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Quantitative Top-Down Proteomics Revealed Staurosporine-induced Proteoform-level Changes in Cancer Cells

ABSTRACT: We conducted a quantitative top-down proteomics analysis on HeLa cells treated with staurosporine to investigate differential expression of proteoforms. Furthermore, we assessed alterations in phosphorylation patterns for intact phosphoproteoforms

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ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Kellye Sutton

LAB HEAD: Si Wu

PROVIDER: PXD048148 | Pride | 2025-01-31

REPOSITORIES: Pride

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			Action	DRS
	20220919_Trishika_DMSO_1_Rep1.mzML	Mzml
	20220919_Trishika_DMSO_1_Rep1_ms2_toppic_proteoform.txt	Txt
	20220919_Trishika_DMSO_1_Rep2.mzML	Mzml
	20220919_Trishika_DMSO_1_Rep2_ms2_toppic_proteoform.txt	Txt
	20220919_Trishika_DMSO_2_Rep1.mzML	Mzml

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Project description:Purpose: Compare the transcriptional profile of staurosporine-treated cells in Neurospora crassa wild type and M-NM-^Tczt-1 (M-NM-^TNCU09974) cells Methods: Conidial suspensions were obtained and 1 x 106 cells/ml incubated in minimal medium for 6 hours (26M-BM-:C, 140 rpm, constant light) followed by the addition of staurosporine (or DMSO) and growth for 1 more hour. Cells were harvested using 0.45 M-NM-<m filters and immediately frozen in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. After digestion of 25 M-NM-<g RNA with TURBO DNAse (Life Technologies), mRNA was purified using Dynabeads oligo(dT) magnetic beads (Life Technologies). The mRNA was chemically fragmented using the Ambion RNA fragmentation kit (Life Technologies). First and second strand cDNA synthesis was achieved using appropriate kits (Life Technologies). The illumina TruSeq kit was employed to generate the cDNA libraries with indexing adapters essentially following the manufacturerM-bM-^@M-^Ys protocol. After purification of the libraries with AMPure XP beads (Roche), the quality of the libraries was analysed in a Agilent 2100 Bioanalyzer. The cDNA libraries were sequenced in a illumina HiSeq2000 and single reads of 50 bp were obtained. Sequencing data was handled essentially with Tophat, Cufflinks and Cuffdiff. Expression levels are presented as Fragments/Reads Per Kilobase of transcript per Million mapped reads (FPKM/RPKM). Results: Transcriptional profiling of staurosporine-treated cells wild type by RNA-sequencing showed that genes encoding the machinery for protein synthesis are enriched among the genes repressed by the drug. Functional category enrichment analyses also show that genes encoding components of the mitochondrial respiratory chain are downregulated by staurosporine, whereas genes involved in endoplasmic reticulum activities are upregulated. In contrast, a staurosporine-treated czt-1 deletion strain is unable to repress the genes for the respiratory chain and to induce the genes related with the endoplasmic reticulum, indicating a role for CZT-1 on the regulation of activity of these organelles. Conclusions: This transcriptional profiling study is framed in a comprehensive study on the role of the novel transcription factor CZT-1 (NCU09974) in Neurospora crassa. Transcriptional profile of wild type and M-NM-^Tczt-1 cells staurosporine-treated cells in Neurospora crassa was compared using illumina HiSeq2000 and single reads of 50 bp.

Project description:Purpose: Compare the transcriptional profile of staurosporine-treated cells in Neurospora crassa wild type cells grown with different availabilities of calcium. Methods: Conidial suspensions were obtained and 1 x 106 cells/ml incubated in minimal medium (with the indicated concentrations of calcium) for 6 hours (26M-BM-:C, 140 rpm, constant light) followed by the addition of staurosporine (or DMSO) and growth for 1 more hour. For the experiments in 0 Ca2+ or 20 mM CaCl2 media, the concentration of KH2PO4 in the 50x VogelM-bM-^@M-^Ys stock solution was limited to 10 mM to avoid precipitation with supplemental calcium. Cells were harvested using 0.45 M-NM-<m filters and immediately frozen in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. After digestion of 25 M-NM-<g RNA with TURBO DNAse (Life Technologies), mRNA was purified using Dynabeads oligo(dT) magnetic beads (Life Technologies). The mRNA was chemically fragmented using the Ambion RNA fragmentation kit (Life Technologies). First and second strand cDNA synthesis was achieved using appropriate kits (Life Technologies). The illumina TruSeq kit was employed to generate the cDNA libraries with indexing adapters essentially following the manufacturerM-bM-^@M-^Ys protocol. After purification of the libraries with AMPure XP beads (Roche), the quality of the libraries was analyzed in a Agilent 2100 Bioanalyzer. The cDNA libraries were sequenced in a illumina HiSeq2000 and single reads of 50 bp were obtained. Sequencing data was handled essentially with Tophat, Cufflinks and Cuffdiff. Expression levels are presented as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Results: Differences in the concentration of extracellularly available Ca2+ result in distinct transcriptional programs. Conclusions: Our transcriptomic analysis provides a reference dataset for future investigations on the role of Ca2+ in fungal biology. Transcriptional profile of staurosporine-treated Neurospora crassa wild type cells growing with different concentrations of calcium was compared using illumina HiSeq2000 and single reads of 50 bp.

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Quantitative Top-Down Proteomics Revealed Staurosporine-induced Proteoform-level Changes in Cancer Cells

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