Dataset Information

NanoTPOT: Enhanced Sample Preparation for Quantitative Na-noproteomic Analysis (revision work)

ABSTRACT: With the ever-growing need for protein-level understanding in pathological research, proteomics researchers thrive to exam-ine detailed proteome dynamics using crucial, yet often limited, primary and clinical samples. Aside from mass spectrometer instrumentation advancement, a single-tube-based high-throughput sample processing workflow is imperative to ensure sen-sitive, quantitative and reproducible protein analysis for these increasingly sophisticated studies. Leveraging the benefits of an acid-cleavable detergent, RapiGest SF Surfactant (Waters Corporation), we developed and optimized a nanoproteomic workflow that we termed Nanogram TMT Processing in One Tube (NanoTPOT). Through the assessment of proteolytic di-gestion, tandem mass tag (TMT) labeling, online and offline fractionation strategies, our optimized workflow effectively eliminated the need for sample desalting and enabled compatible sample processing for mass spectrometry analysis. We further applied the NanoTPOT workflow to examine cellular response to stress caused by dithiothreitol in HeLa cells, where we identified and quantified 6935 and 5474 proteins in TMT 10-plex experiments with one microgram of lysate protein and 2000 sorted HeLa cells (500ng lysate protein) in each channel, respectively. Our workflow has been proven to be an effec-tive alternative for current nanoproteomic sample processing to minimize sample loss in biological and clinical applications.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Yu Lu

LAB HEAD: Yu Lu

PROVIDER: PXD018247 | Pride | 2021-03-16

REPOSITORIES: Pride

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Publications

NanoTPOT: Enhanced Sample Preparation for Quantitative Nanoproteomic Analysis.

Wu Ruilin R Pai Akshat A Liu Lina L Xing Sansi S Lu Yu Y

Analytical chemistry 20200420 9

With the ever-growing need for protein-level understanding in pathological research, proteomics researchers thrive to examine detailed proteome dynamics using crucial, yet often limited, primary and clinical samples. Aside from mass spectrometer instrumentation advancement, a single-tube-based high-throughput sample processing workflow is imperative to ensure sensitive, quantitative, and reproducible protein analysis for these increasingly sophisticated studies. Leveraging the benefits of an aci ...[more]

PMID: 32255623

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Project description:Purpose: Alternative splicing is fundamental for post-transcriptional regulation and proteome diversity. The goals of this study are to compare transcriptome and splicing profiling (RNA-seq) between wild type and prp8a prp8b mutant ovules of the spliceosome subunit and define the molecular signature of prp8a prp8b pollen tube attraction phenotype. Methods: mRNA profiles from mature ovules of 6-weeks-old wild-type (WT) and pre-mRNA processing factor 8 (PRP8Aa prp8bb) Arabidopsis plants were generated by deep sequencing, in triplicates, using Illumina HiSeq4000 100bp paired-end reads. The sequence reads that passed quality filters were were mapped to TAIR10 whole genome and analyzed for differential expression, differential exone usage and intron retention as indicated in DATA PROCESSING PIPELINE section. Results: Using an optimized data analysis workflow, about 15 million sequence read pairs per sample were mapped to the Arabidopsis genome (TAIR10). Approximately 2.9% of the transcripts showed differential expression between the WT and PRP8Aa prp8bb ovules, with a fold change ≥1.5 and p value <0.05. Analysis for differential gene expression, exon usage and intron retention with DESeq2 v1.22.1 and DEXseq v1.28.0 or IRFinder v1.2.3 respectively, uncovered several as yet uncharacterized genes that may contribute to pollen tube attraction and female gametophyte cell fate specification. Conclusions: Our work has uncovered a molecular signature through which PRP8A/PRP8B subunits act redundantly to define male-female signaling competence for successful pollen tube attraction in Arabidopsis. Application of DESeq2 algorithms to our ovule RNA-seq data identified downregulation of over 50 different CRP genes with yet unknown function from the synergid and the central cells including all LURE pollen tube attractants. Whereas use of DEXseq workflow, revealed mis-splicing of key genes involved embryo sac specificiation and genes of the secretory pathway. We concluded that 100bp paired-end RNAseq was a sufficient compromise for detection of differential gene expression and splice isoforms, however, our experiment would have benefited with more number of replicates.

Dataset Information

NanoTPOT: Enhanced Sample Preparation for Quantitative Na-noproteomic Analysis (revision work)

Publications

NanoTPOT: Enhanced Sample Preparation for Quantitative Nanoproteomic Analysis.

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