Tachykinins Processing is Significantly Impaired in PC1 and PC2 Mutant Mouse Spinal Cord S9 Fractions

Saidi, Mouna; Kamali, Soufiane; Ruiz, Alberto Orduna; Beaudry, Francis

doi:10.1007/s11064-015-1720-0

Cited by 8 publications

(7 citation statements)

References 27 publications

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“…Instrument calibration was performed prior to all analyses and mass accuracy was below 1 ppm using Thermo Pierce calibration solution and automated instrument protocol. The analytical method performance was previously well described (Ferland et al, ; Pailleux & Beaudry, ; Pailleux, Vachon, Lemoine, & Beaurdy, ; Saidi, Kamali, Orduna Ruiz, & Beaudry, ; Orduna Ruiz & Beaudry, ) and peptide quantification was performed using peak‐area ratio of light (unlabeled) and heavy (labeled) isotope.…”

Section: Methodsmentioning

confidence: 99%

Neuropeptidomics: Comparison of parallel reaction monitoring and data‐independent acquisition for the analysis of neuropeptides using high‐resolution mass spectrometry

Saidi

Kamali

Beaudry

2019

Biomedical Chromatography

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Targeted peptide quantitation by mass spectrometry is a rapidly emerging field. Traditionally it relied on the development and validation of multiple reaction monitoring assays that could comply with a high level of sensitivity, specificity, accuracy and reproducibility in complex biological samples. However, with the introduction of high‐resolution mass spectrometers, other acquisition modes could provide more comprehensive datasets for identification and quantification but also for in‐depth data mining. The objective of this study was to evaluate two analytical approaches, parallel‐reaction monitoring (PRM) and data‐independent analysis (DIA) using a hybrid Quadrupole–Orbitrap mass spectrometer for the quantification of neuropeptides in animal spinal cord tissues. Mouse spinal cord tissues were harvested, homogenized and neuropeptides extracted using a C18 solid‐phase extraction protocol. Chromatography was achieved using a Thermo Biobasic C8 100 × 1 mm (5 μm) column. The initial mobile phase conditions consisted of acetonitrile and water (both containing 0.1% of formic acid) at a ratio of 5:95. An 11 min linear gradient was applied up to a ratio of 50:50 and maintained for 3 min. The flow rate was fixed at 75 μL/min and 2 μL of sample was injected. Mass spectrometry analyses were performed using a Thermo Q Exactive Plus MS using PRM and DIA approaches. Quantitative data using an isotopic dilution and a label‐free strategy were obtained for both methods and statistically compared. Using both approaches, we were able to clearly detect endogenous neuropeptides. However, with DIA, mass spectra alone could not distinguish Leu‐Enk and Met‐Enk. We used a Bland–Altman plot (Difference plot) to analyze the agreement between both approaches and no systematic bias was observed. Further statistical analyses, including variance analysis, showed more variability in DIA compared with PRM mode. Further analyses were performed using a label‐free approach and confirmed an increase of the variance using a DIA approach.

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Section: Methodsmentioning

confidence: 99%

Neuropeptidomics: Comparison of parallel reaction monitoring and data‐independent acquisition for the analysis of neuropeptides using high‐resolution mass spectrometry

Saidi

Kamali

Beaudry

2019

Biomedical Chromatography

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“…The tissue preparation was performed as we previously described (Saidi et al, 2015;Orduna & Beaudry, 2016). Briefly, three rat spinal cords were pooled and homogenized in a 0.1 M phosphate buffer, pH 7.4 at a ratio of 1:5 (w/v).…”

Section: Tissue Sample Preparationmentioning

confidence: 99%

“…The tachykinin precursor 1 (TAC1) gene encodes the protachykinin-1 protein containing the sequence of four tachykinin peptides, including SP and NKA (Basbaum, 1999), as well as related peptides neuropeptide K (NPK) and neuropeptide gamma (Otsuka & Yoshioka, 1993). The protachykinin-1 protein is cleaved by the action of specific proteases into active neuropeptides by post-translational proteolytic processing during axonal transport (Hook et al, 2008;Saidi & Beaudry, 2017;Saidi, Kamali, Ruiz, & Beaudry, 2015). It is implied that many neuropeptides are synthesized by the actions of proprotein convertases (PCs) Abbreviations: CNS, central nervous system; FCI, furin convertase inhibitor; NK1R, neurokinin 1 receptor; NK2R, neurokinin 2 receptor; NK3R, neurokinin 3 receptor; NKA, neurokinin A; NPK, neuropeptide K; PC, proprotein convertase; PNS, peripheral nervous system; PRM, parallel reaction monitoring; SP, substance P; TAC1, protachykinin-1 protein; TFA, trifluoroacetic acid; TIC, total ion current; XIC, extracted-ion chromatograms.…”

