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Quantitative Proteomic Analysis of Aeromonas hydrophila under Potassium Dichromate Stress

ABSTRACT: This project investigates the proteomic changes in Aeromonas hydrophila under potassium dichromate-induced stress using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) LC-MS/MS strategies. Protein extracts from wild-type and dichromate-treated A. hydrophila cells were enzymatically digested and analyzed using an Orbitrap Fusion Lumos mass spectrometer coupled with an EASY-nLC 1200 system. A total of six biological samples (three replicates per group) were processed. Peptides were separated on a C18 analytical column with a detailed gradient and subjected to both DDA and DIA acquisition. DDA scans were performed at a resolution of 120,000 (full scan) and 30,000 (MS/MS), while DIA acquisition involved 45 variable windows spanning m/z 350–1400. The dataset aims to provide insight into the bacterial response mechanisms under heavy metal stress, particularly chromium exposure, and may serve as a resource for identifying stress-related and metal resistance-associated proteins in Gram-negative bacteria.

ORGANISM(S): Aeromonas Hydrophila Subsp. Hydrophila Atcc 7966

SUBMITTER: Xiangmin Lin

PROVIDER: PXD066345 | iProX | Sat Jul 19 00:00:00 BST 2025

REPOSITORIES: iProX

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Project description:Significant occupational and environmental exposures to hexavalent chromium, a metal with broad toxicity potential in humans, have been reported. In order to understand the mechanisms of dermal toxicity induced by hexavalent chromium, global gene expression profiling of human dermal fibroblasts exposed to a toxic concentration of potassium dichromate was performed. Microarray analysis of the gene expression profile in the fibroblasts treated with potassium dichromate identified significant differential expression of approximately 1,200 transcripts compared with the control cells. Hierarchical cluster analysis of the gene expression profile demonstrated a clear separation of the treated cells from the control group of cells. Functional categorization of the differentially expressed genes identified the enrichment of genes involved in several cellular processes, including apoptosis and oxidative stress, in the fibroblasts exposed to hexavalent chromium. Induction of apoptosis and generation of hydroxyl radicals indicative of oxidative stress in the dermal fibroblasts in response to their exposure to hexavalent chromium were independently confirmed by TUNEL assay and electron spin resonance (ESR) analysis, respectively. The potassium dichromate-induced cytotoxicity, differential gene expression, apoptosis, and oxidative stress were significantly blocked by the addition of ferrous sulfate, an agent known for its ability to reduce hexavalent chromium to the insoluble and therefore impermeable trivalent form, to the cell culture medium. Taken together, our data provide insights into the potential mechanisms underlying the dermal toxicity of hexavalent chromium and suggest a definite role for apoptosis and oxidative stress in Cr(VI)-induced cytotoxicity in human dermal fibroblasts. 16 samples were analyzed in this experiment. Exponentially growing human dermal fibroblasts (3x105 cells) were cultured in T25 cell culture flasks. When the cells were approximately 70% confluent, hexavalent potassium dichromate (5µM) was added to the medium with or without 40µM ferrous sulfate. Following 16 hours of culturing at 37oC, total RNA was isolated for gene expression studies. Details of the samples are: Control 4 Samples: Cr(0)-1, Cr(0)-2, Cr(0)-3, Cr(0)-4 Cr 5 µM 4 Samples: Cr(5)-5, Cr(5)-6, Cr(5)-7, Cr(5)-8 FeSO4 40 µM 4 Samples: Fe(40)-9, Fe(40)-10, Fe(40)-11, Fe(40)-12 Cr 5 µM + FeSO4 40 µM 4 Samples: Cr(5)+Fe(40)-13,Cr(5)+Fe(40)-14, Cr(5)+Fe(40)-15, Cr(5)+Fe(40)-16

Project description:Data independent acquisition-mass spectrometry (DIA-MS) coupled with liquid chromatography is a promising approach for rapid, automatic sampling of MS/MS data in untargeted metabolomics. However, wide isolation windows in DIA-MS generate MS/MS spectra containing a mixed population of fragment ions together with their precursor ions. This precursor-fragment ion map in a comprehensive MS/MS spectral library is crucial for relative quantification of fragment ions uniquely representative of each precursor ion. However, existing reference libraries are not sufficient for this purpose since the fragmentation patterns of small molecules can vary in different instrument setups. Here we developed a bioinformatics workflow called MetaboDIA to build customized MS/MS spectral libraries using a user's own data dependent acquisition (DDA) data and to perform MS/MS-based quantification with DIA data, thus complementing conventional MS1-based quantification. MetaboDIA also allows users to build a spectral library directly from DIA data in studies of a large sample size. Using a marine algae data set, we show that quantification of fragment ions extracted with a customized MS/MS library can provide as reliable quantitative data as the direct quantification of precursor ions based on MS1 data. To test its applicability in complex samples, we applied MetaboDIA to a clinical serum metabolomics data set, where we built a DDA-based spectral library containing consensus spectra for 1829 compounds. We performed fragment ion quantification using DIA data using this library, yielding sensitive differential expression analysis. </br></br> Serum metabolome of 40 age-related macular degeneration patients and 20 control samples was analyzed using untargeted mass spectrometry. We used data dependent acquisition data to build a MS/MS spectral assay library for more than 1,000 compounds and performed targeted extraction of MS2 ion chromatograms from data independent acquisition analysis.

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Quantitative Proteomic Analysis of Aeromonas hydrophila under Potassium Dichromate Stress

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