MethylQuant: a tool for sensitive validation of enzyme-mediated protein methylation sites from heavy-methyl SILAC data

Aidan P. Tay, Vincent Geoghegan, Daniel Yagoub, Marc R. Wilkins, Gene Hart-Smith*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)


The study of post-translational methylation is hampered by the fact that large-scale LC-MS/MS experiments produce high methylpeptide false discovery rates (FDRs). The use of heavy-methyl stable isotope labeling by amino acids in cell culture (heavy-methyl SILAC) can drastically reduce these FDRs; however, this approach is limited by a lack of heavy-methyl SILAC compatible software. To fill this gap, we recently developed MethylQuant. Here, using an updated version of MethylQuant, we demonstrate its methylpeptide validation and quantification capabilities and provide guidelines for its best use. Using reference heavy-methyl SILAC data sets, we show that MethylQuant predicts with statistical significance the true or false positive status of methylpeptides in samples of varying complexity, degree of methylpeptide enrichment, and heavy to light mixing ratios. We introduce methylpeptide confidence indicators, MethylQuant Confidence and MethylQuant Score, and demonstrate their strong performance in complex samples characterized by a lack of methylpeptide enrichment. For these challenging data sets, MethylQuant identifies 882 of 1165 true positive methylpeptide spectrum matches (i.e., >75% sensitivity) at high specificity (<2% FDR) and achieves near-perfect specificity at 41% sensitivity. We also demonstrate that MethylQuant produces high accuracy relative quantification data that are tolerant of interference from coeluting peptide ions. Together MethylQuant's capabilities provide a path toward routine, accurate characterizations of the methylproteome using heavy-methyl SILAC.

Original languageEnglish
Pages (from-to)359-373
Number of pages15
JournalJournal of Proteome Research
Issue number1
Publication statusPublished - 5 Jan 2018
Externally publishedYes


  • post-translational methylation
  • false discovery rate (FDR)
  • proteomics quality control
  • heavy-methyl SILAC
  • metabolic labeling
  • quantitative proteomics software
  • MethylQuant


Dive into the research topics of 'MethylQuant: a tool for sensitive validation of enzyme-mediated protein methylation sites from heavy-methyl SILAC data'. Together they form a unique fingerprint.

Cite this