Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Evaluation of advanced precursor determination for tandem mass tag (TMT)-based quantitative proteomics across instrument platforms

ABSTRACT: Myers SA, Klaeger S, Satpathy S, Viner R, Choi J, Rogers J, Clauser K, Udeshi ND, and Carr SA. 2018, J Prot Res. TMT-based quantitation is a strong modality for quantitative proteomics as samples can be multiplexed, creating large-scale data sets with high precision and minimal missing values. However, co-isolation/co-fragmentation of near isobaric, co-eluting precursor peptide analytes has been well documented to show ratio compression, compromising the accuracy of peptide/protein quantitation. Advanced peak determination (APD) is a new peak picking algorithm that shows improved identification of peak detection in survey scans (MS1) to increase the number of precursors selected for unimolecular dissociation (MS2). To increase the number of these features selected for MS2 APD purposefully selects multiple peptide precursors of very similar m/z that often derive from different proteins - a major source of ratio compression in TMT quantification. Here, we evaluate the effects of various data acquisition parameters combined with APD on ratio compression. We find that data acquisition with APD enabled results in more co-isolated precursors, more mixed spectra, and in turn, fewer peptide spectral matches, especially at standard on-column loads. We conclude that APD should not be utilized for isobaric tagging, MS2-based experiments.

INSTRUMENT(S): Orbitrap Fusion Lumos, QExactive HFX

ORGANISM(S): Saccharomyces Cerevisiae (ncbitaxon:4932)

SUBMITTER: Steven A. Carr

PROVIDER: MSV000083005 | MassIVE | Sun Oct 07 09:41:00 BST 2018

REPOSITORIES: MassIVE

ACCESS DATA

Json Xml

Similar Datasets

Evaluation of a Dual Isolation Width Acquisition (DIWA) method for isobaric labelling ratio decompression

Project description:Stable isotope labelling of peptides using isobaric reagents such as iTRAQ and TMT enables the multiplexed analysis of proteomes with deep quantitative coverage. Isobaric tagging demonstrates high precision but imperfect accuracy due to ratio underestimation caused by co-fragmentation of ions with mass-to-charge ratios within the isolation window of the targeted precursors. Prompted by empirical observations of isobaric-labelled peptide MS2 spectra, we argue that although a very narrow isolation window will result in severe loss of backbone fragment ions, rendering the spectra unsuitable for peptide identification, the reporter ion signals will remain intense enough to generate quantitative information for a significant portion of the spectra. Based on this assumption we have designed a Dual Isolation Width Acquisition (DIWA) method, in which each precursor is first fragmented with HCD using a standard isolation width for peptide identification and preliminary quantification followed by a concomitant MS2 HCD fragmentation using a much narrower isolation width for the acquisition of quantification-only spectra with reduced interference. We leverage the quantitative values obtained by the “narrow” scans to build linear regression models and apply these to decompress the fold-changes measured at the “standard” scans. Here, we evaluate the DIWA method using a two species TMT-6plex model and discuss the potential of this approach.

2019-01-07 | PXD010571 | Pride

Extensive Peptide Fractionation and y1 Ion-based Interference Detection Enable Accurate Quantification by Isobaric Labeling and Mass Spectrometry

Project description:Isobaric labeling quantification by mass spectrometry (MS) has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from co-isolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection and post-MS computational interference correction. We pooled tandem mass tag (TMT) labeled E. coli peptides at 1 : 3 : 10 ratios, and added in ~20-fold more rat peptides as background, followed by the analysis of two dimensional liquid chromatography (LC)-MS/MS. The quantitative interference was systematic investigated and the interference in MS2 scans were estimated by the intensity of contaminated y1 product ions. An algorithm was thus developed to correct reporter ion ratios of tryptic peptides.

2022-02-28 | PXD005797 | Pride

EASI-tag enables accurate multiplexed and interference-free MS2-based proteome quantification

Project description:We developed EASI-tag (Easily Abstractable Sulfoxide-based Isobaric tag), a new generation of amine-derivatizing and sulfoxide-containing isobaric labelling reagents, which dissociate at low collision energy and generate peptide-coupled, interference-free reporter ions with high yield. Efficient isolation of 12C precursors and quantification at the MS2 level enable accurate determination of quantitative differences between multiplexed samples. EASI-tag makes isobaric labeling applicable to any bottom up proteomics application and to benchtop mass spectrometers.

