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Title: Human Gut Microbiota from Autism Spectrum Disorder Promote Behavioral Symptoms in Mice

Abstract

Large-scale phosphoproteomics with coverage of over 10,000 sites of phosphorylation have now been routinely achieved with advanced mass spectrometry (MS)-based workflows. However, accurate targeted MS-based quantification of phosphorylation dynamics, an important direction for gaining quantitative understanding of signaling pathways or networks, has been much less investigated. Herein, we report an assessment of the targeted workflow in the context of signal transduction pathways, using the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) pathway as our model. 43 phosphopeptides from the EGFR–MAPK pathway were selected for the study. The recovery and sensitivity of a workflow consisted of two commonly used enrichment methods, immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO2), combined with selected reaction monitoring (SRM)-MS, were evaluated. The recovery of phosphopeptides by IMAC and TiO2 enrichment was quantified to be 38 ± 5% and 58 ± 20%, respectively, based on internal standards. Moreover, both enrichment methods provided comparable sensitivity from 1-100 g starting peptides. Robust quantification was consistently achieved for most targeted phosphopeptides when starting with 25-100 g peptides. However, the numbers of quantified targets significantly dropped when peptide samples were in the 1-25g range. Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK pathway in Hs578T cellsmore » following 3 ng/mL EGF treatment. The kinetics of phosphorylation clearly revealed early and late phases of phosphorylation, even for very low abundance proteins. These results demonstrate the feasibility of robust targeted quantification of phosphorylation dynamics for specific pathways, even starting with relatively small amounts of protein.« less

Authors:
 [1];  [2];  [3];  [4];  [5]; ORCiD logo [6]; ORCiD logo [6]; ORCiD logo [6];  [7];  [8]; ORCiD logo [6];  [6]; ORCiD logo [6];  [9];  [5];  [5];  [10];  [6];  [7]; ORCiD logo [6] more »;  [5];  [4];  [3];  [5] « less
  1. California Institute of Technology, Pasadena, CA
  2. Califormia Institution of Technology
  3. Arizona State University
  4. University of California
  5. California Institute of Technology
  6. BATTELLE (PACIFIC NW LAB)
  7. University of California, San Diego
  8. University of Southern California
  9. University of Washington
  10. UNIVERSITY OF WASHINGTON
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1544608
Report Number(s):
PNNL-SA-131563
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Cell
Additional Journal Information:
Journal Volume: 177; Journal Issue: 6
Country of Publication:
United States
Language:
English

Citation Formats

Sharon, Gil, Cruz, Nikki J., Kang, Dae Wook, Gandal, Michael, Wang, Bo, Kim, Young-Mo, Zink, Erika M., Casey, Cameron P., Taylor, Bryn C., Lane, Christianne J., Bramer, Lisa M., Isern, Nancy G., Hoyt, David W., Noecker, Cecilia, Sweredoski, Michael J., Moradian, Annie, Borenstein, Elhanan A., Jansson, Janet K., Knight, Rob, Metz, Thomas O., Lois, Carlos, Geschwind, Daniel H., Krajmalnik-Brown, Rosa, and Mazmanian, Sarkis K. Human Gut Microbiota from Autism Spectrum Disorder Promote Behavioral Symptoms in Mice. United States: N. p., 2019. Web. doi:10.1016/j.cell.2019.05.004.
Sharon, Gil, Cruz, Nikki J., Kang, Dae Wook, Gandal, Michael, Wang, Bo, Kim, Young-Mo, Zink, Erika M., Casey, Cameron P., Taylor, Bryn C., Lane, Christianne J., Bramer, Lisa M., Isern, Nancy G., Hoyt, David W., Noecker, Cecilia, Sweredoski, Michael J., Moradian, Annie, Borenstein, Elhanan A., Jansson, Janet K., Knight, Rob, Metz, Thomas O., Lois, Carlos, Geschwind, Daniel H., Krajmalnik-Brown, Rosa, & Mazmanian, Sarkis K. Human Gut Microbiota from Autism Spectrum Disorder Promote Behavioral Symptoms in Mice. United States. doi:10.1016/j.cell.2019.05.004.
Sharon, Gil, Cruz, Nikki J., Kang, Dae Wook, Gandal, Michael, Wang, Bo, Kim, Young-Mo, Zink, Erika M., Casey, Cameron P., Taylor, Bryn C., Lane, Christianne J., Bramer, Lisa M., Isern, Nancy G., Hoyt, David W., Noecker, Cecilia, Sweredoski, Michael J., Moradian, Annie, Borenstein, Elhanan A., Jansson, Janet K., Knight, Rob, Metz, Thomas O., Lois, Carlos, Geschwind, Daniel H., Krajmalnik-Brown, Rosa, and Mazmanian, Sarkis K. Thu . "Human Gut Microbiota from Autism Spectrum Disorder Promote Behavioral Symptoms in Mice". United States. doi:10.1016/j.cell.2019.05.004.
@article{osti_1544608,
title = {Human Gut Microbiota from Autism Spectrum Disorder Promote Behavioral Symptoms in Mice},
author = {Sharon, Gil and Cruz, Nikki J. and Kang, Dae Wook and Gandal, Michael and Wang, Bo and Kim, Young-Mo and Zink, Erika M. and Casey, Cameron P. and Taylor, Bryn C. and Lane, Christianne J. and Bramer, Lisa M. and Isern, Nancy G. and Hoyt, David W. and Noecker, Cecilia and Sweredoski, Michael J. and Moradian, Annie and Borenstein, Elhanan A. and Jansson, Janet K. and Knight, Rob and Metz, Thomas O. and Lois, Carlos and Geschwind, Daniel H. and Krajmalnik-Brown, Rosa and Mazmanian, Sarkis K.},
abstractNote = {Large-scale phosphoproteomics with coverage of over 10,000 sites of phosphorylation have now been routinely achieved with advanced mass spectrometry (MS)-based workflows. However, accurate targeted MS-based quantification of phosphorylation dynamics, an important direction for gaining quantitative understanding of signaling pathways or networks, has been much less investigated. Herein, we report an assessment of the targeted workflow in the context of signal transduction pathways, using the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) pathway as our model. 43 phosphopeptides from the EGFR–MAPK pathway were selected for the study. The recovery and sensitivity of a workflow consisted of two commonly used enrichment methods, immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO2), combined with selected reaction monitoring (SRM)-MS, were evaluated. The recovery of phosphopeptides by IMAC and TiO2 enrichment was quantified to be 38 ± 5% and 58 ± 20%, respectively, based on internal standards. Moreover, both enrichment methods provided comparable sensitivity from 1-100 g starting peptides. Robust quantification was consistently achieved for most targeted phosphopeptides when starting with 25-100 g peptides. However, the numbers of quantified targets significantly dropped when peptide samples were in the 1-25g range. Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK pathway in Hs578T cells following 3 ng/mL EGF treatment. The kinetics of phosphorylation clearly revealed early and late phases of phosphorylation, even for very low abundance proteins. These results demonstrate the feasibility of robust targeted quantification of phosphorylation dynamics for specific pathways, even starting with relatively small amounts of protein.},
doi = {10.1016/j.cell.2019.05.004},
journal = {Cell},
number = 6,
volume = 177,
place = {United States},
year = {2019},
month = {5}
}