Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Optimizing accuracy and depth of protein quantification in experiments using isobaric carriers

ABSTRACT: The isobaric carrier approach, which combines small isobarically-labeled samples with a larger isobarically-labeled carrier sample, is finding diverse applications in ultrasensitive mass-spectrometry analysis of very small samples, such as single cells. To enhance the growing use of isobaric carriers, we characterized the trade-offs of using isobaric carriers in controlled experiments with complex human proteomes. The data indicate that isobaric carriers directly enhances peptide sequence identification without simultaneously increasing the number of protein copies sampled from small samples. The results also indicate strategies for optimizing the amount of isobaric carrier and analytical parameters, such as ion accumulation time, for different priorities such as improved quantification or increased number of identified proteins. Balancing these trade-offs enables adapting isobaric carrier experiments to different applications, such as quantifying proteins from limited biopsies or organoids, building single-cell atlases, or modeling protein networks in single cells. In all cases, the reliability of protein quantification should be estimated and incorporated in all subsequent analysis. We expect that these guidelines will aid in explicit incorporation of the characterized trade-offs in experimental designs and transparent error propagation in data analysis.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Nikolai Slavov

PROVIDER: MSV000086004 | MassIVE | Mon Aug 24 08:53:00 BST 2020

REPOSITORIES: MassIVE

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

A carrier-based quantitative proteomics method applied to biomarker discovery in pericardial fluid

Project description:Data-dependent liquid chromatography tandem mass spectrometry (LC-MS/MS) is challenged by the large concentration range of proteins in plasma and related fluids. We adapted the SCoPE method from single-cell proteomics to pericardial fluid, where a myocardial tissue carrier was used to aid protein quantification. The carrier proteome and patient samples were labeled with distinct isobaric labels, which allowed separate quantification. Undepleted pericardial fluid from patients with type 2 diabetes mellitus and/or heart failure undergoing heart surgery was analyzed with either a traditional LC-MS/MS method or with the carrier proteome. In total, 1398 proteins were quantified with a carrier, compared to 265 without, and a higher proportion of these proteins were of myocardial origin. The number of differentially expressed proteins also increased nearly four-fold. For patients with both heart failure and type 2 diabetes mellitus, pathway analysis of upregulated proteins demonstrated enrichment of immune activation, blood coagulation, and stress pathways. Overall, our work demonstrates the applicability of a carrier for enhanced protein quantification in challenging biological matrices such as pericardial fluid, with potential applications for biomarker discovery.

2024-07-31 | PXD053450 | Pride

OXPHOS bioenergetic compensation does not explain disease penetrance in Leber hereditary optic neuropathy

Project description:Leber hereditary optic neuropathy (LHON) is one of the most common primary mitochondrial diseases. It is caused by point mutations in mitochondrial DNA (mtDNA) genes and in some cases, it can result in irreversible vision loss, primarily in young men. It is currently unknown why LHON mutations affect only some carriers and whether bioenergetic compensation enables asymptomatic carriers to overcome mitochondrial impairment and preserve cellular function. Here, we conducted bioenergetic metabolic assays and RNA sequencing to address this question using male-only, age-matched, m.11778G>A lymphoblasts and primary fibroblasts from both carriers and affected individuals. Our work indicates that OXPHOS bioenergetic compensation in LHON peripheral cells does not explain disease phenotype. We show that complex I impairment is similar in carrier and affected cells, despite a transcriptional downregulation of metabolic pathways including glycolysis in affected cells relative to carriers detected by RNA sequencing. Although we did not detect OXPHOS bioenergetic compensation in carrier cells, our results indicate that affected cells suffer a growth impairment under metabolic challenge compared to carrier cells, which were unaffected by metabolic challenge. If recapitulated in retinal ganglion cells, decreased susceptibility to metabolic challenge in carriers may help preserve metabolic homeostasis in the face of the mitochondrial complex I bioenergetic defect.

2020-02-08 | GSE144914 | GEO

Offset mass carrier proteome improves quantification of multiplexed single cell proteomics

Project description:Multiplexed single cell proteomics by mass spectrometry (scpMS) approaches currently offer the highest throughput as measured by cells analyzed per day. These methods employ isobaric labels and typically a carrier proteome - a sample added at 20-500x the single cell level that improves peptide sampling and identification. Peptides from the carrier and single cell proteomes exist within the same precursor isotopic cluster and are co-isolated for identification and quantification. This represents a challenge as high levels of carrier proteome limit the sampling of peptide ions from single cell samples and can potentially lead to decreased accuracy of quantitative measurements. Here, we address this limitation by introducing a triggered by offset mass acquisition method for scpMS (toma-scpMS) that utilizes a carrier proteome labeled with non-isobaric tags that have the same chemical composition but different mass as the labels used for quantitative multiplexing. Within toma-scpMS the carrier proteome and single cell proteome are separated at the precursor level, enabling separate isolation, fragmentation, and quantitation of the single cell samples. To enable this workflow we implemented a custom data acquisition scheme within inSeqAPI, an instrument application programming interface program, that performed real-time identification of carrier proteome peptides and subsequent triggering of offset single cell quantification scans. We demonstrate that toma-scpMS is more robust to high-levels of carrier proteome and offers superior quantitative accuracy as compared to traditional multiplexed scpMS approaches when similar carrier proteome levels are employed.