2018-06-21 | PXD007165 | Pride

IsobaricQuant enables cross-platform quantification, visualization, and filtering of isobarically-labeled peptides

Project description:In mass spectrometry (MS)-based quantitative proteomics, labeling with isobaric mass tags such as iTRAQ and TMT can substantially improve sample throughput and reduce peptide missing values. Nonetheless, the quantification of labeled peptides tends to suffer from reduced quantitative accuracy due to the co-isolation of co-eluting precursors of similar mass-to-charge. Acquisition approaches such as MS3 level quantification or ion mobility separation address this problem, yet are difficult to audit and limited to expensive instrumentation. Here we introduce IsobaricQuant, an open-source software tool for the quantification, visualization, and filtering of peptides labeled with isobaric mass tags, with specific focus on precursor interference. IsobaricQuant is compatible with MS2- and MS3 level acquisition strategies, has a viewer that allows assessing interference, and provides several scores to aid the filtering of scans with compression. We demonstrate that IsobaricQuant quantifications are accurate by comparing it with commonly used software. We further show that its QC scores can successfully filter scans with reduced quantitative accuracy at MS2- and MS3 level, removing inaccurate peptide quantifications and decreasing protein CVs. Finally, we apply IsobaricQuant to a PISA dataset and show that QC scores improve the sensitivity of the identification of protein targets of a kinase inhibitor. IsobaricQuant is available at https://github.com/Villen-Lab/isobaricquant.

2022-07-11 | PXD034534 | Pride

HYpro16: A Two-Proteome Mixture to Assess Interference in Isobaric Tag-Based Sample Multiplexing Experiments

Project description:Isobaric tagging is a powerful strategy for global proteome profiling. A caveat of isobaric tag-based quantification is “interference” which may be caused by co-eluting peptides that are co-isolated, co-fragmented, and co-analyzed, thereby confounding quantitative accuracy. Here, we present a two-proteome standard that challenges the mass spectrometer to measure a range of protein abundance ratios in a background of potential interference. The HYpro16 standard consists of TMTpro-labeled human peptides at a 1:1 ratio across all channels into which we spike TMTpro-labeled peptides in triplicate at 20:1, 10:1, 4:1, and 2:1 ratios. We showcase the HYpro16 standard by 1) altering the MS2 isolation window width and 2) using different data acquisition methods (hrMS2, SPS-MS3, RTS-MS3). Our data illustrate that wider isolation widths moderately increase TMT signal, the benefits of which are offset by decreased ratio accuracy. We also show that using real-time database searching (RTS)-MS3 resulted in the most accurate ratios. Additionally, the number of quantified yeast proteins using RTS-MS3 approaches that of hrMS2 when using a yeast-specific database for real-time searching. In short, this quality control standard allows for the assessment of multiple quantitative measurements within a single run which can be compared across instruments to benchmark and track performance.

2021-08-16 | PXD020815 | Pride

TKO6: A Peptide Standard to Assess Interference for Unit-Resolved Isobaric Labeling Platforms

Project description:Protein abundance profiling using isobaric labeling is a well-established quantitative mass spectrometry technique. However, ratio distortion resulting from co-isolated and co-fragmented ions - commonly referred to as interference - remains a caveat of this technique. Tribrid mass spectrometers, such as the Orbitrap Fusion and the Orbitrap Fusion Lumos with a triple mass analyzer configuration, facilitate methods (namely SPS-MS3) that can help alleviate interference. Yet, few standards are available to measure interference. Here we introduce the TKO6 standard that assesses ion interference and is designed specifically for data acquired at low (unit) mass resolution. We use TKO6 to compare the degree of interference in MS2 versus MS3-based quantitation methods, data acquisition methods of different lengths, and ion trap-based TMT reporter ion analysis (IT-MS3). We show that the TKO6 standard is a valuable tool for assessing data quality and is particularly useful for benchmarking ion trap-based SPS-MS3 analyses.

