Bottom-Up and Middle-Down Proteomics Have Comparable Accuracies in Defining Histone Post-Translational Modification Relative Abundance and Stoichiometry

Simone Sidoli, Shu Lin, Kelly R. Karch, Benjamin A. Garcia

Research output: Contribution to journalArticle

33 Scopus citations


Histone proteins are key components of chromatin. Their N-terminal tails are enriched in combinatorial post-translational modifications (PTMs), which influence gene regulation, DNA repair, and chromosome condensation. Mass spectrometry (MS)-based middle-down proteomics has emerged as a technique to analyze co-occurring PTMs, as it allows for the characterization of intact histone tails (>50 aa) rather than short (<20 aa) peptides analyzed by bottom-up. However, a demonstration of its reliability is still lacking. We compared results obtained with the middle-down and the bottom-up strategy in calculating PTM relative abundance and stoichiometry. Since bottom-up was proven to have biases in peptide signal detection such as uneven ionization efficiency, we performed an external correction using a synthetic peptide library with known peptide relative abundance. Corrected bottom-up data were used as reference. Calculated abundances of single PTMs showed similar deviations from the reference when comparing middle-down and uncorrected bottom-up results. Moreover, we show that the two strategies provided similar performance in defining accurate PTM stoichiometry. Collectively, we evidenced that the middle-down strategy is at least equally reliable to bottom-up in quantifying histone PTMs. (Figure Presented).

Original languageEnglish (US)
Pages (from-to)3129-3133
Number of pages5
JournalAnalytical Chemistry
Issue number6
StatePublished - Mar 17 2015


ASJC Scopus subject areas

  • Analytical Chemistry

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