Mass spectrometric analysis of the kinetics of in vivo rhodopsin phosphorylation

Protein Science : a Publication of the Protein Society
Kimberly A LeeJ B Hurley

Abstract

On stimulation, rhodopsin, the light-sensing protein in the rod cells of the retina, is phosphorylated at several sites on its C terminus as the first step in deactivation. We have developed a mass spectrometry-based method to quantify the kinetics of phosphorylation at each site in vivo. After exposing either a freshly dissected mouse retina or the eye of an anesthetized mouse to a flash of light, phosphorylation and dephosphorylation reactions are terminated by rapidly homogenizing the retina or enucleated eye in 8 M urea. The C-terminal peptide containing all known phosphorylation sites is cleaved from rhodopsin, partially purified by ultracentrifugation, and analyzed by liquid chromatography coupled with mass spectrometry (LCMS). The mass spectrometer responds linearly to the peptide from 10 fmole to 100 pmole. The relative sensitivity to peptides with zero to five phosphates was determined using purified phosphopeptide standards. High pressure liquid chromatography (HPLC) coupled with tandem mass spectrometry (LCMS/MS) was used to distinguish the three primary sites of phosphorylation, Ser 334, Ser 338, and Ser 343. Peptides monophosphorylated on Ser 334 were separable from those monophosphorylated on Ser 338 and Ser 343 b...Continue Reading

References

Feb 1, 1996·Analytical Chemistry·M WilmM Mann
Jan 1, 1997·Trends in Biochemical Sciences·T Hunter, G D Plowman
Jun 6, 1997·The Journal of Biological Chemistry·L ZhangE R Weiss
Jul 17, 1998·Vision Research·J B HurleyG A Niemi
Jun 9, 1999·Proceedings of the National Academy of Sciences of the United States of America·Y OdaB T Chait
Aug 24, 1999·Pharmacology & Therapeutics·J D Graves, E G Krebs
Nov 26, 1999·Proceedings of the National Academy of Sciences of the United States of America·G D PlowmanT Hunter
Mar 25, 2000·Science·M D AdamsJ C Venter
Apr 3, 2001·Nature Biotechnology·H ZhouR Aebersold

❮ Previous
Next ❯

Citations

Sep 5, 2003·Journal of the American Society for Mass Spectrometry·Lin GuoRichard S Johnson
Apr 27, 2005·The Journal of Biological Chemistry·Zhongyan WangJanis Lem
Sep 13, 2007·The Journal of Biological Chemistry·Sergey A VishnivetskiyVsevolod V Gurevich
Dec 4, 2004·Biological & Pharmaceutical Bulletin·Akira KameiNoriko Takeuchi
May 18, 2007·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Derek H RosenzweigVladlen Z Slepak
Apr 1, 2014·Biochemistry·Joseph R BlasicPhyllis R Robinson
Mar 21, 2009·Journal of Mass Spectrometry : JMS·Jan GropengiesserEberhard Krause
Mar 3, 2007·The EMBO Journal·Susan M HansonVsevolod V Gurevich
Apr 2, 2003·Biophysical Journal·Alessandro PintarSándor Pongor
Dec 28, 2016·International Journal of Molecular Sciences·Marco A Alfonzo-MéndezJ Adolfo García-Sáinz
Oct 16, 2007·Journal of Chromatography. a·Jeongkwon KimRichard D Smith
Aug 30, 2008·Photochemical & Photobiological Sciences : Official Journal of the European Photochemistry Association and the European Society for Photobiology·Hanna FabczakStanisław Fabczak
Jul 20, 2019·Bio-protocol·Sowmya Bekshe LokappaJeannie Chen

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.