Dataset Information

High-throughput proteomics and phosphoproteomics of rat tissues using micro-flow Zeno SWATH

ABSTRACT: We investigated the combined sensitivity of micro-flow liquid chromatography with a ZenoTOF mass spectrometer for high throughput proteomic and phosphoproteomic analysis of rat tissues. Comparing the proteomes acquired using data-independent acquisition (DIA) on the ZenoTOF 7600 with the previous generation TripleTOF 6600, more proteins were quantified using a fifth of the sample load and a third of the instrument time. Zeno SWATH data was evaluated using replicate injections of rat organ digests to compare FragPipe and DIA-NN computational pipelines. FragPipe identified more proteins in 7 of the 8 rat organs, with an extra 12% and 17% observed in heart and muscle tissue respectively. The number of identified peptides per protein were higher with FragPipe and the precision of missing values across replicate injections was more consistent. Single-shot phosphopeptide enrichment from 100 µg rat tissue, without fractionation, was acquired using data-dependent acquisition (DDA) on both instruments. A total of 5,108 phosphosites were quantified with a negligible increase in phosphosites found using the ZenoTOF 7600 relative to the 6600. Using DIA on the ZenoTOF, 8,013 phosphosites were quantified using Spectronaut.

INSTRUMENT(S): SCIEX instrument model

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Spleen, Heart, Testis, Brain, Lung, Liver, Kidney

SUBMITTER: Erin Humphries

LAB HEAD: Phillip J.

PROVIDER: PXD045472 | Pride | 2024-06-02

REPOSITORIES: pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20190717_RRP_SWISS_Canon_Rat_PhosphoSpikeMix.fasta	Fasta
	20220901_EMH_HEK_MS2acc01_100VW_7600_ZS.wiff	Wiff
	20220901_EMH_HEK_MS2acc01_100VW_7600_ZS.wiff.scan	Wiff
	20220901_EMH_HEK_MS2acc01_25VW_7600_ZS.wiff	Wiff
	20220901_EMH_HEK_MS2acc01_25VW_7600_ZS.wiff.scan	Wiff

Items per page:

1 - 5 of 2445

Similar Datasets

Project description:We used phosphoproteomics to compare the responses of the ERK1/2 inhibitors, SCH772984 and GDC0994, and the MKK1/2 inhibitor, trametinib. These are compared with responses to the MKK1/2 inhibitor, selumetinib (AZD6244), previously measured by our lab in the same metastatic melanoma cell line. In three replicate experiments, we quantified a total of 12,805 class I phosphosites on 3,819 proteins in the trametinib-SCH772984-DMSO experiment, and 7,074 class I phosphosites on 2,453 in the GDC0994-SCH772984-DMSO experiment. This included 466 phosphosites that reproducibly decreased in response to at least one inhibitor in the trametinib-SCH772984-DMSO experiment and 414 phosphosites in the GDC0994-SCH772984-DMSO experiment. The results demonstrate linearity in signaling through the MAP kinase pathway. By comparing multiple inhibitors targeted to multiple tiers of protein kinases in the MAPK pathway, we gain insight into regulation and new targets of the oncogenic BRAF driver pathway in cancer cells, and a useful approach for evaluating the specificity of drugs and drug candidates. SILAC Experimental Design Experiment 1 Replicate 1: Heavy – DMSO, Medium – SCH772984, Light – Trametinib Replicate 2: Heavy – SCH772984, Medium – Trametinib, Light – DMSO Replicate 3: Heavy – Trametinib, Medium – DMSO, Light – SCH772984 SILAC Experimental Design Experiment 2 Replicate 1: Heavy – DMSO, Medium – SCH772984, Light – GDC0994 Replicate 2: Heavy – SCH772984, Medium – GDC0994, Light – DMSO Replicate 3: Heavy – GDC0994, Medium – DMSO, Light – SCH772984 File List 1. Zipped MaxQuant search results folder containing index and output folders for each raw file, ‘combined’ output folder, and mqpar.xml MaxQuant search parameters file 2. Individual raw files of phosphopeptide-enriched ERLIC fractions 3. Zipped MaxQuant version used for analysis 4. FASTA file containing Uniprot human identifications 5. Instructions for viewing annotated spectra

Project description:We used phosphoproteomics to compare the responses of the ERK1/2 inhibitors, SCH772984 and GDC0994, and the MKK1/2 inhibitor, trametinib. These are compared with responses to the MKK1/2 inhibitor, selumetinib (AZD6244), previously measured by our lab in the same metastatic melanoma cell line. In three replicate experiments, we quantified a total of 12,805 class I phosphosites on 3,819 proteins in the trametinib-SCH772984-DMSO experiment, and 7,074 class I phosphosites on 2,453 in the GDC0994-SCH772984-DMSO experiment. This included 466 phosphosites that reproducibly decreased in response to at least one inhibitor in the trametinib-SCH772984-DMSO experiment and 414 phosphosites in the GDC0994-SCH772984-DMSO experiment. The results demonstrate linearity in signaling through the MAP kinase pathway. By comparing multiple inhibitors targeted to multiple tiers of protein kinases in the MAPK pathway, we gain insight into regulation and new targets of the oncogenic BRAF driver pathway in cancer cells, and a useful approach for evaluating the specificity of drugs and drug candidates. SILAC Experimental Design Experiment 1 Replicate 1: Heavy – DMSO, Medium – SCH772984, Light – Trametinib Replicate 2: Heavy – SCH772984, Medium – Trametinib, Light – DMSO Replicate 3: Heavy – Trametinib, Medium – DMSO, Light – SCH772984 SILAC Experimental Design Experiment 2 Replicate 1: Heavy – DMSO, Medium – SCH772984, Light – GDC0994 Replicate 2: Heavy – SCH772984, Medium – GDC0994, Light – DMSO Replicate 3: Heavy – GDC0994, Medium – DMSO, Light – SCH772984 File List 1. Zipped MaxQuant search results folder containing index and output folders for each raw file, ‘combined’ output folder, and mqpar.xml MaxQuant search parameters file 2. Individual raw files of phosphopeptide-enriched ERLIC fractions and total protein fractions 3. Zipped MaxQuant version used for analysis 4. FASTA file containing Uniprot human identifications 5. Instructions for viewing annotated spectra

Dataset Information

High-throughput proteomics and phosphoproteomics of rat tissues using micro-flow Zeno SWATH

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets