The Experts below are selected from a list of 2091 Experts worldwide ranked by ideXlab platform
Chia-feng Tsai - One of the best experts on this subject based on the ideXlab platform.
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An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics.
Molecular & cellular proteomics : MCP, 2020Co-Authors: Chia-feng Tsai, William B. Chrisler, Rui Zhao, Ronald J. Moore, Ljiljana Paša-tolić, Sarah M Williams, Kendall Schultz, Karin D. Rodland, Richard D. Smith, Tujin ShiAbstract:Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in Isobaric Labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with Isobaric Labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general
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Abstract 2856: An intelligent boosting to amplify signal with Isobaric Labeling (iBASIL) strategy for precise quantitative single-cell proteomics analysis
Clinical Research (Excluding Clinical Trials), 2020Co-Authors: Chia-feng Tsai, Rui Zhao, Ronald J. Moore, Ljiljana Paša-tolić, Kendall Schultz, Karin D. Rodland, Richard D. Smith, Sarah Francis Watson Williams, Paul D. Piehowski, Tujin ShiAbstract:Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in Isobaric Labeling to increase the sensitivity in MS detection (e.g., our recent boosting to amplify signal with Isobaric Labeling (BASIL) approach) has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have systematically evaluated and optimized the BASIL approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of single cells. This improved, intelligent BASIL (iBASIL) approach enables comprehensive and quantitative single-cell proteomics analysis by carefully controlling the boosting-to-sample ratio and optimizing MS automatic gain control and ion injection time settings. Using standard LC-MS platforms, iBASIL enabled precise quantification of 800-1,200 proteins from single FACS-isolated cells (depending on the cell type). Moreover, coupling iBASIL to nanoscale fractionation enabled identification of >3,000 proteins and reliable quantification of >2,000 proteins while readily separating single cells from 3 different acute myeloid leukemia cell lines. We believe iBASIL has broad utility for comprehensive and precise quantitative single-cell proteomics for systems biology and biomedical research, as well as for comprehensive proteomic analysis of size-limited clinical specimens that cannot be readily accessed by regular proteomics platforms. It also has the potential to be adapted for phosphoproteome analysis of as few as 100 cells. Citation Format: Chia-Feng Tsai, Rui Zhao, Ronald Moore, Sarah Williams, Kendall Schultz, Paul Piehowski, Ljiljana Pasa-Tolic, Karin Rodland, Richard Smith, Tujin Shi, Ying Zhu, Ying Zhu, Tao Liu. An intelligent boosting to amplify signal with Isobaric Labeling (iBASIL) strategy for precise quantitative single-cell proteomics analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2856.
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an improved boosting to amplify signal with Isobaric Labeling ibasil strategy for precise quantitative single cell proteomics
Molecular & Cellular Proteomics, 2020Co-Authors: Chia-feng Tsai, William B. Chrisler, Rui Zhao, Ronald J. Moore, Sarah M Williams, Kendall Schultz, Karin D. Rodland, Richard D. Smith, Ljiljana Pasatolic, Tujin ShiAbstract:Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in Isobaric Labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with Isobaric Labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general <100×) and optimizing MS automatic gain control (AGC) and ion injection time settings in MS/MS analysis (e.g. 5E5 and 300 ms, respectively, which is significantly higher than that used in typical bulk analysis). By coupling with a nanodroplet-based single cell preparation (nanoPOTS) platform, iBASIL enabled identification of ∼2500 proteins and precise quantification of ∼1500 proteins in the analysis of 104 FACS-isolated single cells, with the resulting protein profiles robustly clustering the cells from three different acute myeloid leukemia cell lines. This study highlights the importance of carefully evaluating and optimizing the boosting ratios and MS data acquisition conditions for achieving robust, comprehensive proteomic analysis of single cells.
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High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labeling in a Nanodroplet Sample Preparation Platform
Analytical chemistry, 2019Co-Authors: Maowei Dou, Geremy Clair, Chia-feng Tsai, William B. Chrisler, Ryan L. Sontag, Rui Zhao, Ronald J. Moore, Tao Liu, Ljiljana Paša-tolićAbstract: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...
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Boosting to Amplify Signal with Isobaric Labeling (BASIL) Strategy for Comprehensive Quantitative Phosphoproteomic Characterization of Small Populations of Cells.
Analytical chemistry, 2019Co-Authors: Chia-feng Tsai, Tujin Shi, Paul D. Piehowski, Ercument Dirice, Adam C. Swensen, Jing Chen, Marina A. Gritsenko, Rosalie K. Chu, Richard D. SmithAbstract:Comprehensive phosphoproteomic analysis of small populations of cells remains a daunting task due primarily to the insufficient MS signal intensity from low concentrations of enriched phosphopeptid...
Tujin Shi - One of the best experts on this subject based on the ideXlab platform.
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An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics.
Molecular & cellular proteomics : MCP, 2020Co-Authors: Chia-feng Tsai, William B. Chrisler, Rui Zhao, Ronald J. Moore, Ljiljana Paša-tolić, Sarah M Williams, Kendall Schultz, Karin D. Rodland, Richard D. Smith, Tujin ShiAbstract:Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in Isobaric Labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with Isobaric Labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general
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Abstract 2856: An intelligent boosting to amplify signal with Isobaric Labeling (iBASIL) strategy for precise quantitative single-cell proteomics analysis
Clinical Research (Excluding Clinical Trials), 2020Co-Authors: Chia-feng Tsai, Rui Zhao, Ronald J. Moore, Ljiljana Paša-tolić, Kendall Schultz, Karin D. Rodland, Richard D. Smith, Sarah Francis Watson Williams, Paul D. Piehowski, Tujin ShiAbstract:Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in Isobaric Labeling to increase the sensitivity in MS detection (e.g., our recent boosting to amplify signal with Isobaric Labeling (BASIL) approach) has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have systematically evaluated and optimized the BASIL approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of single cells. This improved, intelligent BASIL (iBASIL) approach enables comprehensive and quantitative single-cell proteomics analysis by carefully controlling the boosting-to-sample ratio and optimizing MS automatic gain control and ion injection time settings. Using standard LC-MS platforms, iBASIL enabled precise quantification of 800-1,200 proteins from single FACS-isolated cells (depending on the cell type). Moreover, coupling iBASIL to nanoscale fractionation enabled identification of >3,000 proteins and reliable quantification of >2,000 proteins while readily separating single cells from 3 different acute myeloid leukemia cell lines. We believe iBASIL has broad utility for comprehensive and precise quantitative single-cell proteomics for systems biology and biomedical research, as well as for comprehensive proteomic analysis of size-limited clinical specimens that cannot be readily accessed by regular proteomics platforms. It also has the potential to be adapted for phosphoproteome analysis of as few as 100 cells. Citation Format: Chia-Feng Tsai, Rui Zhao, Ronald Moore, Sarah Williams, Kendall Schultz, Paul Piehowski, Ljiljana Pasa-Tolic, Karin Rodland, Richard Smith, Tujin Shi, Ying Zhu, Ying Zhu, Tao Liu. An intelligent boosting to amplify signal with Isobaric Labeling (iBASIL) strategy for precise quantitative single-cell proteomics analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2856.
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an improved boosting to amplify signal with Isobaric Labeling ibasil strategy for precise quantitative single cell proteomics
Molecular & Cellular Proteomics, 2020Co-Authors: Chia-feng Tsai, William B. Chrisler, Rui Zhao, Ronald J. Moore, Sarah M Williams, Kendall Schultz, Karin D. Rodland, Richard D. Smith, Ljiljana Pasatolic, Tujin ShiAbstract:Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in Isobaric Labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with Isobaric Labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general <100×) and optimizing MS automatic gain control (AGC) and ion injection time settings in MS/MS analysis (e.g. 5E5 and 300 ms, respectively, which is significantly higher than that used in typical bulk analysis). By coupling with a nanodroplet-based single cell preparation (nanoPOTS) platform, iBASIL enabled identification of ∼2500 proteins and precise quantification of ∼1500 proteins in the analysis of 104 FACS-isolated single cells, with the resulting protein profiles robustly clustering the cells from three different acute myeloid leukemia cell lines. This study highlights the importance of carefully evaluating and optimizing the boosting ratios and MS data acquisition conditions for achieving robust, comprehensive proteomic analysis of single cells.
