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Ubiquitinated peptide identification in tomato

ABSTRACT: In this study, we identified ubiquitinated peptides that changed abundance in the mutant of an E3 ubiquitin ligase gene by coupling a K-(GG) peptide immunoprecipitation with quantitative proteomic analysis. Two independent replicates identified 265 and 236 ubiquitinated peptides, respectively. Seventy-four ubiquitinated peptides were overlapped in both biological replicates. Quantitative analysis revealed that 27 of these ubiquitinated peptides changed abundance significantly (P < 0.05) in the mutant.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Solanum Lycopersicum

TISSUE(S): Pericarp

SUBMITTER: Weihao Wang

LAB HEAD: Guozheng Qin

PROVIDER: PXD018707 | Pride | 2020-11-24

REPOSITORIES: Pride

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Project description:Chronic lymphocytic leukaemia (CLL) is renowned for its variable clinical course and response to therapy, suggesting a role for precision medicine. However, the molecular basis of CLL variability remains incompletely understood. Recent developments in data independent acquisition (DIA) -MS technologies, such as SWATH (Sequential Windowed Acquisition of all THeoretical fragments), provide an opportunity to study the pathophysiology of CLL at the proteome level. This report describes the sample replication and data handling requirements for SWATH-MS analysis of clinical samples. A CLL-specific spectral library compromising over 1,500,000 spectra with digital information for over 150,000 peptides has been generated to enable the quantification of up to 7736 proteins. To assess the reproducibility of the assay, SWATH-MS analysis was performed on 6 cryopreserved CLL patient samples, incorporating biological (IGHV mutational status), sample preparation and MS technical replicates. Quantitative information was obtained for 5169 proteins (<1% FDR) across 54 SWATH-MS acquisitions. These analyses showed that SWATH-MS is a highly reproducible technique. However, replicate sample preparations on different days was identified as the main source of variation. To overcome the effect of variation between sample preparation batches, different computational approaches for batch correction were tested. All approaches successfully removed technical variability whilst retaining proteomic differences between clinical subgroups. Functional enrichment analysis of proteins associated to IGHV mutational status highlighted the importance of metabolic remodelling in the biology of CLL and overlapped significantly with previous studies based on gene expression profiling. Finally, an approach to perform statistical power analysis in proteomics studies was developed. This approach could be used to drive the design of future CLL SWATH-MS studies. These fundamental requirements for DIA approaches should be widely applicable to other clinical proteomics studies.

			Action	DRS
	mix-rep1.group	Other
	mix-rep1.wiff	Wiff
	mix-rep1.wiff.scan	Wiff
	mix-rep2.group	Other
	mix-rep2.wiff	Wiff

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Ubiquitinated peptide identification in tomato

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