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Fibroblast Growth Factor (FGF) 23 and FGF Receptor 4 promote cardiac metabolic remodeling in CKD

ABSTRACT: Frozen mitochondrial pellets were sonicated in SDS followed by centrifugation. 18 micrograms of protein was reduced and alkylated and digested with 1.5 ugs trypsin using an S-Trap micro device (Protifi) and lyophilized. A study pool QC (SPQC) was made from equal volumes of reconstituted samples. 0.75 ug of each sample and replicates of the SPQC (see metadata for run order) were analyzed by LC-MS/MS using a nanoACQUITY UPLC system (Waters) operating in trap-elute configuration with a 90 min analytica gradient of 5-30% MeCN and a 75 um x 25 cm HSS-T3 column. The LC was coupled to a Q-Exactive HF-X high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoFlex ionization source. MS was performed in data-dependent acquisition (DDA) mode with a 120,000 resolution (@ m/z 200) full MS scan from m/z 375 to 1600 followed by a with a target AGC value of 3e6 ions and 50 ms maximum injection time (IT). MS/MS used a Top30 method at 15,000 resolution and with an AGC targeted of 5e4 and max IT of 45 ms, and isolation window of 1.2 m/z and normalized collision energy of 27. Peptides were selected for MS/MS using a minimum AGC target of 2.25e3, charge state filtering (2-5), an apex trigger of 5 to 40 s, and a dynamic exclusion of 20 s.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Alexander Grabner

PROVIDER: MSV000094087 | MassIVE | Wed Feb 14 13:50:00 GMT 2024

REPOSITORIES: MassIVE

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Project description:This study was a proteomic and secretomic survey of the Gram-negative saprophytic bacterium Cellvibio japonicus during growth using fungal polysaccharides or authentic fungal biomass as a sole nutrient source. The purified polysaccharides were beta-glucan (derived from yeast) and alpha chitin (derived from shrimp shells). The authentic fungal biomass came from Saccharomyces cerevisiae and Aspergillus nidulans. The proteome and secretome samples were taken during mid-exponential growth in biological triplicate. Bacterial cells were homogenized via probe sonication and reduced and alkylated followed by trypsin digestion using a suspension trap (S-trap). Supernatants were reduced and alkylated and followed by trypsin digestion via MStern Blotting. An SPQC was made by combining equal volumes of each sample for both sample types. After lyophilization and reconstitution, 2 ul of cell digests and 15 ul of supernatant digests were loaded onto Evotips. Quantitative LC/MS/MS was performed using an Evosep One LC coupled to a Thermo Orbitrap Astral via a Nanospray Flex ionization source. Samples were block randomized and interspersed with the SPQC replicates. The Evosep used a 60 sample-per-day method with a Pepsep 8 cm x 150 um column (1.5 um particle size) with a PepSep Sprayer and stainless steel (30 um) emitter. The MS analysis used a 240,000 resolution precursor ion (MS1) scan from 380-980 m/z, AGC target of 500% and maximum injection time (IT) of 50 ms, collected every 0.6 s in centroid mode. MS/MS was performed using a DIA method with default charge state = 3, precursor mass range of 380-980, 10 m/z isolation windows, AGC target of 500% and maximum IT of 20 ms, and a NCE of 28. An RF lens of 40% was used for MS1 and DIA scans. Raw MS data was converted to .htrms format using HTRMS converter and processed in Spectronaut 19 (Biognosys). A spectral library was built using direct-DIA searches of all DIA analyses using a Cellvibrio japonicus database, downloaded from Uniprot on 10/29/2024 and appended contaminant sequences using FragPipeSearch settings included N-terminal trypsin/P specificity up to 2 missed cleavages; peptide length from 7-52 amino acids with the following modifications: acetyl (N-term) and carbamidomethyl(Cys). For DIA analysis, default extraction, calibration, identification, and protein inference settings were used.

Project description:In the current study, we used a mass spectrometry-based redox proteomics approach to test responses of the cysteine (Cys) proteome to selective disruption of the Trx- and GSH-dependent systems. Auranofin (ARF) was used to inhibit Trx reductase without detectable oxidation of the GSH/GSSG couple, and buthionine sulfoximine (BSO) was used to deplete GSH without detectable oxidation of Trx1. Results for 606 Cys-containing peptides (peptidyl Cys) showed that 36% were oxidized over 1.3-fold by ARF, while BSO-induced oxidation of peptidyl Cys was only 10%. Mean fold oxidation of these peptides was also higher by ARF than BSO treatment. Analysis of potential functional pathways showed that ARF oxidized peptides associated with glycolysis, cytoskeleton remodeling, translation and cell adhesion. Of 60 peptidyl Cys oxidized due to depletion of GSH, 41 were also oxidized by ARF and included proteins of translation and cell adhesion but not glycolysis or cytoskeletal remodeling. Studies to test functional correlates showed that pyruvate kinase activity and lactate levels were decreased with ARF but not BSO, confirming the effects on glycolysis-associated proteins are sensitive to oxidation by ARF. These data show that the Trx system regulates a broader range of proteins than GSH system, support distinct function of Trx and GSH in cellular redox control, and show for the first time in mammalian cells selective targeting peptidyl Cys and biological pathways due to deficient function of the Trx system. ICAT-labeled Cys peptides were analyzed by reverse-phase liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Peptide eluents were monitored in an MS survey scan followed by ten data-dependent MS/MS scans on an LTQ-Orbitrap ion trap mass spectrometer (Thermo Finnigan, San Jose, CA). The LTQ was used to acquire MS/MS spectra (2 m/z isolation width, 35% collision energy, 5,000 AGC target, 200 ms maximum ion time). The Orbitrap was used to collect MS scans (300-1600 m/z, 1,000,000 AGC target, 500 ms maximum ion time, resolution 30,000). All data were converted from .raw files to the .dta format using ExtractMS version 2.0 (Thermo Finnigan, San Jose, CA). The acquired MS/MS spectra were searched against a concatenated target-decoy human RefSeq (release 37 - September 2009 - 38108 target proteins) database of the National Center for Biotechnology Information using the SEQUEST Sorcerer algorithm (version 3.11, SAGE-N) searching parameters included partially tryptic restriction, parent ion mass tolerance (+/- 20 ppm), dynamic modifications of oxidized Met (+15.9949 Da), differential ICAT-modified Cys (+9.0302 Da) and static ICAT modification of Cys (+227.1270 Da). The peptides were classified by charge state and tryptic state (fully and partial) and first filtered by mass accuracy (10 ppm for high-resolution MS), and then dynamically by increasing XCorr and deltaCn values to reduce protein false discovery rate to less than 1%, according to the target-decoy strategy.

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Fibroblast Growth Factor (FGF) 23 and FGF Receptor 4 promote cardiac metabolic remodeling in CKD

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