title: Bottom-up and middle-down proteomics have comparable accuracies in defining histone post-translational modification relative abundance and stoichiometry.
abstract: 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.
authors.affiliation: Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Room 9-124, 3400 Civic Center Boulevard, Building 421, Philadelphia, Pennsylvania 19104, United States.
authors.forename: Simone
authors.initials: S
authors.lastname: Sidoli
authors.affiliation: Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Room 9-124, 3400 Civic Center Boulevard, Building 421, Philadelphia, Pennsylvania 19104, United States.
authors.forename: Shu
authors.initials: S
authors.lastname: Lin
authors.affiliation: Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Room 9-124, 3400 Civic Center Boulevard, Building 421, Philadelphia, Pennsylvania 19104, United States.
authors.forename: Kelly R
authors.initials: KR
authors.lastname: Karch
authors.affiliation: Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Room 9-124, 3400 Civic Center Boulevard, Building 421, Philadelphia, Pennsylvania 19104, United States.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Garcia
mesh_terms: D002853:Chromatography, Liquid; D006367:HeLa Cells; D006801:Humans; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry