title: High resolution parallel reaction monitoring with electron transfer dissociation for middle-down proteomics.
abstract: In recent years, middle-down proteomics has emerged as a popular technique for the characterization and quantification of proteins not readily amenable to typical bottom-up approaches. So far, all high resolution middle-down approaches are done in data-dependent acquisition mode, using both collision-induced dissociation or electron capture/transfer dissociation techniques. Here, we explore middle-down proteomics with electron transfer dissociation using a targeted acquisition mode, parallel reaction monitoring (PRM), on an Orbitrap Fusion. As an example of a highly modified protein, we used histone H3 fractions from untreated and DMSO-treated Murine ErythroLeukemia (MEL) cells. We first determined optimized instrument parameters to obtain high sequence coverage using a synthetic standard peptide. We then setup a combined method of both MS1 scans and PRM scans of the 20 most abundant combinations of methylation and acetylation of the +10 charge state of the N-terminal tail of H3. Weak cation exchange hydrophilic interaction chromatography was used to separate the N-terminal H3 tail, primarily, by its acetylation and, to a secondary degree, by its methylation status, which aided in the interpretation of the results. After deconvolution of the highly charged ions, peaks were annotated to a minimum set of 254 H3 proteoforms in the untreated and treated samples. Upon DMSO treatment, global quantitation changes from the MS1 level show a relative decrease of 2, 3, 4, and 5 acetylations and an increase of 0 and 1 acetylations. A fragment ion map was developed to visualize specific differences between treated and untreated samples. Taken together, the data presented here show that middle-down proteomics with electron transfer dissociation using PRM is a novel, attractive method for the effective analysis and quantification of large and highly modified peptides.
authors.affiliation: †Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman Institute, California Institute of Technology, Pasadena, California 91125, United States.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sweredoski
authors.affiliation: †Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman Institute, California Institute of Technology, Pasadena, California 91125, United States.
authors.forename: Annie
authors.initials: A
authors.lastname: Moradian
authors.affiliation: †Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman Institute, California Institute of Technology, Pasadena, California 91125, United States.|‡Hochschule Weihenstephan-Triesdorf, University of Applied Sciences, Faculty of Biotechnology and Bioinformatic, Am Hofgarten 4, 85354 Freising, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Raedle
authors.affiliation: †Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman Institute, California Institute of Technology, Pasadena, California 91125, United States.|§Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Catarina
authors.initials: C
authors.lastname: Franco
authors.affiliation: †Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman Institute, California Institute of Technology, Pasadena, California 91125, United States.
authors.forename: Sonja
authors.initials: S
authors.lastname: Hess
mesh_terms: D002853:Chromatography, Liquid; D004583:Electrons; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis* / Q000235:genetics; D040901:Proteomics / Q000379:methods*
chemical_list: D010455:Peptides