Journal
ANALYTICAL CHEMISTRY
Volume 87, Issue 6, Pages 3129-3133Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.5b00072
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Funding
- NIH [DP2OD007447, R01GM110174]
- Office of the Director
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [T32GM071339, R01GM110174] Funding Source: NIH RePORTER
- OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH [DP2OD007447] Funding Source: NIH RePORTER
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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 stoichimetry. 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.
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