Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Separation of phospho- and non-phosphopeptides using reverse phase column chromatography

H Ohguro1, K Palczewski

  • 1Department of Ophthalmology, School of Medicine, University of Washington, Seattle 98195, USA.

FEBS Letters
|July 24, 1995
PubMed
Summary

This study introduces a robust C18 reverse-phase chromatography method for separating phosphorylated peptides from their non-phosphorylated counterparts. Heptafluorobutyric acid as a counter-ion significantly enhances this separation, aiding in phosphorylation site analysis.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Guanylate cyclase-activating proteins and retina disease.

Sub-cellular biochemistry·2008
Same author

G protein-coupled receptor drug discovery: implications from the crystal structure of rhodopsin.

Current opinion in drug discovery & development·2003
Same author

Crystal structure of rhodopsin: a template for cone visual pigments and other G protein-coupled receptors.

Biochimica et biophysica acta·2002
Same author

Crystal structure of rhodopsin: a G-protein-coupled receptor.

Chembiochem : a European journal of chemical biology·2002
Same author

Gene transfer mediated by recombinant baculovirus into mouse eye.

Investigative ophthalmology & visual science·2001
Same author

Isomerization of 11-cis-retinoids to all-trans-retinoids in vitro and in vivo.

The Journal of biological chemistry·2001

Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Proteomics

Background:

  • Phosphorylation is a critical post-translational modification regulating numerous cellular processes.
  • Distinguishing phosphorylated peptides from non-phosphorylated forms is essential for studying phosphorylation dynamics.
  • Existing chromatographic methods may not offer sufficient resolution for complex peptide mixtures.

Purpose of the Study:

  • To develop and optimize a chromatographic method for effective separation of phosphorylated peptides.
  • To identify optimal conditions for distinguishing phosphoserine, phosphothreonine, and phosphotyrosine peptides.
  • To enable detailed analysis of phosphorylation sites and kinetics.

Main Methods:

  • Utilized standard C18 reverse-phase high-performance liquid chromatography (RP-HPLC).

Related Experiment Videos

  • Employed water/acetonitrile gradients with various counter-ions.
  • Investigated the impact of heptafluorobutyric acid (HFBA) as a mobile phase additive.

    • Main Results:

    • Achieved excellent separation of phosphorylated peptides from non-phosphorylated peptides using HFBA.
    • Observed retention time differences up to approximately 20 minutes.
    • Demonstrated reliable separation of peptides with differential phosphorylation sites and varying amino acid compositions (acidic/basic phospho-Ser/Thr, phospho-Tyr).

    Conclusions:

    • HFBA-supplemented RP-HPLC provides a powerful tool for phosphopeptide enrichment and analysis.
    • This method facilitates the study of phosphorylation/dephosphorylation kinetics.
    • The technique is valuable for in vivo phosphorylation site mapping.