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Mayer, Ralf; Reh, Sabine; Scholz, Joachim; Maeße, Jens; Berdelmann, Kathrin; Angermüller, Johannes; Stojanov, Krassimir; Thompson, Christiane; Schäfer, Alfred; Braungart, Anna

doi:10.30965/9783657775989

Cited by 16 publications

(6 citation statements)

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“…Tandem mass tag (TMT) isobaric labeling is an approach developed for multiplexed identification and quantification of proteins from multiple different samples in a single LC-MS analysis/run. 27 Individual samples are first labeled with discrete TMT reagents (also referred to as TMT channels) and then combined into a multiplexed set prior to analysis by high-resolution LC-MS. The proteins in each sample can be identified and quantified based on the TMT reporter ions.…”

Section: Introductionmentioning

confidence: 99%

High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labeling in a Nanodroplet Sample Preparation Platform

et al. 2019

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Effective extension of mass spectrometry-based proteomics to single cells remains challenging. Herein we combined microfluidic nanodroplet technology with tandem mass tag (TMT) isobaric labeling to significantly improve analysis throughput and proteome coverage for single mammalian cells. Isobaric labeling facilitated multiplex analysis of single cell-sized protein quantities to a depth of ~1,600 proteins with median CV of 10.9% and correlation coefficient of 0.98. To demonstrate in-depth high throughput single cell analysis, the platform was applied to measure protein expression in 72 single cells from three murine cell populations (epithelial, immune, and endothelial cells) in <2 days instrument time with over 2,300 proteins identified. Principal component analysis grouped the single cells into three distinct populations based on protein expression with each population characterized by well-known cell-type specific markers. Our platform enables high throughput and unbiased characterization of single cell heterogeneity at the proteome level.

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Section: Introductionmentioning

confidence: 99%

High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labeling in a Nanodroplet Sample Preparation Platform

et al. 2019

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“…Quantitative proteome characterization with isobaric reagents (e.g., TMT) is an established and compelling approach for comprehensive biological surveys of protein and posttranslational modification dynamics. [1][2][3][4][5] Significant advancements in these multiplexed workflows include the use of a dedicated quantitative spectrum (MS 3 ), multifragment isolation (multinotch/SPS), and continuously evolving hardware (Orbitrap Fusion family), which have enabled broad applications of these workflows and routine use in discoverydriven experimentation. The standardized LC-MS workflow that incorporates these advancements leverages the widely used data-dependent methodologies to select, fragment, and sequence features of interest.…”

Section: Introductionmentioning

confidence: 99%

“…The IAPI greatly simplifies the communication between a client application and the instrument and provides a robust and approachable method for more intelligent data acquisition. RTS-MS 3 is not restricted by computational hardware or software as it operates on the external data system and implements a comprehensive peptide search in an average of 5 ms per MS 2 . We benchmarked the performance of the RTS-MS 3 approach through a challenging twoproteome model of interference that permits the direct measurement of interference.…”

Section: Introductionmentioning

confidence: 99%

Active Instrument Engagement Combined with a Real-Time Database Search for Improved Performance of Sample Multiplexing Workflows

et al. 2019

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Quantitative proteomics employing isobaric reagents has been established as a powerful tool for biological discovery. Current workflows often utilize a dedicated quantitative spectrum to improve quantitative accuracy and precision. A consequence of this approach is a dramatic reduction in the spectral acquisition rate, which necessitates the use of additional instrument time to achieve comprehensive proteomic depth. This work assesses the performance and benefits of online and real-time spectral identification in quantitative multiplexed workflows. A Real-Time Search (RTS) algorithm was implemented to identify fragment spectra within milliseconds as they are acquired using a probabilistic score and to trigger quantitative spectra only upon confident peptide identification. The RTS-MS 3 was benchmarked against standard workflows using a complex twoproteome model of interference and a targeted 10-plex comparison of kinase abundance profiles. Applying the RTS-MS 3 method provided the comprehensive characterization of a 10-plex proteome in 50% less acquisition time. These data indicate that the RTS-MS 3 approach provides dramatic performance improvements for quantitative multiplexed experiments.

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“…[1][2][3] Isobaric tags, such as iTRAQ (isobaric tags for relative and absolute quantification) and TMT (tandem mass tags) impart a single mass shift at the MS 1 level but yield discrete reporter ions in MS 2 spectra for relative quantification. [4][5][6] The multi-Dalton spacing of mass-difference labels restricts quantitative capacity due to the increased spectral complexity resulting from the multiple isotopic clusters. Isobaric tags provide up to 12-plex analysis, but the reduced quantitative accuracy from ratio distortion caused by precursor coisolation confines its utility.…”

Section: Introductionmentioning

confidence: 99%

High-Resolution Enabled 5-plex Mass Defect-Based N,N-Dimethyl Leucine Tags for Quantitative Proteomics

Zhong

Frost

2019

Anal. Chem.

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Mass defect-based labeling strategy provides high accuracy as an MS 1 -centric quantification method, avoiding the ratio compression that affects isobaric label-based reporter ion quantification. We have developed cost-effective 5-plex mass defect N,N-dimethyl leucine (mdDiLeu) tags for quantification of various biological samples with increased multiplexing at a given resolving power afforded by the addition of mass difference isotopologues. The combination of mass difference and mass defect produces two labeled peak clusters separated by 5 Da in MS 1 spectra that are detected as five isotopic peaks at high resolution with mass difference of 15, 17, and 18 mDa per tag. Synthesis of each of the 5-plex mdDiLeu tags is accomplished by a single straightforward reaction step, making it accessible to any lab. To demonstrate 5-plex mdDiLeu for quantitative proteomics, we perform proof-of-principle experiments of mdDiLeu-labeled Saccharomyces cerevisiae lysate digest on an Orbitrap Fusion Lumos mass spectrometer.

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Leistung

Cited by 16 publications

References 0 publications

High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labeling in a Nanodroplet Sample Preparation Platform

High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labeling in a Nanodroplet Sample Preparation Platform

Active Instrument Engagement Combined with a Real-Time Database Search for Improved Performance of Sample Multiplexing Workflows

High-Resolution Enabled 5-plex Mass Defect-Based N,N-Dimethyl Leucine Tags for Quantitative Proteomics

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