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Proteomics and phosphoproteomics analysis of Haematococcus pluvialis: An improved method to generate and interpret proteomics and phosphoproteomics data of a non-model species

ABSTRACT: Haematococcus pluvialis is a green microalga of commercial interests due to its ability to produce a high value ketocarotenoid, astaxanthin. As a non-model species that lacks a well annotated genome, omics analyses such as transcriptomics and proteomics analysis have often been used together with physiological and biochemical analysis to explore pathways of interest. However, interpretation of these datasets remains challenging. In this work, TMT-based proteomics and phosphoproteomics analyses were conducted on Haematococcus cells grown under favorable conditions (green stage biomass) and high-light stress conditions (red stage biomass). Phosphoproteins were enriched using titanium dioxide before LC-MS/MS analysis. Our proteomics and phosphoproteomics analyses identified 1394 proteins and 569 phosphosites on 366 phosphoproteins, respectively. Of these, 1315 proteins and 396 phosphosites on 314 phosphoproteins were quantifiable, among which 370 proteins and 121 phosphosites on 94 phosphoproteins were differentially expressed. Using an improved analysis pipeline that combines Blast2GO, KEGG, and DAVID to analyze differentially expressed proteins and phosphoproteins, total identified proteins increased from 255 to 322 and total identified phosphoproteins increased from 59 to 70, which were 26.28% and 18.64%, respectively, higher than with the UniProt analysis alone. Using this pipeline, a previously uncharacterized protein and phosphoprotein were identified as an ATPase subunit B and a phosphofructokinase, respectively, and further confirmed with translated genomic and transcriptomic data. This work provides the first example of phosphoproteomics analysis in H. pluvialis, while the proteomics and phosphoproteomics analysis pipelines described here may be useful to analyze omics data from other non-model algal species.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Haematococcus Lacustris

SUBMITTER: Kyarii Ramarui

LAB HEAD: Yantao Li

PROVIDER: PXD054975 | Pride | 2024-11-20

REPOSITORIES: Pride

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Project description:We compared the expression profile of TTHA1939 deletion mutant strain of Thermus thermophils HB8 with that of wild-type. The mutant strain grown in minimum essential medium (CS medium) exhibited growth arrest and cellular aggregation during the logarithm growth phase. In this phase, 99 genes were suppressed and 63 genes were activated for the mutant strain. The suppressed genes included genes of ribosomal proteins, TCA cycle, amino acid biosynthesis, fatty acid biosynthesis, cobalamin biosynthesis, transporters, and cell division including dnaB and ftsZ. The activated genes included genes of nitrogen metabolism, stress proteins, and proteases. We suggested that these transcriptional alterations in the mutant cell were induced by the ppGpp accumulation which was prevented by ppGpp degradation activity of TTHA1939 in WT cell. Keywords: gene deletion, minimum essential medium, logarithm growth phase Wild-type and the mutant strains were precultured in rich medium (TR medium) for two times at 70 oC and then subcultured to minimum essential medium (CS medium). These cells were harvested during the logarithm growth phase (800 minutes from the start of culture). Total RNA were extracted from each strain and used for the cDNA synthesis. For the biological replication, above experiments were performed four times independently. The cDNA fragments were labeled with biotin-dideoxy UTP, using terminal transferase according to the manufacturerâs instructions (ENZO Biochem. Inc., Farmingdale, NY). The 3â-terminal-labeled cDNA (2 micro g) was hybridized to a TTHB8401 GeneChip (Affymetrix, Santa Clara, CA). The Probe Array was scanned with a GeneArray Scanner (Affymetrix), and then, the image data was scaled to the target intensity by one-step Tukeyâs biweight algorithm using GeneChip Operating software, version 1.0 (Affymetrix, Santa Clara, CA). The data analysis was performed by using GeneSpring GX (Agilent Tech.). The genes which had detection call of âpresenceâ more than 4 times from 8 samples were used for following analysis. The normalized intensities for the mutant strains were calculated by using the intensities for wild-type strains of each data set, and then, the average of these intensities were used. The genes which produced P value < 0.1 in the Studentâs t-test were extracted. Among them, the genes whose expression level was different more than 2 fold between two strains were considered as significant.

			Action	DRS
	Deamidation_NQ_Sites.txt	Txt
	Oxidation_M_Sites.txt	Txt
	Phospho_STY_Sites.txt	Txt
	TMT16_Phospho.raw	Raw
	TMT16_Total.raw	Raw

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Proteomics and phosphoproteomics analysis of Haematococcus pluvialis: An improved method to generate and interpret proteomics and phosphoproteomics data of a non-model species

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