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Boosting to Amplify Signal with Isobaric Labeling (BASIL) Strategy for Comprehensive Quantitative Phosphoproteomic Characterization of Small Populations of Cells.
Analytical chemistry, 2019Co-Authors: Chia-feng Tsai, Tujin Shi, Paul D. Piehowski, Ercument Dirice, Adam C. Swensen, Jing Chen, Marina A. Gritsenko, Rosalie K. Chu, Richard D. SmithAbstract:Comprehensive phosphoproteomic analysis of small populations of cells remains a daunting task due primarily to the insufficient MS signal intensity from low concentrations of enriched phosphopeptid...
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Boosting to Amplify Signal with Isobaric Labeling (BASIL) Strategy for Comprehensive Quantitative Phosphoproteomic Characterization of Small Populations of Cells
2019Co-Authors: Chia-feng Tsai, Tujin Shi, Paul D. Piehowski, Ercument Dirice, Adam C. Swensen, Jing Chen, Marina A. Gritsenko, Rosalie K. Chu, Richard D. SmithAbstract:Comprehensive phosphoproteomic analysis of small populations of cells remains a daunting task due primarily to the insufficient MS signal intensity from low concentrations of enriched phosphopeptides. Isobaric Labeling has a unique multiplexing feature where the “total” peptide signal from all channels (or samples) triggers MS/MS fragmentation for peptide identification, while the reporter ions provide quantitative information. In light of this feature, we tested the concept of using a “boosting” sample (e.g., a biological sample mimicking the study samples but available in a much larger quantity) in multiplexed analysis to enable sensitive and comprehensive quantitative phosphoproteomic measurements with
Richard D. Smith - One of the best experts on this subject based on the ideXlab platform.
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An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics.
Molecular & cellular proteomics : MCP, 2020Co-Authors: Chia-feng Tsai, William B. Chrisler, Rui Zhao, Ronald J. Moore, Ljiljana Paša-tolić, Sarah M Williams, Kendall Schultz, Karin D. Rodland, Richard D. Smith, Tujin ShiAbstract:Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in Isobaric Labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with Isobaric Labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general
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Abstract 2856: An intelligent boosting to amplify signal with Isobaric Labeling (iBASIL) strategy for precise quantitative single-cell proteomics analysis
Clinical Research (Excluding Clinical Trials), 2020Co-Authors: Chia-feng Tsai, Rui Zhao, Ronald J. Moore, Ljiljana Paša-tolić, Kendall Schultz, Karin D. Rodland, Richard D. Smith, Sarah Francis Watson Williams, Paul D. Piehowski, Tujin ShiAbstract:Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in Isobaric Labeling to increase the sensitivity in MS detection (e.g., our recent boosting to amplify signal with Isobaric Labeling (BASIL) approach) has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have systematically evaluated and optimized the BASIL approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of single cells. This improved, intelligent BASIL (iBASIL) approach enables comprehensive and quantitative single-cell proteomics analysis by carefully controlling the boosting-to-sample ratio and optimizing MS automatic gain control and ion injection time settings. Using standard LC-MS platforms, iBASIL enabled precise quantification of 800-1,200 proteins from single FACS-isolated cells (depending on the cell type). Moreover, coupling iBASIL to nanoscale fractionation enabled identification of >3,000 proteins and reliable quantification of >2,000 proteins while readily separating single cells from 3 different acute myeloid leukemia cell lines. We believe iBASIL has broad utility for comprehensive and precise quantitative single-cell proteomics for systems biology and biomedical research, as well as for comprehensive proteomic analysis of size-limited clinical specimens that cannot be readily accessed by regular proteomics platforms. It also has the potential to be adapted for phosphoproteome analysis of as few as 100 cells. Citation Format: Chia-Feng Tsai, Rui Zhao, Ronald Moore, Sarah Williams, Kendall Schultz, Paul Piehowski, Ljiljana Pasa-Tolic, Karin Rodland, Richard Smith, Tujin Shi, Ying Zhu, Ying Zhu, Tao Liu. An intelligent boosting to amplify signal with Isobaric Labeling (iBASIL) strategy for precise quantitative single-cell proteomics analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2856.
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an improved boosting to amplify signal with Isobaric Labeling ibasil strategy for precise quantitative single cell proteomics
Molecular & Cellular Proteomics, 2020Co-Authors: Chia-feng Tsai, William B. Chrisler, Rui Zhao, Ronald J. Moore, Sarah M Williams, Kendall Schultz, Karin D. Rodland, Richard D. Smith, Ljiljana Pasatolic, Tujin ShiAbstract:Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in Isobaric Labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with Isobaric Labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general <100×) and optimizing MS automatic gain control (AGC) and ion injection time settings in MS/MS analysis (e.g. 5E5 and 300 ms, respectively, which is significantly higher than that used in typical bulk analysis). By coupling with a nanodroplet-based single cell preparation (nanoPOTS) platform, iBASIL enabled identification of ∼2500 proteins and precise quantification of ∼1500 proteins in the analysis of 104 FACS-isolated single cells, with the resulting protein profiles robustly clustering the cells from three different acute myeloid leukemia cell lines. This study highlights the importance of carefully evaluating and optimizing the boosting ratios and MS data acquisition conditions for achieving robust, comprehensive proteomic analysis of single cells.
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Boosting to Amplify Signal with Isobaric Labeling (BASIL) Strategy for Comprehensive Quantitative Phosphoproteomic Characterization of Small Populations of Cells.
Analytical chemistry, 2019Co-Authors: Chia-feng Tsai, Tujin Shi, Paul D. Piehowski, Ercument Dirice, Adam C. Swensen, Jing Chen, Marina A. Gritsenko, Rosalie K. Chu, Richard D. SmithAbstract:Comprehensive phosphoproteomic analysis of small populations of cells remains a daunting task due primarily to the insufficient MS signal intensity from low concentrations of enriched phosphopeptid...
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Boosting to Amplify Signal with Isobaric Labeling (BASIL) Strategy for Comprehensive Quantitative Phosphoproteomic Characterization of Small Populations of Cells
2019Co-Authors: Chia-feng Tsai, Tujin Shi, Paul D. Piehowski, Ercument Dirice, Adam C. Swensen, Jing Chen, Marina A. Gritsenko, Rosalie K. Chu, Richard D. SmithAbstract:Comprehensive phosphoproteomic analysis of small populations of cells remains a daunting task due primarily to the insufficient MS signal intensity from low concentrations of enriched phosphopeptides. Isobaric Labeling has a unique multiplexing feature where the “total” peptide signal from all channels (or samples) triggers MS/MS fragmentation for peptide identification, while the reporter ions provide quantitative information. In light of this feature, we tested the concept of using a “boosting” sample (e.g., a biological sample mimicking the study samples but available in a much larger quantity) in multiplexed analysis to enable sensitive and comprehensive quantitative phosphoproteomic measurements with
Steven P. Gygi - One of the best experts on this subject based on the ideXlab platform.
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a triple knockout Isobaric Labeling quality control platform with an integrated online database search
Journal of the American Society for Mass Spectrometry, 2020Co-Authors: Jeremy P Gygi, Steven P. Gygi, Ramin Rad, Jose Navarreteperea, Simon Younesi, Joao A. PauloAbstract:Sample multiplexing using Isobaric tagging is a powerful strategy for proteome-wide protein quantification. One major caveat of Isobaric tagging is ratio compression that results from the isolation...
