A direct, ratiometric, and quantitative MALDI-MS assay for protein methyltransferases and acetyltransferases

Analytical Biochemistry
Stacie L RichardsonRong Huang

Abstract

Protein methylation and acetylation play important roles in biological processes, and misregulation of these modifications is involved in various diseases. Therefore, it is critical to understand the activities of the enzymes responsible for these modifications. Herein we describe a sensitive method for ratiometric quantification of methylated and acetylated peptides via MALDI-MS by direct spotting of enzymatic methylation and acetylation reaction mixtures without tedious purification procedures. The quantifiable detection limit for peptides with our method is approximately 10 fmol. This is achieved by increasing the signal-to-noise ratio through the addition of NH4H2PO4 to the matrix solution and reduction of the matrix α-cyanohydroxycinnamic acid concentration to 2 mg/ml. We have demonstrated the application of this method in enzyme kinetic analysis and inhibition studies. The unique feature of this method is the simultaneous quantification of multiple peptide species for investigation of processivity mechanisms. Its wide buffer compatibility makes it possible to be adapted to investigate the activity of any protein methyltransferase or acetyltransferase.

References

Oct 16, 2004·The Journal of Biological Chemistry·Debasis PatnaikSriharsa Pradhan
Jun 17, 2005·Analytical Biochemistry·Evys CollazoRaymond C Trievel
Aug 9, 2005·Methods : a Companion to Methods in Enzymology·Christopher E Berndsen, John M Denu
Apr 18, 2006·Journal of the American Society for Mass Spectrometry·Kenneth D GreisGregory F Davis
Feb 27, 2007·Cell·Bradley E BernsteinEric S Lander
Feb 27, 2007·Cell·Peter A Jones, Stephen B Baylin
Feb 12, 2008·The Journal of Biological Chemistry·Ted M Lakowski, Adam Frankel
Feb 26, 2008·Current Opinion in Genetics & Development·Jane MellorDavid Clynes
Aug 18, 2009·Langmuir : the ACS Journal of Surfaces and Colloids·Deenan SanthiyaSouvik Maiti
Sep 19, 2009·Analytical Biochemistry·Ted M LakowskiAdam Frankel
Dec 22, 2009·Nature Structural & Molecular Biology·John R HortonXiaodong Cheng
Jan 30, 2010·Science·Cheol-Sang HwangAlexander Varshavsky
Nov 16, 2010·Developmental Cell·María Berdasco, Manel Esteller
Jan 29, 2011·Epigenetics : Official Journal of the DNA Methylation Society·Lauren P BlairAlan J Tackett
Oct 6, 2011·Journal of Biomolecular Screening·Abdellah Allali-HassaniMasoud Vedadi
Mar 13, 2012·Trends in Biochemical Sciences·Kristian K StarheimThomas Arnesen
Jul 10, 2012·Biochemistry·Janusz J PetkowskiIan G Macara
Oct 2, 2012·Analytical Biochemistry·Till F SchäberleGabriele M König
Apr 19, 2013·Journal of the American Society for Mass Spectrometry·Justyna RechthalerGünter Allmaier
May 15, 2013·ChemMedChem·Srikanth KudithipudiAlbert Jeltsch
Jul 3, 2013·Proceedings of the National Academy of Sciences of the United States of America·Aaron O BaileyDaniel R Foltz
Aug 6, 2013·Nature Structural & Molecular Biology·Glen LiszczakRonen Marmorstein
Aug 28, 2013·Journal of Proteome Research·Xiaoxia DaiYinsheng Wang
Oct 17, 2013·Molecular & Cellular Proteomics : MCP·Ailan GuoMichael J Comb
Mar 17, 2015·The Journal of Biological Chemistry·Stacie L RichardsonRong Huang

❮ Previous
Next ❯

Citations

Nov 28, 2018·Chembiochem : a European Journal of Chemical Biology·Rong Huang
Aug 5, 2020·Scientific Reports·Giordano ProiettiJasmin Mecinović
Jan 14, 2021·International Journal of Molecular Sciences·Yi-Hsun HoRong Huang
Apr 12, 2021·Current Opinion in Chemical Biology·Krystal DiazRong Huang
Mar 19, 2019·Journal of Medicinal Chemistry·Dongxing ChenRong Huang
Jul 28, 2020·Current Protein & Peptide Science·Madhuri JayathirthaCostel C Darie

❮ 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.