Dataset Information

Isobaric Matching between Runs and Novel PSM-Level Normalization in MaxQuant Strongly Improve Reporter Ion-Based Quantification.

ABSTRACT: Isobaric labeling has the promise of combining high sample multiplexing with precise quantification. However, normalization issues and the missing value problem of complete n-plexes hamper quantification across more than one n-plex. Here, we introduce two novel algorithms implemented in MaxQuant that substantially improve the data analysis with multiple n-plexes. First, isobaric matching between runs makes use of the three-dimensional MS1 features to transfer identifications from identified to unidentified MS/MS spectra between liquid chromatography-mass spectrometry runs in order to utilize reporter ion intensities in unidentified spectra for quantification. On typical datasets, we observe a significant gain in MS/MS spectra that can be used for quantification. Second, we introduce a novel PSM-level normalization, applicable to data with and without the common reference channel. It is a weighted median-based method, in which the weights reflect the number of ions that were used for fragmentation. On a typical dataset, we observe complete removal of batch effects and dominance of the biological sample grouping after normalization. Furthermore, we provide many novel processing and normalization options in Perseus, the companion software for the downstream analysis of quantitative proteomics results. All novel tools and algorithms are available with the regular MaxQuant and Perseus releases, which are downloadable at http://maxquant.org.

SUBMITTER: Yu SH

PROVIDER: S-EPMC7586393 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Isobaric Matching between Runs and Novel PSM-Level Normalization in MaxQuant Strongly Improve Reporter Ion-Based Quantification.

Yu Sung-Huan SH Kyriakidou Pelagia P Cox Jürgen J

Journal of proteome research 20200916 10

Isobaric labeling has the promise of combining high sample multiplexing with precise quantification. However, normalization issues and the missing value problem of complete n-plexes hamper quantification across more than one n-plex. Here, we introduce two novel algorithms implemented in MaxQuant that substantially improve the data analysis with multiple n-plexes. First, isobaric matching between runs makes use of the three-dimensional MS1 features to transfer identifications ...[more]

PMID: 32892627

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Project description:We report the development of novel reagents for cell-level protein quantification, referred to as Caltech isobaric tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT). Click chemistry, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), is applied to generate a gas-phase cleavable linker suitable for the formation of reporter ions. Upon collisional activation, the 1,2,3-triazole ring constructed by CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the nucleophilic displacement occurs in competition with formation of b- and y-type backbone fragment ions regardless of the amino acid side chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded precursor, allyl-d(5)-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping with common peptide MS/MS fragments as well as the low mass cutoff problems inherent in ion trap mass spectrometers. The applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems.

Dataset Information

Isobaric Matching between Runs and Novel PSM-Level Normalization in MaxQuant Strongly Improve Reporter Ion-Based Quantification.

Publications

Isobaric Matching between Runs and Novel PSM-Level Normalization in MaxQuant Strongly Improve Reporter Ion-Based Quantification.

Similar Datasets

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