Quantification of H3K27 methylation using heavy peptides as standards

ABSTRACT: To demonstrate the usability of our SIL peptides (JPT GmbH)on an MS application, 4 standard peptides were synthesized at 95% purity and each spiked at an amount 133 fmol into protein extracts of wild type HEK cells and EZH2-knockout cells, respectively. The spiked peptides in this experiment are histone H3 K27-containing heavy-arginine versions of the peptide ARKSAPATGGVKKPH-R, where K27 was synthetically modified to carry either no methylation, or one or two or three (me3) methyl groups, respectively. Previously to trypsination, the HEK samples were spiked with the 4 SIL peptides. Because the standards have 10 Da greater mass given by the heavy R*, they can be distinguished from their light natural versions in the MS1 spectra. Furthermore, the standards were each spiked at a concentration of 133 fmol and thus, their MS intensities were used to normalize the intensities of the endogenous peptides and quantify them in absolute terms.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Victor Solis  

LAB HEAD: Axel Imhof

PROVIDER: PXD018321 | Pride | 2020-04-23

REPOSITORIES: Pride