Browse
Submit Data
Databases
API
Help

Dataset Information

393 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Protein turnover measurement using selected reaction monitoring-mass spectrometry (SRM-MS) C4PR_LIV

ABSTRACT: Protein turnover represents an important mechanism in the functioning of cells, with deregulated synthesis and degradation of proteins implicated in many diseased states. Therefore, proteomics strategies to measure, with high confidence, turnover rates are of vital importance to understanding many biological processes. In this study, the more widely used approach of non-targeted precursor ion signal intensity (MS1) quantification is compared to selected reaction monitoring (SRM), a data acquisition strategy that records data for specific peptides, to determine if improved quantitative data would be obtained using a targeted quantification approach. Using mouse liver as a model system, turnover measurement of four tricarboxylic acid cycle proteins was performed using both MS1 and SRM quantification strategies. SRM outperformed MS1 in terms of sensitivity and selectivity of measurement, allowing more confident determination of protein turnover rates. SRM data is acquired using cheaper and more widely available tandem quadrupole mass spectrometers, making the approach accessible to a larger number of researchers than MS1 quantification, which is best performed on high mass resolution instruments. SRM acquisition is ideally suited to focussed studies where the turnover of tens of proteins is measured, making it applicable in determining the dynamics of proteins complexes and complete metabolic pathways.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Hepatocyte, Liver

SUBMITTER: Stephen Holman

LAB HEAD: Robert J. Beynon

PROVIDER: PXD004413 | Pride | 2016-08-10

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

Comparison of targeted mass spectrometry techniques with an immunoassay: a case study for HSP90 alpha

Project description:We developed 2D-LC-MS/MS-based SRM and PRM assays to measure HSP90 alpha in serum and compared the results to a commercially available immunoassay (ELISA). Serum samples were trypsin-digested and fractionated by SCX chromatography prior to SRM and PRM measurements. PRM data obtained by high-resolution mass spectrometry correlated better with ELISA measurements than SRM data measured on a triple quadrupole mass spectrometer. While all three methods (SRM, PRM, ELISA) were able to quantify HSP90 alpha in serum at the ng/mL level, the use of PRM on a high-resolution mass spectrometer reduced variation. To rule out that the observed differences in SRM and PRM are due to different mass spectrometry systems, the SCX fractions were measured on the same high-resolution instrument (Orbitrap) in the ion trap mode (IT-PRM); such measurements showed that intense co-eluting signals were present in the SRM method, but these interfering peaks were eliminated in the high-resolution PRM mode. Thus, this report shows that it is possible to measure ng/mL levels of HSP90 alpha in a reproducible, selective and sensitive way using PRM. This opens up the possibility to quantify low levels of multiple proteins in complex samples based on a fractionation strategy on tryptic peptides followed by PRM.

2018-07-05 | PXD006615 | Pride

Identification of human blood plasma proteins in shotgun proteomics

Project description:The study focuses on identification of human blood plasma proteins based on the analysis of MS1 and MS2 data with the use of crude unlabelled synthetic (CUS) peptides for guaranteed recording of their MS2 spectra and subsequent verification of the protein identifications by the SRM method. The search performed and subsequent verification of the protein identifications by the selected reaction monitoring method of had identified 17 proteins in human blood plasma, which corresponded to 17 out of the 19 CUS peptides used.

2019-06-26 | PXD007580 | Pride

QUANTITATIVE SUBCELLULAR PROTEOMICS OF THE ORBITOFRONTAL CORTEX OF SCHIZOPHRENIA PATIENTS

Project description:We analyzed orbitofrontal cortex post-mortem brain tissue from patients. We generated an organellar proteome map from different subcellular fractions such as mitochondria (MIT), crude nuclear fraction (NUC) and cytoplasm (CYT) with the aim to monitor the subproteome changes using iTRAQ labelling for relative quantification and Selected Reaction Monitoring (SRM) for targeted quantification.

2019-10-10 | PXD014284 | Pride

Comparison of targeted mass spectrometry techniques with an immunoassay: a case study for HSP90 alpha

2022-03-10 | PXD006569 | Pride

Quantification of Peptide 2 (RLLWENGNL) concentration in the mouse plasma

Project description:Diabetes mellitus (DM) causes the change in the components of the plasma, which may induce tissue and organ injuries, including the heart and kidney. In the diabetic model db/db mice, intravenous injection of purified dipeptidyl peptidase III (DPPIII) attenuated the heart and kidney damages provoked by DM without alteration in blood glucose level. This led us to hypothesize that there might be peptide(s) that are involved in the progression of the DM-mediated damages and are cleaved by DPPIII. To explore the peptides, the whole peptidomics analysis (shotgun peptidomics) was conducted using tandem mass spectrometry, in which peptides derived from the plasma of PBS-infused C57BL/6 mice, PBS-infused db/db mice, and DPPIII-treated db/db mice were compared. The raw mass spectrometry data were deposited to the ProteomeXchange with the dataset identifier PXD025440. Together with the mass spectrometry analysis and other experiments, we identified a peptide named Peptide 2 (AA sequence: RLLWENGNL) as a substrate of DPPIII. Peptide 2 is a part of C3f (53% identical), and C3f can act as an anaphylatoxin like C3a. To quantify the amount in plasma derived from the plasma of PBS-infused C57BL/6 mice, PBS-infused db/db mice, and DPPIII-treated db/db mice, selected reaction monitoring (SRM) assay was performed. All of the chromatograms and quantified data obtained by SRM from the three mouse groups were shown in the Analyst formated and PDF files.

