TAG-TMTpro, a Hyperplexing Quantitative Approach for High-Throughput Proteomic Studies

Wu, Zhiyong; Shen, Yi; Zhang, Xumin

doi:10.1021/acs.analchem.2c02099

Cited by 7 publications

(21 citation statements)

References 23 publications

(48 reference statements)

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“…Nonetheless, as shown in Figures S14 and S15, fewer proteins and peptides are identified in combinatorial analysis, possibly due to the loss of identification in low-abundant peaks with lower intensities (Figure S16). The consistent observations were also reported in our previous study …”

Section: Results and Discussionsupporting

confidence: 93%

“…The consistent observations were also reported in our previous study. 12 We next examined the quantitative performance of each labeling in the combinatorial analysis (Figures 5, S17, and S18). For IBT16, the experimental ratios were observed with nearly the same accuracy and higher precision compared to that in the independent analysis (Figure 3D), indicating that the unique channels could guarantee the quantitative performance even in more complex samples.…”

Section: Combination Of the Three Isobaric Reagents Couldmentioning

confidence: 99%

“…Very recently, we reported a versatile TAG-TMTpro approach by introduction of different amino acid tags before TMTpro labeling, avoiding the reaction selectivity problem and tripling the quantitative capacity of TMTpro 18-plex reagent to 54-plex. 12 Another type of hyperplexing approach is realized by employing different isobaric reagents in one experiment. Recently, 27-plex or 29-plex quantification has been achieved by combining TMT 11-plex and TMTpro 16-plex/18-plex reagents.…”

mentioning

confidence: 99%

“…These approaches mainly rely on the selective amine reaction at different pH conditions, which are often not ideal as expected. Very recently, we reported a versatile TAG-TMTpro approach by introduction of different amino acid tags before TMTpro labeling, avoiding the reaction selectivity problem and tripling the quantitative capacity of TMTpro 18-plex reagent to 54-plex …”

mentioning

confidence: 99%

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Hyperplexing Approaches for up to 45-Plex Quantitative Proteomic Analysis

Xiang

Huang

et al. 2023

Anal. Chem.

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Isobaric labeling has emerged as an indispensable quantitative proteomic approach for its unprecedented multiplexing capacity in a single analysis. Currently, different hyperplexing approaches have been developed to meet the demand for the increasing sample size in large-scale cohort analysis. In this report, we present a tribrid hyperplexing approach by the combinatorial use of three types of isobaric reagents, a novel isobaric tag 16-plex (IBT16) reagent and the widely used tandem mass tag (TMT; TMT11) and TMTpro (TMT18) reagents. After the determination of labeling efficiency and the optimization of testing conditions, we systematically evaluated the identification and quantification performance of the three labeling reagents in both independent and combinatorial manners using the mixtures of E. coli and HeLa peptides with different ratios. Our results reveal that the three reagents are quite similar in all testing aspects despite some differences, and the combination use of the three reagents could expand the multiplexing capacity to up to 45-plex. Furthermore, we conclude the advantages of IBT16 in the combination use and the preferred combinations for different practical applications. Data are available via ProteomeXchange with identifier PXD037498.

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Section: Results and Discussionsupporting

confidence: 93%

Section: Combination Of the Three Isobaric Reagents Couldmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Hyperplexing Approaches for up to 45-Plex Quantitative Proteomic Analysis

Xiang

Huang

et al. 2023

Anal. Chem.

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“…Quantitative proteomics aims to measure relative and absolute proteome changes across various experimental conditions to expand understanding of disease, mechanistic pathways, and biological processes . Enhanced multiplexing technologies such as N -hydroxysuccinimide-ester tandem labeling in one pot (NETLOP), 29-plex tandem mass tag (TMT), TAG-TMTpro, combined precursor isotopic labeling and isobaric tagging (cPILOT), and others − enable high-throughput proteomics experiments by combining more than one multiplexing technology into a single experiment. For example, NETLOP combines isotopic and isobaric versions of TMT tags to achieve up to 48-plex labeling, TAG-TMTpro combines amino acid labeling with TMT isobaric tags to achieve up to 45-plex labeling, and cPILOT combines isotopic dimethylation and TMT or dimethyl leucine (DiLeu) isobaric tags to achieve up to 42-plex labeling. − ,,, cPILOT can encompass multiple conditions such as tissue types, time points, and genotypes and has been broadly applied to study aging, Alzheimer’s disease, ,, and post-translational modifications .…”

Section: Introductionmentioning

confidence: 99%

Evaluating cPILOT Data toward Quality Control Implementation

Bowser

Patterson

Robinson

2023

J. Am. Soc. Mass Spectrom.

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Multiplexing enables the monitoring of hundreds to thousands of proteins in quantitative proteomics analyses and increases sample throughput. In most mass-spectrometry-based proteomics workflows, multiplexing is achieved by labeling biological samples with heavy isotopes via precursor isotopic labeling or isobaric tagging. Enhanced multiplexing strategies, such as combined precursor isotopic labeling and isobaric tagging (cPILOT), combine multiple technologies to afford an even higher sample throughput. Critical to enhanced multiplexing analyses is ensuring that analytical performance is optimal and that missingness of sample channels is minimized. Automation of sample preparation steps and use of quality control (QC) metrics can be incorporated into multiplexing analyses and reduce the likelihood of missing information, thus maximizing the amount of usable quantitative data. Here, we implemented QC metrics previously developed in our laboratory to evaluate a 36-plex cPILOT experiment that encompassed 144 mouse samples of various tissue types, time points, genotypes, and biological replicates. The evaluation focuses on the use of a sample pool generated from all samples in the experiment to monitor the daily instrument performance and to provide a means for data normalization across sample batches. Our results show that tracking QC metrics enabled the quantification of ∼7000 proteins in each sample batch, of which ∼70% had minimal missing values across up to 36 sample channels. Implementation of QC metrics for future cPILOT studies as well as other enhanced multiplexing strategies will help yield high-quality data sets.

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102-Plex Approach for Accurate and Multiplexed Proteome Quantification

Wu,

Huang,

Huang

et al. 2024

Anal. Chem.

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Hyperplexing approaches have been aimed to meet the demand for large-scale proteomic analyses. Currently, the analysis capacity has expanded to up to 54 samples within a single experiment by utilizing different isotopic and isobaric reagent combinations. In this report, we propose a super multiplexed approach to enable the analysis of up to 102 samples in a single experiment, by the combination of our recently developed TAG-TMTpro and TAG-IBT16 labeling. We systematically investigated the identification and quantification performance of the 102-plex approach using the mixtures of E. coli and HeLa peptides. Our results revealed that all labeling series demonstrated accurate and reliable quantification performance. The combination of TAG-IBT16 and TAG-TMTpro approaches expands the multiplexing capacity to 102 plexes, providing a more multiplexed quantification method for even larger-scale proteomic analysis. Data are available via ProteomeXchange with the identifier PXD042398.

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TAG-TMTpro, a Hyperplexing Quantitative Approach for High-Throughput Proteomic Studies

Cited by 7 publications

References 23 publications

Hyperplexing Approaches for up to 45-Plex Quantitative Proteomic Analysis

Hyperplexing Approaches for up to 45-Plex Quantitative Proteomic Analysis

Evaluating cPILOT Data toward Quality Control Implementation

102-Plex Approach for Accurate and Multiplexed Proteome Quantification

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