Region-specific bioconversion of dynorphin neuropeptide detected by in situ histochemistry and MALDI imaging mass spectrometry

• Published in: Peptides (New York, N.Y. : 1980) N.Y. : 1980), 2017-01, Vol.87, p.20-27
• Main Authors: Bivehed, Erik, Strömvall, Robert, Bergquist, Jonas, Bakalkin, Georgy, Andersson, Malin
• Format: Article
• Language: English
• Subjects: Animals; Bioconversion; Brain - metabolism; Cysteine Endopeptidases - chemistry; Cysteine Endopeptidases - metabolism; Dynorphin; Dynorphins - antagonists & inhibitors; Dynorphins - chemistry; Dynorphins - isolation & purification; Dynorphins - metabolism; Endorphins - antagonists & inhibitors; Endorphins - chemistry; Endorphins - isolation & purification; Endorphins - metabolism; Enzyme; Enzyme inhibitor; Glycopeptides - administration & dosage; Histochemistry; Humans; MALDI imaging mass spectrometry; Mass spectrometry; Neuropathic pain; Neuropeptide; Neuropeptides - antagonists & inhibitors; Neuropeptides - chemistry; Neuropeptides - isolation & purification; Neuropeptides - metabolism; Oligopeptides - administration & dosage; Parkinson Disease - metabolism; Parkinson Disease - pathology; Parkinson's disease; Protease Inhibitors - administration & dosage; Rats; Salivary Proteins and Peptides - administration & dosage; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 0196-9781; 1873-5169; 1873-5169
• DOI: 10.1016/j.peptides.2016.11.006

[Display omitted] •Combining in situ histochemistry with MALDI imaging MS.•Unbiased in that no labeling required.•Detects the neuropeptide substrate and many metabolic fragments in a single experiment.•Can be used in imaging mode for localization of enzymatic activity or profiling mode for fast screening of enzyme inhibitors.•Versatile method that can be adapted for many purposes. Brain region-specific expression of proteolytic enzymes can control the biological activity of endogenous neuropeptides and has recently been targeted for the development of novel drugs, for neuropathic pain, cancer, and Parkinson’s disease. Rapid and sensitive analytical methods to profile modulators of enzymatic activity are important for finding effective inhibitors with high therapeutic value. Combination of in situ enzyme histochemistry with MALDI imaging mass spectrometry allowed developing a highly sensitive method for analysis of brain-area specific neuropeptide conversion of synthetic and endogenous neuropeptides, and for selection of peptidase inhibitors that differentially target conversion enzymes at specific anatomical sites. Conversion and degradation products of Dynorphin B as model neuropeptide and effects of peptidase inhibitors applied to native brain tissue sections were analyzed at different brain locations. Synthetic dynorphin B (2pmol) was found to be converted to the N-terminal fragments on brain sections whereas fewer C-terminal fragments were detected. N-ethylmaleimide (NEM), a non-selective inhibitor of cysteine peptidases, almost completely blocked the conversion of dynorphin B to dynorphin B(1–6; Leu-Enk-Arg), (1–9), (2–13), and (7–13). Proteinase inhibitor cocktail, and also incubation with acetic acid displayed similar results. Bioconversion of synthetic dynorphin B was region-specific producing dynorphin B(1–7) in the cortex and dynorphin B (2–13) in the striatum. Enzyme inhibitors showed region- and enzyme-specific inhibition of dynorphin bioconversion. Both phosphoramidon (inhibitor of the known dynorphin converting enzyme neprilysin) and opiorphin (inhibitor of neprilysin and aminopeptidase N) blocked cortical bioconversion to dynorphin B(1–7), wheras only opiorphin blocked striatal bioconversion to dynorphin B(2–13). This method may impact the development of novel therapies with aim to strengthen the effects of endogenous neuropeptides under pathological conditions such as chronic pain. Combining histochemistry and MALDI imaging MS is a powerful