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 ...

Cpe fat ͞Cpe fat mice have a naturally occurring point mutation within the carboxypeptidase E gene that inactivates this enzyme, leading to an accumulation of many neuroendocrine peptides containing C-terminal basic residues. These processing intermediates can be readily purified on an anhydrotrypsin affinity resin. Using MS to obtain molecular mass and partial sequence information, more than 100 peptides have been identified. These peptides represent fragments of 16 known secretory pathway proteins, including proenkephalin, proopiomelanocortin, protachykinins A and B, chromogranin A and B, and secretogranin II. Many of the identified peptides represent previously uncharacterized fragments of the precursors. For example, 12 of the 13 chromogranin B-derived peptides found in the present study have not been previously reported. Of these 13 chromogranin B-derived peptides, only five contain consensus cleavage sites for prohormone convertases at both the C and N termini. Two distinct chromogranin B-derived peptides result from cleavage at Trp-Trp bonds, a site not typically associated with neuropeptide processing. An RIA was used to confirm that one of these peptides, designated WE-15, exists in wild-type mouse brain, thus validating the approach to identify peptides in Cpe fat ͞Cpe fat mice. These ''orphan'' peptides are candidate ligands for orphan G protein-coupled receptors. In addition, the general technique of using affinity chromatography to isolate endogenous substrates from a mutant organism lacking an enzyme should be applicable to a wide range of enzymesubstrate systems.peptide processing ͉ carboxypeptidase E ͉ carboxypeptidase D ͉ chromogranin ͉ secretogranin

Determining the relative levels of neuropeptides in two samples is important for many biological studies. An efficient, sensitive and accurate technique for relative quantitative analysis involves tagging the peptides in the two samples with isotopically distinct labels, pooling the samples and analyzing them using liquid chromatography/mass spectrometry (LC/MS). In this study, we compared two different sets of isotopic tags for analysis of endogenous mouse pituitary peptides: succinic anhydride with either four hydrogens or deuteriums and [3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)propyl]trimethylammonium chloride with either nine hydrogens or deuteriums. These two labels react with amines and impart either a negative charge (succinyl) or a positive charge (4-trimethylammoniumbutyryl (TMAB)). Every endogenous mouse pituitary peptide labeled with the light TMAB reagent eluted from the C18 reversed-phase column at essentially the same time as the corresponding peptide labeled with the heavy reagent. Most of the peptides labeled with succinyl groups also showed co-elution of the heavy- and light-labeled forms on LC/MS. The mass difference between the heavy and light TMAB reagents (9 Da per label) was larger than that of the heavy and light succinyl labels (4 Da per label), and for some peptides the larger mass difference provided more accurate determination of the relative abundance of each form. Altogether, using both labels, 82 peptides were detected in Cpe(fat/fat) mouse pituitary extracts. Of these, only 16 were detected with both labels, 41 were detected only with the TMAB label and 25 were detected only with the succinyl label. A number of these peptides were de novo sequenced using low-energy collisional tandem mass spectrometry. Whereas the succinyl group was stable to the collision-induced dissociation of the peptide, the TMAB-labeled peptides lost 59 Da per H9 TMAB group. Several peptides identified in this analysis represent previously undescribed post-translational processing products of known pituitary prohormones. In conclusion, both succinyl and TMAB isotopic labels are useful for quantitative peptidomics, and together these two labels provide more complete coverage of the endogenous peptides.

Neuropeptides perform a large variety of functions as intercellular signaling molecules. While most proteomic studies involve digestion of the proteins with trypsin or other proteases, peptidomics studies usually analyze the native peptide forms. Neuropeptides can be studied by using mass spectrometry for identification and quantitation. In many cases, mass spectrometry provides an understanding of the precise molecular form of the native peptide, including post-translational cleavages and other modifications. Quantitative peptidomics studies generally use differential isotopic tags to label two sets of extracted peptides, as done with proteomic studies, except that the Cys-based reagents typically used for quantitation of proteins are not suitable because most peptides lack Cys residues. Instead, a number of amine-specific labels have been created and some of these are useful for peptide quantitation by mass spectrometry. In this review, peptidomics techniques are discussed along with the major findings of many recent studies and future directions for the field.