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DIA/SWATH-MS analysis of Halobacterium salinarum NRC-1 collected at four time points of batch culture growth

ABSTRACT: Data-Independent Acquisition (DIA) is a mass spectrometry-based method to reliably identify and reproducibly quantify large fractions of a target proteome. The peptide-centric data analysis strategy employed in DIA requires a priori generated spectral assay libraries. Such assay libraries allow to extract quantitative data in a targeted approach and have been generated for human, mouse, zebrafish, E. coli and few other organisms. However, a spectral assay library for the extreme halophilic archaeon Halobacterium salinarum NRC-1, a model organism that contributed to several notable discoveries, is not publicly available yet. Here, we report a comprehensive spectral assay library to measure 2,563 of 2,646 annotated H. salinarum NRC-1 proteins. We demonstrate the utility of this library by measuring global protein abundances over time under standard growth conditions. The H. salinarum NRC-1 library includes 21,074 distinct peptides representing 97% of the predicted proteome and provides a new, valuable resource to confidently measure and quantify any protein of this archaeon.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Halobacterium Salinarum

TISSUE(S): Cell Culture

SUBMITTER: Ulrike Kusebauch

LAB HEAD: Robert L.

PROVIDER: PXD042774 | Pride | 2023-09-21

REPOSITORIES: Pride

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			Action	DRS
	HSalinarumLysate_HaloIonLibraryVNG_SW100_OneOmics_data_matrix_unfiltered.xlsx	Xlsx
	HSalinarumLysate_HaloIonLibraryVNG_SW100_Spectronaut_Peptide_Report_C4.xls	Xls
	HSalinarumLysate_HaloIonLibraryVNG_SW100_Spectronaut_Protein_Report_C4.xls	Xls
	HSalinarumLysate_directDIA_Spectronaut_Peptide_Report_C4.xls	Xls
	HSalinarumLysate_directDIA_Spectronaut_Protein_Report_C4.xls	Xls

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Project description:Experimentally mapped transcriptome structure of H. salinarum NRC-1 by hybridizing total RNA (including RNA species <200 nt) to genome-wide high-density tiling arrays (60 mer probes with 40 nt overlap between contiguous probes). H. salinarum NRC-1 presents a number of interesting switches in metabolism during growth due to complex changes in EFs including pH, oxygen, nutrition, etc. While most single perturbations (radiation, oxygen, metals, etc.) affect the expression of only ~10% of all genes (Baliga et al, 2004; Kauret al , 2006; Whitehead et al, 2006), the changes during growth resulted in differential regulation of a significantly higher proportion of genes (~63%, 1,518 genes). These conditions enabled the investigation of a wider transcriptional landscape, which includes not only modulation of transcript levels, but also extensive changes in transcriptome structure. We observed altered transcription start sites (TSSs), transcription termination site (TTSs), operon organizations and differential regulation of putative ncRNAs. By integrating hybridization signals with dynamic growth-related changes, we estimated the probability that each tiling array probe was complementary to a transcribed region, mapped locations of putative transcript boundaries and identified 1,574 TSSs and 1,952 TTSs for most genes with some transcriptional variation. In sum, TSSs were assigned to 64% (1,156 singletons and 544 genes in 203 operons) of all annotated genes and TTSs were assigned to 1,114 genes and 202 operons. We were also able to identify 5' and 3' UTRs and revise start sites for 61 genes and 12 operons. H. salinarum NRC-1 growth curve experiments were conducted in CM media, in a water bath incubator at 37oC with agitation of 125 rpm. Reference samples were cultured under standard growth conditions (Baliga and DasSarma, 1999), at mid-log phase (OD600 = ~0.6), as well as all strains used for ChIP-chip experiments (Facciotti et al., 2007). Whole-genome high resolution tiling arrays for H. salinarum NRC-1 were designed with e-Array (Agilent Technologies), using strand specific 60mer probes tiled every 20nt for the main chromosome (NC_002607) and every 21nt for the plasmids pNRC200 (NC_002608) and pNRC100 (NC_001869), consisting a total of 244K probes, including manufacturersâ?? controls. Microarrays were printed by Agilent technologies and hybridized to total RNA, which was isolated using mirVana miRNA Isolation kit (Ambion) and direct labeled with Alexa547 and Alexa647 dyes (Kreatech) (Baliga et al., 2004). We used direct chemical labeling of RNA (Baliga et al., 2004) to avoid enzymatic labeling artifacts (Perocchi et al., 2007) and enable strand-specific signals for transcribed segments. Hybridization and washing were performed according to array manufacturerâ??s instructions. Arrays were scanned in ScanArray (Perkin Elmer) and spot-finding was performed using Feature Extraction (Agilent Technologies). Two biological replicates were sampled and dye-flip experiments were conducted for each sample. Resulting intensities were quantile normalized across all experiments. Log ratios were calculated for each probe (growth curve sample/reference). Reference RNA signals were normalized by sequence content using a linear model similar to that of (Johnson et al., 2006). Interactive visualization of the data was performed in the Gaggle Genome Browser (Bare et al., in preparation), available at http://baliga.systemsbiology.net/regulatory_logic/.

