Next Article in Journal
The Dark Side of the Moon: The Right Ventricle
Previous Article in Journal
Multiple Roles of Pitx2 in Cardiac Development and Disease
Article Menu

Export Article

Open AccessArticle
J. Cardiovasc. Dev. Dis. 2017, 4(4), 17; doi:10.3390/jcdd4040017

Strategy for Identification of Phosphorylation Levels of Low Abundance Proteins in Vivo for Which Antibodies Are not Available

1
Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Kanagawa 251-8555 Fujisawa, Japan
2
Cardiovascular and Metabolic Drug Discovery Unit, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Kanagawa 251-8555 Fujisawa, Japan
*
Author to whom correspondence should be addressed.
Received: 24 August 2017 / Revised: 22 September 2017 / Accepted: 30 September 2017 / Published: 8 October 2017
(This article belongs to the Special Issue Proteomics and Protein Post-Translational Modification)
View Full-Text   |   Download PDF [1605 KB, uploaded 11 October 2017]   |  

Abstract

Protein function is mainly modulated by dynamic reversible or irreversible post-translational modifications. Among them, the identification of protein phosphorylation sites and changes in phosphorylation levels in vivo are of considerable interest for a better understanding of the protein function. Thus, effective strategies for the quantitative determination of phosphorylation degrees for low abundant proteins, for which antibodies are not available, are required in order to evaluate the functional regulation of proteins attributed to phosphorylation. In this study, we used the heart β1-adrenergic receptor (Adrb1) as a model protein and developed FLAG-Adrb1 knock-in mice, in which the FLAG tag was inserted at the N-terminus of Adrb1. The phosphorylation sites and levels of Adrb1 in the heart were elucidated by immuno-affinity purification followed by quantitative mass spectrometry analysis using ion intensity ratio of the phosphorylated peptide versus corresponding unphosphorylated peptide. The phosphorylation levels at Ser274 and Ser462 of Adrb1 were approximately 0.25 and 0.0023. This effective strategy should be useful for not only analyzing site-specific phosphorylation levels of target proteins, but also quantifying the expression levels of proteins of interest when appropriate antibodies are not available. View Full-Text
Keywords: β1-adrenergic receptor; immuno-affinity purification; knock-in mice; phosphorylation β1-adrenergic receptor; immuno-affinity purification; knock-in mice; phosphorylation
Figures

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

Supplementary materials

Scifeed alert for new publications

Never miss any articles matching your research from any publisher
  • Get alerts for new papers matching your research
  • Find out the new papers from selected authors
  • Updated daily for 49'000+ journals and 6000+ publishers
  • Define your Scifeed now

SciFeed Share & Cite This Article

MDPI and ACS Style

Hayashi, K.; Yamashita, R.; Takami, R.; Matsui, T.; Gotou, M.; Nishimoto, T.; Kobayashi, H. Strategy for Identification of Phosphorylation Levels of Low Abundance Proteins in Vivo for Which Antibodies Are not Available. J. Cardiovasc. Dev. Dis. 2017, 4, 17.

Show more citation formats Show less citations formats

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Related Articles

Article Metrics

Article Access Statistics

1

Comments

[Return to top]
J. Cardiovasc. Dev. Dis. EISSN 2308-3425 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top