Dataset Information

Analysis of Reproducibility of Proteome Coverage and Quantitation Using Isobaric Mass Tags (iTRAQ and TMT)

ABSTRACT: This study aimed to compare the depth and reproducibility of total proteome and differentially expressed protein coverage in technical duplicates and triplicates using iTRAQ 4-plex, iTRAQ 8-plex and TMT 6-plex reagents.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Phaeosphaeria Nodorum Sn15 (ncbitaxon:321614)

SUBMITTER: Richard J Lipscombe

PROVIDER: MSV000079594 | MassIVE | Thu Mar 17 21:59:00 GMT 2016

SECONDARY ACCESSION(S): PXD003807

REPOSITORIES: MassIVE

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Publications

Analysis of Reproducibility of Proteome Coverage and Quantitation Using Isobaric Mass Tags (iTRAQ and TMT).

Casey Tammy M TM Khan Javed M JM Bringans Scott D SD Koudelka Tomas T Takle Pari S PS Downs Rachael A RA Livk Andreja A Syme Robert A RA Tan Kar-Chun KC Lipscombe Richard J RJ

Journal of proteome research 20161207 2

This study aimed to compare the depth and reproducibility of total proteome and differentially expressed protein coverage in technical duplicates and triplicates using iTRAQ 4-plex, iTRAQ 8-plex, and TMT 6-plex reagents. The analysis was undertaken because comprehensive comparisons of isobaric mass tag reproducibility have not been widely reported in the literature. The highest number of proteins was identified with 4-plex, followed by 8-plex and then 6-plex reagents. Quantitative analyses revea ...[more]

PMID: 28152591

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Project description:The recent development within high-throughput technologies for expression profiling has allowed for parallel analysis of transcriptomes and proteomes in biological systems such as comparative analysis of transcript and protein levels of tissue regulated genes. Until now, such studies of have only included microarray or short length sequence tags for transcript profiling. Furthermore, most comparisons of transcript and protein levels have been based on absolute expression values from within the same tissue and not relative expression values based on tissue ratios. Presented here is a novel study of two porcine tissues based on integrative analysis of data from expression profiling of identical samples using cDNA microarray, 454-sequencing and iTRAQ-based proteomics. Sequence homology identified 2.541 unique transcripts that are detectable by both microarray hybridizations and 454-sequencing of 1.2 million cDNA tags. Both transcript-based technologies showed high reproducibility between sample replicates of the same tissue, but the correlation across these two technologies was modest. Thousands of genes being differentially expressed were identified with microarray. Out of the 306 differentially expressed genes, identified by 454-sequencing, 198 (65 %) were also found by microarray. The relationship between the regulation of transcript and protein levels was analyzed by integrating iTRAQ-based proteomics data. Protein expression ratios were determined for 354 genes, of which 148 could be mapped to both microarray and 454-sequencing data. A comparison of the expression ratios from the three technologies revealed that differences in transcript and protein levels across heart and muscle tissues are positively correlated. We show that the reproducibility within cDNA microarray and 454-sequencing is high, but that the agreement across these two technologies is modest. We demonstrate that the regulation of transcript and protein levels across identical tissue samples is positively correlated when the tissue expression ratios are used for comparison. The results presented are of interest in systems biology research in terms of integration and analysis of high-throughput expression data from mammalian tissues. Keywords: tissue comparison, platform comparison Tissue samples of heart (HEA) and Longissimus dorsi (LDO) were prepared by pooling from five healthy Hampshire gilts at age four to six months and division into six sub-samples, three for each tissue named HEA1, HEA2, HEA3, LDO1, LDO2 and LDO3. The exact same six tissue samples were used for subsequent expression profiling with cDNA microarray, 454-sequencing and iTRAQ-based proteomics. A reference sample for the cDNA microarray experiment and iTRAQ-based proteomics was constructed by combining the six samples. In the microarray experiment, three cDNA microarray slides were used per sample.

Dataset Information

Analysis of Reproducibility of Proteome Coverage and Quantitation Using Isobaric Mass Tags (iTRAQ and TMT)

Publications

Analysis of Reproducibility of Proteome Coverage and Quantitation Using Isobaric Mass Tags (iTRAQ and TMT).

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