title: Sensitive and Precise Characterization of Combinatorial Histone Modifications by Selective Derivatization Coupled with RPLC-EThcD-MS/MS.
abstract: Deciphering the combinatorial histone codes has been a long-standing interest in the epigenetics field, which requires the reliable and robust characterization of the post-translational modifications (PTMs) coexisting on histones. To this end, weak cation exchange hydrophilic interaction liquid chromatography is commonly used in middle-down liquid chromatography-mass spectrometry approaches for online separation of variously modified histone peptides. Here we provide a novel strategy that combines the selective histone peptide derivatization using N-hydroxysuccinimide propionate ester with reversed-phase liquid chromatography (RPLC) for the robust, sensitive, and reliable characterization of combinatorial histone PTMs. Derivatization amplifies the subtle physical differences between similarly modified histone peptides, thereby allowing baseline separation of these peptides by standard RPLC. Also, the sensitivity of MS is enhanced greatly by derivatization due to the increased peptide hydrophobicity and concentrated charge-state envelope during electrospray ionization. Furthermore, we systematically optimized the dual electron transfer and higher energy collision dissociation and achieved near-complete peptide sequence coverage in MS/MS spectra, allowing highly precise and reliable PTM identification. Using this method, we identified 311 and 293 combinations of histone H3 PTMs from the lymphoma cells Karpas-422 with/without drug treatment, confirming the advantages of our method in serving as a platform for profiling combinatorial histone PTMs.
authors.affiliation: Xuhui Central Hospital, Shanghai Clinical Center, Chinese Academy of Sciences , Shanghai 200031, China.
authors.forename: Rijing
authors.identifier: 0000-0002-1844-158X
authors.initials: R
authors.lastname: Liao
authors.affiliation: Novartis Institutes for BioMedical Research (China) Co., Ltd. , 4218 Jinke Road, Shanghai 201203, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Zheng
authors.affiliation: Thermo Fisher Scientific (China) Co., Ltd. , Building 6, No. 27 Xin Jinqiao Road, Shanghai 201206, China.
authors.forename: Aiying
authors.initials: A
authors.lastname: Nie
authors.affiliation: Novartis Institutes for BioMedical Research (China) Co., Ltd. , 4218 Jinke Road, Shanghai 201203, China.
authors.forename: Shaolian
authors.initials: S
authors.lastname: Zhou
authors.affiliation: Novartis Institutes for BioMedical Research (China) Co., Ltd. , 4218 Jinke Road, Shanghai 201203, China.
authors.forename: Haibing
authors.initials: H
authors.lastname: Deng
authors.affiliation: Novartis Institutes for BioMedical Research (China) Co., Ltd. , 4218 Jinke Road, Shanghai 201203, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Department of Chemistry, Fudan University , 220 Handan Road, Shanghai 200433, China.
authors.forename: Pengyuan
authors.initials: P
authors.lastname: Yang
authors.affiliation: Novartis Institutes for BioMedical Research (China) Co., Ltd. , 4218 Jinke Road, Shanghai 201203, China.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Novartis Institutes for BioMedical Research (China) Co., Ltd. , 4218 Jinke Road, Shanghai 201203, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Tan
authors.affiliation: Novartis Institutes for BioMedical Research (China) Co., Ltd. , 4218 Jinke Road, Shanghai 201203, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Qi
authors.affiliation: Xuhui Central Hospital, Shanghai Clinical Center, Chinese Academy of Sciences , Shanghai 200031, China.
authors.forename: Jiaxi
authors.initials: J
authors.lastname: Wu
authors.affiliation: Novartis Institutes for BioMedical Research (China) Co., Ltd. , 4218 Jinke Road, Shanghai 201203, China.
authors.forename: En
authors.initials: E
authors.lastname: Li
authors.affiliation: Novartis Institutes for BioMedical Research (China) Co., Ltd. , 4218 Jinke Road, Shanghai 201203, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Yi
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D056148:Chromatography, Reverse-Phase; D042421:Histone Code / Q000235:genetics; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008223:Lymphoma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013388:Succinimides / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D006657:Histones; D010455:Peptides; D013388:Succinimides; C001426:N-hydroxysuccinimide
keywords: EThcD; LC−MS; combinatorial histone modifications; derivatization; middle-down approach; reversed-phase chromatography