Method for the affinity purification of covalently linked peptides following cyanogen bromide cleavage of proteins

Analytical Chemistry
Tujin ShiGerold Schmitt-Ulms

Abstract

The low resolution structure of a protein can sometimes be inferred from information about existing disulfide bridges or experimentally introduced chemical crosslinks. Frequently, this task involves enzymatic digestion of a protein followed by mass spectrometry-based identification of covalently linked peptides. To facilitate this task, we developed a method for the enrichment of covalently linked peptides following the chemical cleavage of a protein. The method capitalizes on the availability of homoserine lactone moieties at the C-termini of cyanogen bromide cleavage products which support selective conjugation of affinity tags. The availability of two C-termini within covalently linked peptides allows for the conjugation of two distinct affinity tags and thereby enables subsequent removal of unmodified peptides by tandem affinity chromatography. Here, we demonstrate the stepwise implementation of this method using a polyhistidine tag and a biotin tag for the selective two-step purification of covalently linked cyanogen bromide fragments from increasingly complex protein samples. The method is independent of the nature of the covalent bond, is adaptable to fully denaturing conditions, and requires only low picomole quantities...Continue Reading

References

Nov 1, 1973·The Biochemical Journal·D R Eyre, M J Glimcher
Nov 20, 1969·Biochemical and Biophysical Research Communications·K A PiezW T Butler
Oct 1, 1996·Nucleic Acids Research·O N JensenD F Barofsky
May 17, 2000·Proceedings of the National Academy of Sciences of the United States of America·M M YoungG Dollinger
Jun 24, 2000·Nature·A Pandey, M Mann
Apr 26, 2001·Proceedings of the National Academy of Sciences of the United States of America·Y ItohH G Khorana
Jan 23, 2002·Rapid Communications in Mass Spectrometry : RCM·Kara M PearsonHenry M Fales
Jun 11, 2002·Methods : a Companion to Methods in Enzymology·Henning UrlaubReinhard Lührmann
Sep 18, 2002·The Journal of Biological Chemistry·Thomas TavernerRichard J Simpson
Sep 19, 2002·Protein Science : a Publication of the Protein Society·Jaap W BackAnton O Muijsers
Dec 7, 2002·Trends in Biochemical Sciences·Patrick Aloy, Robert B Russell
Dec 31, 2002·Current Opinion in Structural Biology·Frank Sobott, Carol V Robinson
Jan 1, 2003·Journal of Molecular Biology·Jason LanmanPeter E Prevelige
Feb 27, 2003·Journal of the American Chemical Society·Michelle Trester-ZedlitzTom W Muir
Jun 5, 2003·Matrix Biology : Journal of the International Society for Matrix Biology·Yoshito MatsuiDavid R Eyre
May 18, 2004·Journal of the American Society for Mass Spectrometry·Gregory B HurstStephen J Kennel
Jul 28, 2004·Journal of the American Society for Mass Spectrometry·Bill X HuangChhabil Dass
Mar 16, 2005·Nature Biotechnology·Scott M BrittainEric C Peters
May 20, 2005·Molecular & Cellular Proteomics : MCP·Evgeniy V PetrotchenkoChristoph H Borchers
Feb 8, 2006·Biophysical Journal·Edi Gabellieri, Giovanni B Strambini
Sep 2, 2006·Journal of Proteome Research·Harsha P GunawardenaScott A McLuckey
Nov 17, 2006·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Chihiro SatoTakeshi Iwatsubo
Feb 3, 2007·Journal of Proteome Research·Xiaoting TangJames E Bruce
Mar 14, 2007·Journal of the American Society for Mass Spectrometry·Christof LenzHenning Urlaub
Jun 28, 2007·Nature Reviews. Molecular Cell Biology·Anne-Claude GingrasRuedi Aebersold
Nov 5, 2008·Methods in Molecular Biology·Henning UrlaubReinhard Lührmann
Jun 23, 2009·Methods in Molecular Biology·Bingwen LuJohn R Yates

❮ Previous
Next ❯

Citations

Jan 17, 2013·Rapid Communications in Mass Spectrometry : RCM·Julien RoeserRainer P H Bischoff
Aug 10, 2011·Bioconjugate Chemistry·Thomas FrickeRudolf K Allemann

❮ 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.