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A Versatile Isobaric Tag Enables Proteome Quantification in Data University of Groningen A Versatile Isobaric Tag Enables Proteome Quantification in Data-Dependent and Data- Independent Acquisition Modes Tian, Xiaobo; de Vries, Marcel P; Permentier, Hjalmar P; Bischoff, Rainer Published in: Analytical Chemistry DOI: 10.1021/acs.analchem.0c03858 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2020 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Tian, X., de Vries, M. P., Permentier, H. P., & Bischoff, R. (2020). A Versatile Isobaric Tag Enables Proteome Quantification in Data-Dependent and Data-Independent Acquisition Modes. Analytical Chemistry, 92(24), 16149-16157. https://doi.org/10.1021/acs.analchem.0c03858 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 24-09-2021 This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes. pubs.acs.org/ac Article A Versatile Isobaric Tag Enables Proteome Quantification in Data- Dependent and Data-Independent Acquisition Modes Xiaobo Tian, Marcel P. de Vries, Hjalmar P. Permentier, and Rainer Bischoff* Cite This: Anal. Chem. 2020, 92, 16149−16157 Read Online ACCESS Metrics & More Article Recommendations *sı Supporting Information ABSTRACT: 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, presumably because current approaches lead to more complex MS2 spectra, severe ratio distortion, or to a reduction in quantification accuracy and precision. Herein, we present a versatile acetyl-alanine-glycine (Ac-AG) tag that conceals quanti- tative 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 identical, the Ac-AG tag is compatible with both DDA and DIA. By isolating the monoisotopic peak of the precursor ion in DDA, intensities of the peptide-coupled reporter ions represent the relative ratios between constituent samples, whereas in DIA, the ratio can be inferred after deconvoluting the peptide-coupled reporter ion isotopes. 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 this complex proteomics background, 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. ■ INTRODUCTION well as the throughput of quantitative proteomics in the DDA 2,3 Advancements in liquid chromatography and mass spectrom- mode. Isobaric strategies can be further classified into three Downloaded via 84.241.196.236 on February 8, 2021 at 15:11:40 (UTC). etry over the last decades have led to the maturation and categories: (1) reporter-ion-based quantification (e.g., widespread application of a variety of mass-spectrometry-based 2,4,5 6,7 proteome quantification approaches. Label-free quantification TMT and iTRAQ ); (2) peptide fragment ion based quantification, including isobaric peptide termini labeling is hampered by the effect of changing ionization efficiencies − (IPTL)-based approaches;8 10 (3) peptide-coupled reporter- See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. − between LC MS runs and the stochastic nature of precursor fi 3,11 12 ion selection in the data-dependent acquisition (DDA) mode. ion-based quanti cation, like TMTc and EASI tag. Multiplex stable isotope labeling circumvents these issues and Among these, the reporter-ion-based methods (TMT and has been widely applied to label samples derived from different iTRAQ) are most widely used because of their high experimental conditions. Analysis of multiple samples labeled multiplexing capacity and well-developed data-processing with distinct isotopes in a single LC−MS run leads to more software. However, it has become increasingly apparent that fi 1 reporter-ion-based quantification methods suffer from ratio reliable quanti cation. As reviewed by Arul and Robinson, 3,11,13,14 isotope-label-based quantification methods can be classified distortion caused by peptide cofragmentation in which fi the produced reporter ions from different peptides are into MS1 quanti cation, which is usually based on isotopic fl labeling resulting in different precursor masses of the same indistinguishable resulting in intensities that no longer re ect peptide derived from different samples, and MS2 quantifica- tion, which generally relies on isobaric labeling, resulting in Received: September 11, 2020 identical precursor masses while quantifying based on fragment Accepted: November 19, 2020 ions. Isobaric labeling strategies quantify constituent proteins Published: December 1, 2020 of mixed samples by distinct fragment ions upon MS/MS without increasing the complexity of the MS1 spectra. These methods significantly improve the accuracy and precision as © 2020 American Chemical Society https://dx.doi.org/10.1021/acs.analchem.0c03858 16149 Anal. Chem. 2020, 92, 16149−16157 Analytical Chemistry pubs.acs.org/ac Article Figure 1. Schematic view of the Ac-AG approach. (A) Isobaric labeling steps; (B) LC−MS/MS of a mixture of triplex-labeled samples with DDA or DIA approaches; (C) functional design of the triplex-labeled Ac-AG-PNP tag (13C isotope locations are marked with asterisks). the ratios between the peptides and thus lead to inaccurate recently introduced EASI tag12 utilizes a sulfoxide-based tag protein ratios. Various solutions to alleviate the ratio distortion to increase the formation efficiency of peptide-coupled problem have been described, such as narrowing the width of reporter ions and applied an asymmetric isolation window to the precursor ion isolation window,3,11 gas-phase purifica- specifically isolate the monoisotopic peak to further simplify tion,14 and MultiNotch MS3,13,15 but these methods are not the isotope envelope of peptide-coupled reporter ions. widely used as they rely on specific, state-of-the-art Besides the commonly applied DDA strategy, data- instrumentation, use scan routines that are difficult to optimize independent acquisition (DIA) approaches are becoming and control, or require MS3 scanning leading to a longer cycle increasingly popular because they avoid the stochastic nature time. Peptide fragment ion based quantification, an approach of DDA with its bias toward fragmenting higher intensity that is not affected by cofragmentation,8 has thus attracted peptide ions and the resulting missing value problem between attention: a growing number of IPTL-based approaches have runs.19 In contrast to DDA, isolation of precursors for been reported, such as Triplex-QITL,16 SMD-IPTL,10 and m- fragmentation in DIA does not rely on the peak intensities pIDL.17 In these methods, every labeling channel has a set of in MS1; instead, all precursors present within the isolation distinct fragment ions resulting in more precise quantification. window will be fragmented and analyzed together. As a result, However, the increased complexity18 of MS2 spectra requires the problem of missing values of specific peptides between custom-made data analysis tools, which has restricted wide- injections in a sample batch is largely avoided and spread use. In contrast to the two methods mentioned above, reproducibility is improved. On the other hand, MS2 spectra quantifying proteins based on a precursor-specific, peptide- are more complex as the fragment ions derived from multiple, coupled reporter ion is not affected by peptide cofragmenta- coisolated precursor ions are present in the same spectrum, tion nor does it increase the complexity of the MS2 spectra. which makes analyzing DIA data more challenging than DDA This approach was first proposed by Wühr et al.11 in 2012, but data. It is worth noting that, fueled by improvements in the two limitations leading tounsatisfactoryquantification scan rate and resolution in mass spectrometry and the − accuracy and precision were pointed out: (1) the modest development of sophisticated data processing algorithms,19 21 efficiency of forming peptide-coupled reporter ions and (2) the the analysis of DIA data has been improved over the last complicated isotope envelope of the peptide-coupled reporter decade, especially with respect to the reproducibility from run ions. The improved TMTc+ approach3 applied a narrow to run.20 precursor ion isolation window of 0.4 Th rather than the However, DIA-based methods are not suitable for most generally used 2 Th to select the monoisotopic peak, which current multiplexing approaches, which makes them inferior in significantly improved the quantification accuracy. The terms of throughput to DDA-based methods, such as those 16150 https://dx.doi.org/10.1021/acs.analchem.0c03858 Anal. 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