Section: Introductionmentioning

confidence: 99%

“…It was demonstrated that PC1/3 and PC2 recognize motives composed of either a pair of basic amino acids (KR-, RR-, RK-and KK-) or a single arginine (R) residue (only PC2; Seidah et al, 2008). The role of PCs in the maturation of the protachykinin-1 protein was recently suggested (Saidi et al, 2015;Saidi & Beaudry, 2017). As shown in Figure 1, the primary sequence of the protein suggests that PC1/3 and PC2 play an important role in the release of SP and NKA.…”

Section: Introductionmentioning

confidence: 99%

“…The tachykinin precursor 1 (TAC1) gene encodes the protachykinin‐1 protein containing the sequence of four tachykinin peptides, including SP and NKA (Basbaum, ), as well as related peptides neuropeptide K (NPK) and neuropeptide gamma (Otsuka & Yoshioka, ). The protachykinin‐1 protein is cleaved by the action of specific proteases into active neuropeptides by post‐translational proteolytic processing during axonal transport (Hook et al, ; Saidi & Beaudry, ; Saidi, Kamali, Ruiz, & Beaudry, ). It is implied that many neuropeptides are synthesized by the actions of proprotein convertases (PCs) and endopeptidases during the axonal transport (Harrison & Geppetti, ; Hook et al, ).…”

Section: Introductionmentioning

confidence: 99%

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Characterization of neuropeptide K processing in rat spinal cord S9 fractions using high‐resolution quadrupole–Orbitrap mass spectrometry

Salem

Nkambeu

Beaudry

2018

Biomedical Chromatography

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Tachykinins are a family of pronociceptive neuropeptides with a specific role in pain and inflammation. Several mechanisms regulate endogenous tachykinins levels, including the differential expression of protachykinin mRNA and the controlled secretion of tachykinin peptides from neurons. We suspect that proteolysis regulates extracellular neuropeptide K (NPK) and neurokinin A (NKA) concentrations and NPK is a precursor of NKA. Here, we provide evidence that proteolysis controls NPK and NKA levels in the spinal cord, leading to the formation of active C-terminal peptide fragments. Using high-resolution mass spectrometry, specific tachykinin fragments were identified and characterized. The metabolic stability in rat spinal cord fractions of NPK and NKA was very short, resulting in half-lives of 1.9 and 2.2 min respectively. Following the degradation of NPK, several C-terminal fragments were identified, including NPK , NKA, NKA , NKA , NKA and NKA , which conserve affinity for the neurokinin 2 receptor but also for the neurokinin 1 receptor. Interestingly, the same fragments were identified following the degradation of NKA. A specific proprotein convertases inhibitor was used and showed a significant reduction in the rate of formation of NKA, providing strong evidence that proprotein convertase is involved in C-terminal processing of NPK in the spinal cord, leading to the formation of NKA.

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Deciphering the Role of EGL-3 for Neuropeptides Processing in Caenorhabditis elegans Using High-Resolution Quadrupole–Orbitrap Mass Spectrometry

et al. 2018

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Neuropeptides are derived from large and inactive proteins which require endoproteolytic processing for the biosynthesis of the bioactive peptides. The maturation of pro-neuropeptide to neuropeptide is believed to be performed by ortholog pro-protein convertase EGL-3 in Caenorhabditis elegans (C. elegans). Furthermore, ortholog of Cathepsin L, CPL-1 are found in C. elegans and can potentially cleave paired basic amino acids at the N-terminal suggesting the presence of both pathways. The objective of this study was to decipher the role of EGL-3 in the proteolysis of FMRF amide-related peptides (FLPs) or neuropeptide-like proteins (NLPs) using synthetic surrogate peptides based on a universal enzymatic cleavage pattern published by Schechter and Berger and used widely in enzymology. The results show evidence that proteolysis controls FLP-21 and NLP-8 related neuropeptide levels in C. elegans. Surrogate peptides were degraded rapidly when exposed to C. elegans S9 fractions leading to the formation of specific peptide fragments related to EGL-3 and CPL-1 pathway. The results suggest that CPL-1 pathway does not compensate for the loss of the EGL-3 pathway. Proteolysis of pro-neuropeptides associated to FLP-21 and NLP-8 in elg-3 mutants are severely hampered leading to a lack of mature bioactive neuropeptides.

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Tachykinins Processing is Significantly Impaired in PC1 and PC2 Mutant Mouse Spinal Cord S9 Fractions

Cited by 8 publications

References 27 publications

Neuropeptidomics: Comparison of parallel reaction monitoring and data‐independent acquisition for the analysis of neuropeptides using high‐resolution mass spectrometry

Neuropeptidomics: Comparison of parallel reaction monitoring and data‐independent acquisition for the analysis of neuropeptides using high‐resolution mass spectrometry

Characterization of neuropeptide K processing in rat spinal cord S9 fractions using high‐resolution quadrupole–Orbitrap mass spectrometry

Deciphering the Role of EGL-3 for Neuropeptides Processing in Caenorhabditis elegans Using High-Resolution Quadrupole–Orbitrap Mass Spectrometry

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