2024-08-16 | MSV000095629 | MassIVE

Single Cell Proteomics and the Carrier Proteome Effect

Project description:Single cell proteomics by mass spectrometry (SCoPE-MS) is a recently introduced method that utilizes isobaric labels to quantify multiplexed single cell proteomes. While this technique has generated great excitement, the technologies underlying SCoPE-MS - isobaric labels and mass spectrometry - comprise technical limitations with the potential to unfavorably impact data quality and biological interpretation. These limitations are due to the carrier proteome, a sample added at 25-500x single cell proteomes to enable peptide identifications. Here, we perform SCoPE-MS experiments with increasing amounts of carrier proteome and evaluate quantitative accuracy as it relates to mass analyzer dynamic range, multiplexing level, and number of ions sampled. We demonstrate that an increase in carrier proteome level requires a concomitant increase in the number of ions sampled to maintain quantitative accuracy – we term this the carrier proteome effect. Based on our findings, we provide guidance on experimental design, data collection, and data analysis to limit the impact of the carrier proteome effect within SCoPE-MS measurements.

2020-12-16 | PXD016573 | Pride

An intelligent Boosting to Amplify Signal with Isobaric Labeling (iBASIL) strategy for precise quantitative single-cell proteomics

Project description:Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells, In particular the concept of using boosting/carrier channels in isobaric labeling to increase the sensitivity in MS detection (e.g., our recent boosting to amplify signal with isobaric labeling (BASIL) approach1) has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have systematically evaluated and optimized the BASIL approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of single cells. This improved, intelligent BASIL (iBASIL) approach enables comprehensive and quantitative single-cell proteomics analysis by carefully controlling the boosting-to-sample ratio and optimizing MS automatic gain control and ion injection time settings. Using standard LC-MS platforms, iBASIL enabled precise quantification of >1,000 proteins from 0.1 ng of peptide (equivalent to a single cell), or ~800 proteins from single FACS-isolated MCF10A cells. Moreover, coupling iBASIL to nanoscale fractionation enabled quantification of>1,438 proteins while readily separating 3 different cell lines using single-cell-equivalent peptides. We believe iBASIL has broad utility for comprehensive and precise quantitative single-cell proteomics for systems biology and biomedical research, as well as for comprehensive proteomic analysis of size-limited clinical specimens that cannot be readily accessed by regular proteomics platforms.

2020-03-05 | PXD016057 | JPOST Repository

An improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) strategy for precise quantitative single-cell proteomics

Project description:Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in isobaric labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with isobaric labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g., in general <100x) and optimizing MS automatic gain control (AGC) and ion injection time settings in MS/MS analysis (e.g., 5E5 and 300 ms, respectively, which is significantly higher than that used in typical bulk analysis). Using standard LC-MS platforms, iBASIL enabled identification of ~2,500 proteins and precise quantification of ~1,500 proteins in the analysis of 104 FACS-isolated single cells, with the resulting protein profiles robustly clustering the cells from three different acute myeloid leukemia cell lines. This study highlights the importance of carefully evaluating and optimizing the boosting and MS data acquisition conditions for achieving robust, comprehensive proteomic analysis of single cells.

2020-03-05 | PXD017626 | JPOST Repository

Single amino acid variant

Project description:We performed deep proteomic profiling down to as few as 9 Panc-1 cells using sample fractionation, TMT multiplexing and carrier/reference strategy. Off line fractionation of the TMT-labeled sample pooled with TMT-labeled carrier Panc-1 whole cell proteome was achieved using alkaline reversed phase spin columns. The fractionation in conjunction with carrier/reference proteome allowed us to detect 47,414 unique peptides derived from 6261 proteins which provided a sufficient coverage to search for single amino acid variants (SAAVs)related to cancer.

2020-05-26 | PXD011471 | Pride

A deeper look at carrier proteome effects for single-cell proteomics

Project description:We probe the carrier proteome effects in single cell proteomics with mixed species TMTpro-labeled samples. We demonstrate that carrier proteomes, while increasing overall identifications, dictate which proteins are identified. We show that quantitative precision and signal intensity are limited at high carrier levels, hindering the recognition of regulated proteins. Guidelines for optimized mass spectrometry acquisition parameters and best practices for fold-change or protein copy number-based comparisons are provided.

2022-02-24 | PXD027742 | Pride

brca1_brca2_controls_Vs_universal_RNA

Project description:Comparison of the expression profiles of normal cells from 10 BRCA1 and 10 BRCA2 mutation carriers to 10 non-carrier control samples after radiation induced DNA damage Keywords: dose response

2006-02-01 | GSE3382 | GEO

Expression data from tumor and normal epithelium derived from BRCA1-mutation carriers

Project description:Microarrays were used to determine relative global gene expression changes in WT and BRCA1-mutation carrier breast epithelium as well as tumors created from WT and BRCA1-mutation carrier breast epithelial cells. Total RNA was isolated from freshly dissociated mammary epithelilal cells obtained from disease-free prophylactic masectomy tissues of 4 different BRCA1-mutation carriers or 4 different reduction mammoplasty tissues from non-mutation carriers. Total RNA was also isolated from fresh tumor tissues derived from in vivo transformed human mammary epithelial cells created from cells obtained from WT or BRCA1-muation carrires. Dissociated mammary epithelial cells were transduced with lentiviruses encoding mutant p53, mutant ras, mutant PI3K and cyclin D1 and injected into in humanized glands. There were four tumor tissues isolated for each genetic background.

2011-02-04 | E-GEOD-25835 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

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