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TKO6: A Peptide Standard To Assess Interference for Unit-Resolved Isobaric Labeling Platforms.
Journal of proteome research, 2018Co-Authors: Joao A. Paulo, José Navarrete-perea, Sanjukta Guha Thakurta, Steven P. GygiAbstract:Protein abundance profiling using Isobaric Labeling is a well-established quantitative mass spectrometry technique. However, ratio distortion resulting from coisolated and cofragmented ions, commonly referred to as interference, remains a drawback of this strategy. Tribrid mass spectrometers, such as the Orbitrap Fusion and the Orbitrap Fusion Lumos with a triple mass analyzer configuration, facilitate methods (namely, SPS-MS3) that can help alleviate interference. However, few standards are available to measure interference and thereby aid in method development. Here we introduce the TKO6 standard that assesses ion interference and is designed specifically for data acquired at low (unit) mass resolution. We use TKO6 to compare interference in MS2- versus MS3-based quantitation methods, data acquisition methods of different lengths, and ion-trap-based tandem mass tag reporter ion analysis (IT-MS3) with conventional Orbitrap-based analysis (OT-MS3). We show that the TKO6 standard is a valuable tool for assessing quantification accuracy in Isobaric-tag-based analyses.
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Improved Method for Determining Absolute Phosphorylation Stoichiometry Using Bayesian Statistics and Isobaric Labeling
Journal of proteome research, 2017Co-Authors: Matthew L. M. Lim, Jonathon J. O’brien, Joao A. Paulo, Steven P. GygiAbstract:Phosphorylation stoichiometry, or occupancy, is one element of phosphoproteomics that can add useful biological context (Gerber et al. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 6940–5). We previously developed a method to assess phosphorylation stoichiometry on a proteome-wide scale (Wu et al. Nat. Methods 2011, 8, 677–83). The stoichiometry calculation relies on identifying and measuring the levels of each nonphosphorylated counterpart peptide with and without phosphatase treatment. The method, however, is problematic in that low stoichiometry phosphopeptides can return negative stoichiometry values if measurement error is larger than the percent stoichiometry. Here, we have improved the stoichiometry method through the use of Isobaric Labeling with 10-plex TMT reagents. In this way, five phosphatase treated and five untreated samples are compared simultaneously so that each stoichiometry is represented by five ratio measurements with no missing values. We applied the method to determine basal stoichiom...
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A Triple Knockout (TKO) Proteomics Standard for Diagnosing Ion Interference in Isobaric Labeling Experiments
Journal of The American Society for Mass Spectrometry, 2016Co-Authors: Joao A. Paulo, Jeremy D. O’connell, Steven P. GygiAbstract:Isobaric Labeling is a powerful strategy for quantitative mass spectrometry-based proteomic investigations. A complication of such analyses has been the co-isolation of multiple analytes of similar mass-to-charge resulting in the distortion of relative protein abundance measurements across samples. When properly implemented, synchronous precursor selection and triple-stage mass spectrometry (SPS-MS3) can reduce the occurrence of this phenomenon, referred to as ion interference. However, no diagnostic tool is available currently to rapidly and accurately assess ion interference. To address this need, we developed a multiplexed tandem mass tag (TMT)-based standard, termed the triple knockout (TKO). This standard is comprised of three yeast proteomes in triplicate, each from a strain deficient in a highly abundant protein (Met6, Pfk2, or Ura2). The relative abundance patterns of these proteins, which can be inferred from dozens of peptide measurements can demonstrate ion interference in peptide quantification. We expect no signal in channels where the protein is knocked out, permitting maximum sensitivity for measurements of ion interference against a null background. Here, we emphasize the need to investigate further ion interference-generated ratio distortion and promote the TKO standard as a tool to investigate such issues. Graphical Abstract ᅟ
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Generation of Multiple Reporter Ions from a Single Isobaric Reagent Increases Multiplexing Capacity for Quantitative Proteomics
Analytical chemistry, 2015Co-Authors: Craig R. Braun, Steven P. Gygi, Gregory H. Bird, Martin Wühr, Brian K. Erickson, Ramin Rad, Loren D. Walensky, Wilhelm HaasAbstract:Isobaric Labeling strategies for mass spectrometry-based proteomics enable multiplexed simultaneous quantification of samples and therefore substantially increase the sample throughput in proteomics. However, despite these benefits, current limits to multiplexing capacity are prohibitive for large sample sizes and impose limitations on experimental design. Here, we introduce a novel mechanism for increasing the multiplexing density of Isobaric reagents. We present Combinatorial Isobaric Mass Tags (CMTs), an Isobaric Labeling architecture with the unique ability to generate multiple series of reporter ions simultaneously. We demonstrate that utilization of multiple reporter ion series improves multiplexing capacity of CMT with respect to a commercially available Isobaric Labeling reagent with preserved quantitative accuracy and depth of coverage in complex mixtures. We provide a blueprint for the realization of 16-plex reagents with 1 Da spacing between reporter ions and up to 28-plex at 6 mDa spacing usin...
