title: Quantitative Mass Spectrometry Reveals that Intact Histone H1 Phosphorylations are Variant Specific and Exhibit Single Molecule Hierarchical Dependence.
abstract: Breast cancer was the second leading cause of cancer related mortality for females in 2014. Recent studies suggest histone H1 phosphorylation may be useful as a clinical biomarker of breast and other cancers because of its ability to recognize proliferative cell populations. Although monitoring a single phosphorylated H1 residue is adequate to stratify high-grade breast tumors, expanding our knowledge of how H1 is phosphorylated through the cell cycle is paramount to understanding its role in carcinogenesis. H1 analysis by bottom-up MS is challenging because of the presence of highly homologous sequence variants expressed by most cells. These highly basic proteins are difficult to analyze by LC-MS/MS because of the small, hydrophilic nature of peptides produced by tryptic digestion. Although bottom-up methods permit identification of several H1 phosphorylation events, these peptides are not useful for observing the combinatorial post-translational modification (PTM) patterns on the protein of interest. To complement the information provided by bottom-up MS, we utilized a top-down MS/MS workflow to permit identification and quantitation of H1 proteoforms related to the progression of breast cells through the cell cycle. Histones H1.2 and H1.4 were observed in MDA-MB-231 metastatic breast cells, whereas an additional histone variant, histone H1.3, was identified only in nonneoplastic MCF-10A cells. Progressive phosphorylation of histone H1.4 was identified in both cell lines at mitosis (M phase). Phosphorylation occurred first at S172 followed successively by S187, T18, T146, and T154. Notably, phosphorylation at S173 of histone H1.2 and S172, S187, T18, T146, and T154 of H1.4 significantly increases during M phase relative to S phase, suggesting that these events are cell cycle-dependent and may serve as markers for proliferation. Finally, we report the observation of the H1.2 SNP variant A18V in MCF-10A cells.
authors.affiliation: From the ‡Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, 32310;
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.affiliation: §Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, Ohio, College of Medicine and Arthur G. James Comprehensive Cancer Center, Columbus, Ohio, 43210;
authors.forename: Michael E
authors.initials: ME
authors.lastname: Hoover
authors.affiliation: ¶Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida, 32306.
authors.forename: Xibei
authors.initials: X
authors.lastname: Dang
authors.affiliation: ¶Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida, 32306.
authors.forename: Alan A
authors.initials: AA
authors.lastname: Shomo
authors.affiliation: From the ‡Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, 32310;
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Guan
authors.affiliation: From the ‡Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, 32310; ¶Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida, 32306.
authors.forename: Alan G
authors.initials: AG
authors.lastname: Marshall
authors.affiliation: §Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, Ohio, College of Medicine and Arthur G. James Comprehensive Cancer Center, Columbus, Ohio, 43210; nyoung@magnet.fsu.edu freitas.5@osu.edu.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Freitas
authors.affiliation: From the ‡Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, 32310; nyoung@magnet.fsu.edu freitas.5@osu.edu.
authors.forename: Nicolas L
authors.initials: NL
authors.lastname: Young
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005260:Female; D006657:Histones / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D020641:Polymorphism, Single Nucleotide; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006657:Histones