MassIVE MSV000095252

Partial Public PXD053648

Proteomic Investigation of Neurotrophic trans-Banglene Reveals Potential Link to Iron Homeostasis

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Description

Neurodegenerative diseases remain an area where the causative disease mechanisms, with few exceptions, are poorly understood and which are very resistant to modern therapeutic advances. Current FDA-approved medications result in modest improvements in quality of life measures and no increase in life expectancy. In this context, insight into cellular elements that dictate neuronal survival and growth versus degradation or death (the shared feature of all neurodegenerative disorders), is valuable. Molecular agents that modulate these responses can be uniquely beneficial in such inquiries because of their ease of administration, frequent robustness in biological media, and their potential for pharmacokinetic optimization or derivatization in the hands of a medicinal chemist. Further, investigation of the mechanism of action for small molecule natural products can lead to unexpected insights, such as the serendipitous discovery of the mTOR protein (mammalian target of rapamycin), a central controller of cell metabolism. We performed a global proteomic analysis of t-BG's effects in PC-12 cells and compared it to the impact of natural nerve growth factor (NGF), a neurotrophin protein which causes similar phenotypic changes in PC-12 cells. To our surprise, while we gained insight into classic pro-growth, pro-differentiation, and pro-survival pathways, we also observed changes in iron-binding proteins in the cell that were unanticipated. Since iron dyshomeostasis is an increasingly recognized element of neurodegenerative disease, particularly in early stages of Alzheimer’s and Parkinson’s disease,10 the potential connection between neurotrophic activity and altered iron homeostasis is quite notable. Cell pellets were lysed in 4% SDS 100mM tris pH 6.8 and total protein concentration was estimated with BCA assay. Approximately 20 micrograms of total protein was reduced and alkylated with dithiothreitol and iodoacetamide (PMID12724530) and loaded onto 10% SDS-PAGE. Each sample's lane was separated into 5 fractionations and the proteins were digested out of the gels using MS-grade trypsin (PMID8779443). Resulting peptides were cleaned with C-18 STop And Go Extraction (STAGE) tips (PMID 12498253) using 40% (v/v) acetonitrile in 0.1% (v/v) formic acid as the elution buffer. Peptide concentration was estimated with NanoDrop One (Thermo Scientific) to load equal amounts of total peptides on Impact II Qtof (Bruker Daltonics) coupled to easy nLC 1200 (Thermo Scientific) using a in-house constructed column set up consisting a fritted 2-cm-long trap column made with 100-?m-inner diameter fused silica and 5 ?m Aqua C-18 beads (Phenomenex, cat 04A-4331) packing material, and a 30-50 cm-long analytical column with an integrated spray tip (6-8 ?m-diameter opening, pulled on a P-2000 laser puller from Sutter Instruments) made with 75-?m-inner diameter fused silica and 1.9 ?m-diameter Reprosil-Pur C-18-AQ beads (Dr. Maisch, cat r119.aq.0003) packing material. The column was heated to 50degC with another in-house constructed column heater, and was used for 60 min sample separation using the acquisition settings described in PMID 32539747. Four biological replicates of the treatment sets were processed and each sample was analyzed twice for duplicate technical replicates. The samples were randomized before injection. The resulting data were searched on MaxQuant version 1.6.7.0 (PMID27809316) using Uniprot's rat proteome and common contaminants. Label-free quantitation, and match-between-run options were enabled using fixed modification of carbamidomethylation of cysteines, and variable modifications of oxidation of methionines and acetylation of protein N-termini. Specific proteolytic cleavages after arginine and lysine with up to 2 missed cleavages were set. The data were filtered for 1% false discovery at protein, peptide and PSM levels using revert decoy search mode. [doi:10.25345/C5NC5SQ4Z] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: proteomics ; LC-MSMS ; Neurotrophic trans-Banglene ; neurodegenerative diseases

Contact

Principal Investigators:
(in alphabetical order) 		Leonard Foster, University of British Columbia, Canada
Submitting User: kyungmee

Publications

Gunawardana PBW, Gohil K, Moon KM, Foster LJ, Williams FJ.
Proteomic Investigation of Neurotrophic trans-Banglene Reveals Potential Link to Iron Homeostasis.
Mol Neurobiol. Epub 2025 Mar 14.

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