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

Winter, Sebastian Virreira; Meier, Florian; Wichmann, Christoph; Cox, Juergen; Mann, Matthias; Meissner, Felix

doi:10.1038/s41592-018-0037-8

Cited by 96 publications

(80 citation statements)

References 26 publications

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“…G) The EASI‐tag developed by Virreira Winter et al. similarly fragments comparatively easily . The “reporter” part of the EASI‐tag is a neutral loss.…”

Section: Multiplexed Proteomics With Isobaric Labelingmentioning

confidence: 99%

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A Review on Quantitative Multiplexed Proteomics

2019

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Over the last few decades, mass spectrometry‐based proteomics has become an increasingly powerful tool that is now able to routinely detect and quantify thousands of proteins. A major advance for global protein quantification was the introduction of isobaric tags, which, in a single experiment, enabled the global quantification of proteins across multiple samples. Herein, these methods are referred to as multiplexed proteomics. The principles, advantages, and drawbacks of various multiplexed proteomics techniques are discussed and compared with alternative approaches. We also discuss how the emerging combination of multiplexing with targeted proteomics might enable the reliable and high‐quality quantification of very low abundance proteins across multiple conditions. Lastly, we suggest that fusing multiplexed proteomics with data‐independent acquisition approaches might enable the comparison of hundreds of different samples without missing values, while maintaining the superb measurement precision and accuracy obtainable with isobaric tag quantification.

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“…G) The EASI‐tag developed by Virreira Winter et al. similarly fragments comparatively easily . The “reporter” part of the EASI‐tag is a neutral loss.…”

Section: Multiplexed Proteomics With Isobaric Labelingmentioning

confidence: 99%

“…Another sulfoxide‐based tag was recently developed by Virreira Winter et al., known as the easily abstractable sulfoxide‐based isobaric (EASI) tag (Scheme G) . The EASI‐tag also contains an asymmetric sulfoxide bond that is cleaved at relatively low energy.…”

Section: Multiplexed Proteomics With Isobaric Labelingmentioning

confidence: 99%

A Review on Quantitative Multiplexed Proteomics

2019

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“…It is likely that a newer generation of the ThermoFisher Tribrid instruments (such as the Orbitrap Eclipse) would offset this difference in number of proteins identified compared to MS2 quantitation. However, this lack of improvement, combined with the efficient clustering capabilities of MS2 level data, also suggests that the TMT co-isolation effect is not strong enough to negatively affect our readouts; nevertheless, alternative tagging technologies such as EASI-tags could be explored in future work to determine their compatibility with scMS (Winter et al, 2018). The suitability of TMT for this workflow is further supported by the fact that we can use the raw intensities as provided by Proteome Discoverer for our computational analyses, and are therefore not subject to normalization and correction effects that are sometimes opted to include when using TMT.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Single-Cell Proteomics as a Tool to Characterize Cellular Hierarchies

Schoof

Furtwängler

Üresin

et al. 2019

Preprint

101

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Key words: acute myeloid leukemia / computational biology / proteomics / single-cell approaches / tandem mass tags (TMT)Final character count: 47,782 AbstractIn recent years, cellular life science research has experienced a significant shift, moving away from conducting bulk cell interrogation towards single-cell analysis. It is only through single cell analysis that a complete understanding of cellular heterogeneity, and the interplay between various cell types that are fundamental to specific biological phenotypes, can be achieved. Single-cell assays at the protein level have been predominantly limited to targeted, antibody-based methods. However, here we present an experimental and computational pipeline, which establishes a comprehensive single-cell mass spectrometry-based proteomics workflow.By exploiting a leukemia culture system, containing functionally-defined leukemic stem cells, progenitors and terminally differentiated blasts, we demonstrate that our workflow is able to explore the cellular heterogeneity within this aberrant developmental hierarchy. We show our approach is capable to quantifying hundreds of proteins across hundreds of single cells using limited instrument time. Furthermore, we developed a computational pipeline (SCeptre), that effectively clusters the data and permits the extraction of cell-specific proteins and functional pathways. This proof-of-concept work lays the foundation for future global single-cell proteomics studies. Duployez et al, 2019;Ng et al, 2016), their accuracy has proven limitation when used as a proxy for protein levels (Vogel & Marcotte, 2012; Khan et al, 2013). Therefore, to gain a thorough understanding of what occurs in a cell at the protein level, on a global scale, MSbased approaches are the sole way to accomplish this. Being the cellular workhorses, there is much knowledge to be gained from mechanisms occurring at the protein level, either through enzyme activity, post-translational modifications or protein degradation/proteolysis; hence the great need for protein level approaches at the single-cell level.A few years ago, a novel type of flow cytometry was established; by combining traditional flow cytometry workflows with mass spectrometry, a new analysis method termed Mass Cytometry was developed, more commonly referred to as CyTOF (Newell et al, 2012;Bodenmiller et al, 2012). This allows the simultaneous readout of tens of markers simultaneously, allowing single-cell analysis of pre-defined sets of proteins or posttranslational modifications (PTMs). This method, however, relies heavily on previously

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“…We note that the CII issue has been reduced, though not eliminated, with newer generation Orbitrap MS instruments, where the improved source and quadrupole have enhanced the signal to noise ratio 22 . Furthermore new isobaric tagging methods have been developed which claim to be CII free 27 , however their multiplexing capability is currently limited to a 6-plex.…”

Section: Discussionmentioning

confidence: 99%

Optimising multi-batch TMT analysis to mitigate inflation of missing values, false positives and diminished inter batch accuracy

Brenes

Hukelmann

Bensaddek

et al. 2018

Preprint

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Multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent, TMT in particular. Here we used a large iPSC proteomic experiment with twenty-four 10-plex TMT batches to evaluate the effect of integrating multiple TMT batches within a single analysis. We reveal a significant inflation rate of missing protein and peptide values and show that precision decreases as multiple batches are integrated. Additionally, we explore the effect of false positives using Y chromosome specific peptides as an internal control to quantify the effect of co-isolation interference, as well as primary and secondary reporter ion interference.Based on the results we suggest solutions to mitigate these effects. We show using a reference line can increase precision by normalising the quantification across batches and we propose experimental designs that minimise the effect of cross population reporter ion interference.

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

Cited by 96 publications

References 26 publications

A Review on Quantitative Multiplexed Proteomics

A Review on Quantitative Multiplexed Proteomics

Quantitative Single-Cell Proteomics as a Tool to Characterize Cellular Hierarchies

Optimising multi-batch TMT analysis to mitigate inflation of missing values, false positives and diminished inter batch accuracy

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