-Knockout Mice and C57BL/6 Wild-Type Mice as Revealed by Peptidomic Analysis

Castro

Berti²,

Russo³

et al. 2010

AAPS J

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Abstract. Cells produce and use peptides in distinctive ways. In the present report, using isotope labeling plus semi-quantitative mass spectrometry, we evaluated the intracellular peptide profile of TAP1/β2m −/− (transporter associated with antigen-processing 1/ß2 microglobulin) double-knockout mice and compared it with that of C57BL/6 wild-type animals. Overall, 92 distinctive peptides were identified, and most were shown to have a similar concentration in both mouse strains. However, some peptides showed a modest increase or decrease (~2-fold), whereas a glycine-rich peptide derived from the C-terminal of neurogranin (KGPGPGGPGGAGGARGGAGGGPSGD) showed a substantial increase (6-fold) in TAP1/β2m −/− mice. Thus, TAP1 and β2microglobulin have a small influence on the peptide profile of neuronal tissue, suggesting that the presence of peptides derived from intracellular proteins in neuronal tissue is not associated with antigens of the class I major histocompatibility complex. Therefore, it is possible that these intracellular peptides play a physiological role.

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Relative quantitation of peptides in wild‐type and Cpe^fat/fat mouse pituitary using stable isotopic tags and mass spectrometry

Cited by 80 publications

References 66 publications

Mass spectrometry-based discovery of circadian peptides

Mass spectrometry-based discovery of circadian peptides

Quantitative Neuropeptidomics of Microwave-irradiated Mouse Brain and Pituitary

Similar Intracellular Peptide Profile of TAP1/β2 Microglobulin Double-Knockout Mice and C57BL/6 Wild-Type Mice as Revealed by Peptidomic Analysis

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