Neuropeptidomics Strategies for Specific and Sensitive Identification of Endogenous Peptides

Fälth, Maria; Sköld, K.; Svensson, Marcus; Nilsson, Anna; Fenyö, David; Andrén, Per E.

doi:10.1074/mcp.m700016-mcp200

Cited by 48 publications

(65 citation statements)

References 59 publications

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“…For example, the enkephalin neuropeptides (met-enk-RF and met-enk-RSL) were clearly detected in the stabilized samples and almost undetectable in the snap-frozen samples. Interestingly, the isotopically labeled neuropeptides added as internal standards were only detected in stabilized tissue, aside from the labeled protein fragment stathmin (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) which was detected independent of sample treatment, indicating ex vivo proteolytic activity and the relative stability of the stathmin (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) fragment.…”

Section: Resultsmentioning

confidence: 99%

“…Mice in a fourth sample group (n ) 4) were killed by focused microwave irradiation (1.4 s at 4.5-5 kW, Muromachi Kikai, Tokyo, Japan) which denature the proteins before dissection and homogenization. A microtip ultrasonicator (Vibra-Cell, Sonics & Materials) was used to homogenize the tissue in 5× the sample weight of 0.25% HAc extraction solution containing 40 mM of isotopically labeled stathmin (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), met-enkephalin, neurotensin, substance P (1-11), and substance P (1-7) as internal standards. The crude extract was centrifuged at 20 000g for 30 min at 4°C to pellet cell debris and ultrafiltered using a 10 kDa centrifugal cutoff filter (Microcon YM-10, Millipore) to isolate the peptide content.…”

Section: Methodsmentioning

confidence: 99%

“…The identification of phosphorylated peptides was done from MS/MS spectra where one of the three most abundant peaks in each spectrum corresponded to a neural loss of a phosphate group. 17 These spectra were searched against SwePep precursor 18 allowing for phosphorylations of serine, threonine and tyrosine and all hits were manually verified.…”

Section: Methodsmentioning

confidence: 99%

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Heat Stabilization of the Tissue Proteome: A New Technology for Improved Proteomics

et al. 2009

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After tissue or body fluid sampling, proteases and other protein-modifying enzymes can rapidly change composition of the proteome. As a direct consequence, analytical results will reflect a mix of in vivo proteome and ex vivo degradation products. Vital information about the presampling state may be destroyed or distorted, leading to variation between samples and incorrect conclusions. Sample stabilization and standardization of sample handling can reduce or eliminate this problem. Here, a novel tissue stabilization system which utilizes a combination of heat and pressure under vacuum was used to stop degradation in mouse brain tissue immediately after sampling. It was found by biochemical assays that enzymatic activity was reduced to background levels in stabilized samples. Western blot analysis confirmed that post-translational phosphorylations of analyzed proteins were stable and conserved for up to 2 h at room temperature and that peptide extracts were devoid of abundant protein degradation fragments. The combination of reduced complexity and proteolytic inactivation enabled mass spectrometric identification of several neuropeptides and endogenous peptides including modified species at higher levels compared to nonstabilized samples. The tissue stabilizing system ensures reproducible and rapid inactivation of enzymes. Therefore, the system provides a powerful improvement to proteomics by greatly reducing the complexity and dynamic range of the proteome in tissue samples and enables enhanced possibilities for discovery and analysis of clinically relevant protein/peptide biomarkers.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Heat Stabilization of the Tissue Proteome: A New Technology for Improved Proteomics

et al. 2009

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“…A homology search is unreliable for short amino acid or nucleotide sequences, and special techniques are frequently needed for both experimental and computational analyses. (62) Currently, the prediction of protein coding sequences is made by computational programs having certain artificial limitations: in particular, arbitrary length threshold (e.g. CDS should be greater than 100 codons).…”

Section: Types Of Alternative Open Reading Framesmentioning

confidence: 99%

Alternative translation start sites and hidden coding potential of eukaryotic mRNAs

2008

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SummaryIt is widely suggested that a eukaryotic mRNA typically contains one translation start site and encodes a single functional protein product. However, according to current points of view on translation initiation mechanisms, eukaryotic ribosomes can recognize several alternative translation start sites and the number of experimentally verified examples of alternative translation is growing rapidly. Also, the frequent occurrence of alternative translation events and their functional significance are supported by the results of computational evaluations. The functional role of alternative translation and its contribution to eukaryotic proteome complexity are discussed.

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“…The protein is submitted to in silico cleavage according to the following template: (K/R)X m (K/R)X k (K/R)X n (K/R), where X is any amino acid, m and n correspond to 0, 2, 4, 6, and k corresponds to 3-50. The residues X k represent the predicted neuropeptide sequence [23,24] (See the workflow presented in Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

Neuropeptide alterations in the tree shrew hypothalamus during volatile anesthesia

Fouillen

Petruzziello

Veit

et al. 2013

Journal of Proteomics

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Neuropeptides are critical signaling molecules, involved in the regulation of diverse physiological processes including energy metabolism, pain perception and brain cognitive state. Prolonged general anesthesia has an impact on many of these processes, but the regulation of peptides by general anesthetics is poorly understood. In this study, we present an in-depth characterization of the hypothalamic neuropeptides of the tree shrew during volatile isoflurane/nitrous oxide anesthesia administered accompanying a neurosurgical procedure. Using a predicted-peptide database and hybrid spectral analysis, we first identified 85 peptides from the tree shrew hypothalamus. Differential analysis was then performed between control and experimental group animals. The levels of 12 hypothalamic peptides were up-regulated following prolonged general anesthesia. Our study revealed for the first time that several neuropeptides, including alpha-neoendorphin and somatostatin-14, were altered during general anesthesia. Our study broadens the scope for the involvement of neuropeptides in volatile anesthesia regulation, opening the possibility for investigating the associated regulatory mechanisms.

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Neuropeptidomics Strategies for Specific and Sensitive Identification of Endogenous Peptides

Cited by 48 publications

References 59 publications

Heat Stabilization of the Tissue Proteome: A New Technology for Improved Proteomics

Heat Stabilization of the Tissue Proteome: A New Technology for Improved Proteomics

Alternative translation start sites and hidden coding potential of eukaryotic mRNAs

Neuropeptide alterations in the tree shrew hypothalamus during volatile anesthesia

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