2019-03-07 | PXD011653 | Pride

iTRAQ LLC-PK1 CnI - Sum of peak intensities outperforms peak area integration in iTRAQ protein expression measurement by LC-MS/MS using a TripleTOF 5600+ platform

Project description:In the field of quantitative proteomics, the Isobaric Tags for Relative & Absolute Quanti-tation (iTRAQ) technology has demonstrated efficacy for proteome monitoring despite its lack of a consensus for data handling. In this study, after peptide and protein identification, we com-pared the widespread quantitation method based on the calculation of MS/MS reporter ion peaks areas ratios (Protein Pilot) to the alternative method based on the calculation of ratios of the sum of peak intensities (jTRAQx (Quant)) and we processed output data with the in-house Customi-zable iTRAQ Ratios Calculator (CiR-C) algorithm. Quantitation based on peak area ratios dis-played no significant linear correlation with Western blot quantitation. On the contrary, quantita-tion based on the sum of peak intensities displayed a significant linear association with Western blot quantitation (non-zero slope; Pearson correlation coefficient test, r = 0.2962, p = 0.0099 **) with an average bias of 0.08747 ± 0.5004 and 95% Limits of Agreement from - 0.8932 to 1.068. We proposed the Mascot-jTRAQx-CiR-C strategy as a simple yet powerful data processing ad-junct to the iTRAQ technology

2019-08-19 | PXD007891 | Pride

Extending the Separation Space with Trapped Ion Mobility Spectrometry Improves the Accuracy of Isobaric Tag-based Quantitation in Proteomic LC/MS/MS

Project description:Two-dimensional separation by nano-LC and trapped ion mobility spectrometry (TIMS) prior to Q-TOF tandem mass spectrometry significantly improves the accuracy of isobaric tag-based quantitation in proteome analysis without the need for additional measurement time for TIMS insertion. The obtained peak capacity of up to 3,300 per hour in LC/TIMS reduced the co-isolation of precursor ions at the quadrupole analyzer, resulting in more accurate ratios of reporter ions derived from isobaric tags in product ion spectra obtained at the TOF analyzer. We also found that TIMS with a narrow quadrupole isolation window could reduce the ratio compression effect at least as effectively as the synchronous precursor selection method using MS3 scans without compromising sensitivity or coverage. Our results suggest that the short-gradient LC/TIMS/Q/TOF system is an excellent platform for high-throughput proteomics studies.

2020-05-22 | PXD018650 | JPOST Repository

MS2-affinity purification coupled with RNA sequencing (MAPS) reveals RybB sRNA targetome

Project description:Despite the overwhelming information about sRNAs, one of the biggest challenges in the sRNA field is characterizing sRNA targetomes. Thus, we develop a novel method to identify RNAs that interact with a specific sRNA, regardless of the type of regulation (positive or negative) or targets (mRNA, tRNA, sRNA). This method is called MAPS: MS2 affinity purification coupled with RNA sequencing. As proof of principle, we identified RNAs bound to RybB, a well-characterized E. coli sRNA. Identification of RNAs co-purified with MS2-RybB in a rne131 ΔrybB strain. RybB (without MS2) was used as control

2015-04-16 | E-GEOD-66518 | biostudies-arrayexpress

MS2-affinity purification coupled with RNA sequencing (MAPS) reveals RyhB sRNA targetome

Project description:Despite the overwhelming information about sRNAs, one of the biggest challenges in the sRNA field is characterizing sRNA targetomes. Thus, we develop a novel method to identify RNAs that interact with a specific sRNA, regardless of the type of regulation (positive or negative) or targets (mRNA, tRNA, sRNA). This method is called MAPS: MS2 affinity purification coupled with RNA sequencing. As proof of principle, we identified RNAs bound to RyhB, a well-characterized E. coli sRNA. Identification of RNAs co-purified with MS2-RyhB in a rne131 ?ryhB strain. RyhB (without MS2) was used as control

2015-04-16 | E-GEOD-66519 | biostudies-arrayexpress

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