On-bead chemical synthesis and display of phosphopeptides for affinity pull-down proteomics

Chembiochem : a European Journal of Chemical Biology
Malene BrandtKnud J Jensen

Abstract

We describe a new method for phosphopeptide proteomics based on the solid-phase synthesis of phosphopeptides on beads suitable for affinity pull-down experiments. Peptide sequences containing the Bad Ser112 and Ser136 phosphorylation motifs were used as bait in affinity pull-down experiments to determine their ability to bind 14-3-3 proteins. Support-bound peptides were assembled directly on the solid support (PEGA) by standard solid-phase synthesis through a BAL-type handle. The peptides were varied in length and sequence. This synthetic strategy also allowed introduction of a soft electrophile (aldehyde) at the C terminus for potential activity-based proteomics. The synthetic support-bound Bad phosphopeptides were able to pull down 14-3-3zeta. Furthermore, Bad phosphopeptides bound endogenous 14-3-3 proteins, and all seven members of the 14-3-3 family were identified by mass spectrometry. In control experiments, none of the unphosphorylated Bad peptides bound transfected 14-3-3zeta or endogenous 14-3-3. We conclude that the combined synthesis and display of phosphopeptides on-bead is a fast and efficient method for affinity pull-down proteomics.

References

Sep 1, 1990·International Journal of Peptide and Protein Research·J M LacombeA A Pavia
Jun 3, 2000·Annual Review of Pharmacology and Toxicology·H FuS C Masters
Aug 29, 2000·The Journal of Peptide Research : Official Journal of the American Peptide Society·P M BalseV J Hruby
May 29, 2001·Biopolymers·J S McMurrayM L Campbell
Oct 25, 2001·The Journal of Organic Chemistry·Gregory T. BourneMark L. Smythe
Jun 28, 2002·Cancer Cell·Douglas R Green, Gerard I Evan
Apr 18, 2003·Brazilian Journal of Medical and Biological Research = Revista Brasileira De Pesquisas Médicas E Biológicas·M Rosenquist
Jun 18, 2003·Chemical Communications : Chem Comm·Alessandra BassoSabine L Flitsch
Oct 7, 2003·Annual Review of Biochemistry·Heng ZhuMichael Snyder
Dec 18, 2003·The Journal of Biological Chemistry·Waltraud X Schulze, Matthias Mann
Dec 20, 2003·Combinatorial Chemistry & High Throughput Screening·Harald Schröder
Dec 26, 2003·Chembiochem : a European Journal of Chemical Biology·Anna E Speers, Benjamin F Cravatt
Jun 25, 2004·Molecular & Cellular Proteomics : MCP·Kevin WilhelmsenPeter van der Geer

❮ Previous
Next ❯

Citations

Dec 6, 2012·Journal of Separation Science·Irene MessanaMassimo Castagnola
Mar 18, 2009·Chemical Reviews·Ulrik BoasKnud J Jensen

❮ 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.