Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

Abstract

Chromatin is the structural framework that packages DNA into chromosomes within the nucleus of a cell (2). Histones comprise the principal protein component of chromatin and are involved in the regulation of gene expression (3,4). This epigenetic regulation is achieved through complex patterns of post-translational modifications (PTMs), 1 the incorporation of histone variants, and through controlled histone proteolysis (5-10). Comprehensive characterization of histones by mass spectrometry (MS) has proven technically difficult for a number of reasons. Traditional methods (bottom-up MS) of sequence determination and PTM site localization are not practical. Histone N-terminal regions are rich in lysine and arginine residues, and thus proteolysis using trypsin generates peptides that are too small or that are poorly retained on reversephase HPLC C18 resins for subsequent MS detection (11). With the advent of electron transfer dissociation (ETD) and more efficient electron capture dissociation fragmentation methods, which are better suited for larger, more highly charged peptides (12, 13), several studies utilizing other endoproteases to generate longer peptides have emerged (14 -16). Although these methodologies do well to preserve the combinatorial PTM profiles of histone tails, in some cases it is still impossible to identify the proteoforms from which these peptides originate. This is why analyzing histones intact, as they exist in the cells from which they are derived, is the best method for identifying unique histone proteoforms.The results of several recent studies involving top-down analyses of histones highlight the complexity of the histone From the ‡Department

The Role of Electron Transfer Dissociation in Modern Proteomics

“…The ETD/PTR technique proved extremely valuable in middle down analyses of antibodies, 243 and they further extended its utility in a recent top-down characterization of histones by incorporating parallel ion parking 35 , 306 during the PTR reactions to specifically control reaction of precursor ions to remain in a targeted product m / z range. 237 The value of these strategies is clear for analysis of large peptides and proteins, and we expect this technology will be widely implemented in top-down experiments in coming years. Unsurprisingly, similar approaches have recently been extended to combinations of UVPD and PTR reactions for top-down analyses of denatured and native proteins.…”

Modulation of Protein Fragmentation Through Carbamylation of Primary Amines

“…While measurement of the accurate mass of a protein is a crucial first step, complete characterization of a proteoform (i.e., a unique molecular form of a protein including its mutations and specific post-translational modifications) requires much more information about the sequence, as well as the identity, number, and position of modifications [4]. There are several established methods to activate and dissociate intact proteins; collisionally activated dissociation (CAD [5], beam-type higher energy collision dissociation (HCD [6, 7]), and electron-based methods (most commonly electron transfer dissociation (ETD [8, 9]), have been used for the most significant high throughput top-down studies. Ultraviolet photodissociation (UVPD) is the newest activation method that has been developed for the analysis of intact proteins [10–16].…”

Identification and Quantification of Proteoforms by Mass Spectrometry

“…Therefore, it is common for fragment ion signals to fall below detection limits. Techniques such as charge‐reduction and ion‐parking have been used to simplify spectra and enhance sensitivity. However, sensitivity for identification of proteoforms diminishes severely beyond 30 kDa …”