Novel mass spectrometric method for phosphorylation quantification using cerium oxide nanoparticles and tandem mass tags.
The stoichiometry of protein phosphorylation significantly impacts protein function. The development of quantitative techniques in mass spectrometry has generated the ability to systematically monitor the regulation levels of various proteins. This study reports an integrated methodology using cerium oxide nanoparticles and isobaric tandem mass tag (TMT) labeling to assess absolute stoichiometries of protein phosphorylation. This protocol was designed to directly measure the dephosphorylation levels for a known phosphorylation site, therefore allowing for quantification of phosphosites. Both the accuracy and precision of the method were verified using standard peptides and protein tryptic digests. This novel method was then applied to quantify phosphorylations on eukaryotic initiation factor 3H (eIF3H), a protein integral to overall eukaryotic protein translation initiation. To date, this is the first report of assessment of protein phosphorylation quantification on eIF3.
Department of Molecular and Cellular Biology, University of California, Davis, One Shields Avenue, Davis, California 95616, USA.
Weitao
W
Jia
Armann
A
Andaya
Julie A
JA
Leary
D002563:Cerium / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D039621:Eukaryotic Initiation Factor-3 / Q000378:metabolism; D053768:Metal Nanoparticles / Q000737:chemistry*; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D039621:Eukaryotic Initiation Factor-3; D010748:Phosphopeptides; D002563:Cerium; C030583:ceric oxide