Project description:Oxidative stress (OS) results from genetic defects or stressful environmental challenges that cause unchecked production of reactive oxygen species (ROS). OS has been implicated in many diseases due to its wide ranging damage to nucleic acids, proteins and lipids. Using Halobacterium salinarum NRC-1, an extremophile that thrives under conditions of excessive OS, we have constructed a systems model for oxidative stress response (OSR) by integrating transcriptional changes induced by treatments with H2O2 and paraquat, functional associations inferred through comparative genomics, and de novo discovered cis-regulatory motifs. Together with phenotypic analysis of mutant strains this has revealed a multi-tiered OS management program to transcriptionally coordinate three peroxidase/catalase enzymes, two superoxide dismutases, production of rhodopsins, carotenoids, and gas vesicles, metal trafficking, and various other aspects of metabolism. Remarkably, a significant fraction of this OSR was accurately recapitulated by a model that was previously constructed from cellular responses to diverse environmental perturbations –this constitutes the general stress response component. Notwithstanding this observation, comparison of the two models has identified the coordination of frontline defense and repair systems by regulatory mechanisms that are triggered uniquely by severe OS and not by other environmental stressors, including sub-inhibitory levels of redox-active metals, extreme changes in oxygen tension, and a sub-lethal dose of g rays. Mid-log phase cultures of H. salinarum NRC-1 (2 biological replicates) grown in flasks were treated with either sub-lethal concentrations of 25mM H2O2 or 4mM PQ and incubated with shaking for up to 240 min. For each treatment, two time courses were run to determine the transcriptional responses to (1) constant stress and (2) recovery of H. salinarum NRC-1 to H2O2 and PQ. During constant stress, culture aliquots (~4ml) were collected over a time course (-1, 5, 10, 20, 40, 80 and 160 minutes), centrifuged (16000g, 90 seconds) and flash frozen. For recovery, cells were first treated for 2-hours with either 25 mM H2O2 or 4mM PQ, recovered by centrifugation, washed and re-suspended in CM. Cultures were returned to the incubator with shaking and samples were taken at 0, 10, 20, 30, 40, 50, 60, 120, and 240 minutes and processed as described previously. Analysis of temporal transcriptional changes (-1, 0, 5, 10, 20, 40, 80, 160 and 320m) to sub-inhibitory concentrations of PQ (0.25mM) was also characterized. Total RNA was prepared and labeled with Alexa547 and Alexa647 dyes. Intensities were normalized between the Alexa 546 and Alexa 647 channels by scaling all intensities in one channel such that the 75th percentile in each channel was made equal.

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DIA/SWATH-MS analysis of Halobacterium salinarum NRC-1 collected at four time points of batch culture growth

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