Dataset Information

A Versatile Isobaric Tag Enables Proteome Quantification in Data Dependent and Data Independent Acquisition Mode

ABSTRACT: Quantifying proteins based on peptide-coupled reporter-ions is a leading multiplexed quantitative strategy in proteomics that significantly alleviates the problem of ratio distortion caused by peptide co-fragmentation, as commonly observed in other reporter-ion based approaches, such as TMT and iTRAQ. Fueled by improvements in mass spectrometry and data processing, data-independent acquisition (DIA) is an attractive alternative to data dependent acquisition (DDA) due to its better reproducibility. While multiplexed labeling is widely used in DDA, it is rarely used in DIA, presumably because current approaches lead to more complex MS2 spectra or to a reduction in quantification accuracy and precision. Herein, we present a versatile acetyl-alanine-glycine (Ac-AG) tag which conceals quantitative information in isobarically labeled peptides and reveals it upon tandem MS in the form of peptide-coupled reporter-ions. Since the peptide-coupled reporter-ion is precursor-specific while fragment ions of the peptide backbone originating from different labeling channels are same, the Ac-AG tag is compatible with both the widely adopted DDA as well as with the DIA mode. By isolating the monoisotopic peak of the precursor ion in DDA, intensities of the peptide-coupled reporter-ions simply represent the relative ratios between constituent samples. While in DIA, the ratio can be inferred after deconvoluting peptide-coupled reporter-ions. The proteome quantification capability of the Ac-AG tag was demonstrated by triplex labeling of a yeast proteome spiked with bovine serum albumin (BSA) over a 10-fold dynamic range. Within a complex proteomics background, the BSA spiked at 1:5:10 ratios was detected at ratios of 1.00 : 4.87 : 10.13 in DDA and 1.16 : 5.20 : 9.64 in DIA.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Xiaobo Tian

LAB HEAD: Rainer Bischoff

PROVIDER: PXD021187 | Pride | 2021-09-09

REPOSITORIES: Pride

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Publications

A Versatile Isobaric Tag Enables Proteome Quantification in Data-Dependent and Data-Independent Acquisition Modes.

Tian Xiaobo X de Vries Marcel P MP Permentier Hjalmar P HP Bischoff Rainer R

Analytical chemistry 20201130 24

Quantifying proteins based on peptide-coupled reporter ions is a multiplexed quantitative strategy in proteomics that alleviates the problem of ratio distortion caused by peptide cofragmentation, as commonly observed in other reporter-ion-based approaches, such as TMT and iTRAQ. Data-independent acquisition (DIA) is an attractive alternative to data-dependent acquisition (DDA) due to its better reproducibility. While multiplexed labeling is widely used in DDA, it is rarely used in DIA, presumabl ...[more]

PMID: 33256395

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Project description:Data independent acquisition-mass spectrometry (DIA-MS) coupled with liquid chromatography is a promising approach for rapid, automatic sampling of MS/MS data in untargeted metabolomics. However, wide isolation windows in DIA-MS generate MS/MS spectra containing a mixed population of fragment ions together with their precursor ions. This precursor-fragment ion map in a comprehensive MS/MS spectral library is crucial for relative quantification of fragment ions uniquely representative of each precursor ion. However, existing reference libraries are not sufficient for this purpose since the fragmentation patterns of small molecules can vary in different instrument setups. Here we developed a bioinformatics workflow called MetaboDIA to build customized MS/MS spectral libraries using a user's own data dependent acquisition (DDA) data and to perform MS/MS-based quantification with DIA data, thus complementing conventional MS1-based quantification. MetaboDIA also allows users to build a spectral library directly from DIA data in studies of a large sample size. Using a marine algae data set, we show that quantification of fragment ions extracted with a customized MS/MS library can provide as reliable quantitative data as the direct quantification of precursor ions based on MS1 data. To test its applicability in complex samples, we applied MetaboDIA to a clinical serum metabolomics data set, where we built a DDA-based spectral library containing consensus spectra for 1829 compounds. We performed fragment ion quantification using DIA data using this library, yielding sensitive differential expression analysis. </br></br> Serum metabolome of 40 age-related macular degeneration patients and 20 control samples was analyzed using untargeted mass spectrometry. We used data dependent acquisition data to build a MS/MS spectral assay library for more than 1,000 compounds and performed targeted extraction of MS2 ion chromatograms from data independent acquisition analysis.

Dataset Information

A Versatile Isobaric Tag Enables Proteome Quantification in Data Dependent and Data Independent Acquisition Mode

Publications

A Versatile Isobaric Tag Enables Proteome Quantification in Data-Dependent and Data-Independent Acquisition Modes.

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