2022-02-17 | PXD025469 | Pride

A Combined Shotgun and Targeted Mass Spectrometry Strategy for Breast Cancer Biomarker Discovery

Project description:Metastasizing breast cancer is an incurable disease. Thus, it is important to find proteins responsible for tumor dissemination, which may be used as biomarkers or treatment targets. In this study, we established the combination of a non-targeted LC-MS/MS discovery and a targeted LC-SRM verification workflow for improved discovery and validation of biomarkers. We collected 80 breast tumors, stratified for estrogen receptor status and development of distant recurrence (DR+/-). After enrichment of N-glycosylated proteins, label-free LC-MS/MS was performed on all of the individual tumors in triplicates. In total, 1515 glycopeptides from 778 proteins were identified, used to create a map of the breast cancer N-glycosylated proteome. Based on the breast cancer N-glycosylated proteome map we constructed a 92-plex targeted label-free LC-SRM panel, enriched for interesting proteins, and analyzed the samples for the selected proteins, resulting in 10 proteins differentially regulated between DR+/DR- tumors. We also compared the LC-SRM results to clinically reported HER2 status. 17 of 18 patients could be classified the same way, demonstrating the clinical accuracy of LC-SRM. In conclusion, we demonstrated the use of a combined non-targeted LC-MS/MS and targeted LC-SRM strategy, at large scale on clinical samples, and identified 10 proteins as potential biomarkers.

2015-06-16 | PXD001685 | Pride

Dietary heme stimulates epithelial cell turnover by downregulating feedback inhibitors of proliferation in murine colon

Project description:This SuperSeries is composed of the following subset Series: GSE27847: Dietary heme stimulates epithelial cell turnover by downregulating feedback inhibitors of proliferation in murine colon (part 1) GSE27848: Dietary heme stimulates epithelial cell turnover by downregulating feedback inhibitors of proliferation in murine colon (part 2) Refer to individual Series

2011-10-01 | E-GEOD-27849 | biostudies-arrayexpress

QUANTITATIVE SUBCELLULAR PROTEOMICS OF THE ORBITOFRONTAL CORTEX OF SCHIZOPHRENIA PATIENTS

Project description:We analyzed OFC post-mortem brain tissue from patients classified as residual schizophrenia according to the DMS-IV manual. In this classification, the patients do not manifest prominent positive psychotic symptoms but prevail characteristic disturbance of negative symptoms. Through the comparison of patient´s samples against a pool of control subjects without any history of psychiatric illness we generated an organellar proteome map from different subcellular fractions such as mitochondria (MIT), crude nuclear fraction (NUC) and cytoplasm (CYT) with the aim to monitor the subproteome changes using iTRAQ labelling for relative quantification and Selected Reaction Monitoring (SRM) for targeted quantification.

2019-10-07 | PXD014350 | Pride

A Selected Reaction Monitoring Approach for Validating Candidate Biomarkers

Project description:We here describe a Selected Reaction Monitoring (SRM)-based approach for the discovery and validation of peptide biomarkers for cancer. The first stage of this approach is the direct identification of candidate peptides through comparison of proteolytic peptides derived from the plasma of cancer patients or healthy individuals. Several hundred candidate peptides were identified through this method, providing challenges for choosing and validating the small number of peptides that might prove diagnostically useful. To accomplish this validation, we used two-dimensional chromatography coupled with Selected Reaction Monitoring of candidate peptides. We applied this approach, called SAFE-SRM (for Sequential Analysis of Fractionated Eluates by Selected Reaction Monitoring) to plasma from cancer patients and discovered two peptides encoded by the PPIA (peptidyl-prolyl cis-trans isomerase A) gene whose abundance was increased in the plasma of ovarian cancer patients. At optimal thresholds, elevated levels of at least one of these two peptides was detected in 43 (68.3%) of 63 women with ovarian cancer but in none of 50 healthy controls. In addition to providing a potentially new biomarker for ovarian cancer, this approach is generally applicable to the discovery of proteins and peptides characteristic of various disease states.

2017-12-11 | PXD008094 | Pride

comprehensive SAAVs detection in PANC-1 cell line

Project description:Three different strategies were applied for SAAVs detection. We predicted deleterious SAAVs using online AssVar and confirm 3 SAAVs using SRM

2021-09-09 | PXD017449 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data