Ljiljana Paša-tolić - One of the best experts on this subject based on the ideXlab platform.
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An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics.
Molecular & cellular proteomics : MCP, 2020Co-Authors: Chia-feng Tsai, William B. Chrisler, Rui Zhao, Ronald J. Moore, Ljiljana Paša-tolić, Sarah M Williams, Kendall Schultz, Karin D. Rodland, Richard D. Smith, Tujin ShiAbstract:Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in Isobaric Labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with Isobaric Labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general
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Abstract 2856: An intelligent boosting to amplify signal with Isobaric Labeling (iBASIL) strategy for precise quantitative single-cell proteomics analysis
Clinical Research (Excluding Clinical Trials), 2020Co-Authors: Chia-feng Tsai, Rui Zhao, Ronald J. Moore, Ljiljana Paša-tolić, Kendall Schultz, Karin D. Rodland, Richard D. Smith, Sarah Francis Watson Williams, Paul D. Piehowski, Tujin ShiAbstract:Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in Isobaric Labeling to increase the sensitivity in MS detection (e.g., our recent boosting to amplify signal with Isobaric Labeling (BASIL) approach) has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have systematically evaluated and optimized the BASIL approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of single cells. This improved, intelligent BASIL (iBASIL) approach enables comprehensive and quantitative single-cell proteomics analysis by carefully controlling the boosting-to-sample ratio and optimizing MS automatic gain control and ion injection time settings. Using standard LC-MS platforms, iBASIL enabled precise quantification of 800-1,200 proteins from single FACS-isolated cells (depending on the cell type). Moreover, coupling iBASIL to nanoscale fractionation enabled identification of >3,000 proteins and reliable quantification of >2,000 proteins while readily separating single cells from 3 different acute myeloid leukemia cell lines. We believe iBASIL has broad utility for comprehensive and precise quantitative single-cell proteomics for systems biology and biomedical research, as well as for comprehensive proteomic analysis of size-limited clinical specimens that cannot be readily accessed by regular proteomics platforms. It also has the potential to be adapted for phosphoproteome analysis of as few as 100 cells. Citation Format: Chia-Feng Tsai, Rui Zhao, Ronald Moore, Sarah Williams, Kendall Schultz, Paul Piehowski, Ljiljana Pasa-Tolic, Karin Rodland, Richard Smith, Tujin Shi, Ying Zhu, Ying Zhu, Tao Liu. An intelligent boosting to amplify signal with Isobaric Labeling (iBASIL) strategy for precise quantitative single-cell proteomics analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2856.
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High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labeling in a Nanodroplet Sample Preparation Platform
Analytical chemistry, 2019Co-Authors: Maowei Dou, Geremy Clair, Chia-feng Tsai, William B. Chrisler, Ryan L. Sontag, Rui Zhao, Ronald J. Moore, Tao Liu, Ljiljana Paša-tolićAbstract: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...
