Enhanced Characterization of Histones Using 193 nm Ultraviolet Photodissociation and Proton Transfer Charge Reduction

Abstract

Top-down characterization of histones, proteins that are critical participants in an array of DNA-dependent processes, offers the potential to examine the relationship between histone structure and mechanisms of genetic regulation. Mapping patterns of post-translational modifications (PTMs) of histones requires extensive backbone cleavages to bracket the sites of mass shifts corresponding to specific PTMs. Ultraviolet photodissociation (UVPD) causes substantial fragmentation of proteins, which is well-suited for PTM localization, but the resulting spectra are congested with fragment ions that may have overlapping isotopic distributions that confound deconvolution. Gas-phase proton transfer charge reduction (PTCR) decreases the charge states of highly charged ions, thus alleviating this congestion and facilitating the identification of additional sequence-determining and PTM-localizing fragment ions. By integrating UVPD with PTCR for histone proteoform analyses, sequence coverages up to 91% were achieved for calf thymus histone H4 containing acetylation marks at the N-terminus and Lys12 as well as a dimethylation at Arg3. UVPD-PTCR exhibited large gains in characterization for other histones, such as histone H2A, increasing the sequence coverage from 59 to 77% for monoacetylated H2A.

Characterization of Unbranched Ubiquitin Tetramers by Combining Ultraviolet Photodissociation with Proton Transfer Charge Reduction Reactions

“…In this study, we characterize six unbranched ubiquitin tetramers using UVPD and UVPD + PTCR. The sequence, linkage patterns, and nomenclature of all six tetramers, including Ub-6 Ub-6 Ub-6 Ub, Ub-1 1 Ub-1 1 Ub- 1 1 Ub, Ub- 29 Ub- 29 Ub- 29 Ub, Ub-33 Ub- 33 Ub- 33 Ub, Ub-48 Ub-48 Ub-48 Ub, and Ub-63 Ub-63 Ub-63 Ub (K6-, K11-, K29-, K33-, K48-, and K63-linked tetramers, respectively), are illustrated in Scheme S1. 34 The ESI mass spectra of the six isomeric tetramers exhibit broad charge state distributions typical of denatured proteins and yield the same monoisotopic mass (Figure S1).…”

“…37−41 To alleviate this issue, gas-phase proton transfer charge reduction (PTCR) have been performed on UVPD product ions. 33,37 Reacting large highly charged UVPD product ions with reagent anions produces charge-reduced fragment ions and disperses them across a larger m/z domain, thus decreasing the overlap in the isotopic distributions. 33,37 This process also increases the S/N ratios of large PTM-localizing fragment ions, thus improving their deconvolution and identification, hence increasing the sequence coverage of larger proteins.…”

“…33,37 Reacting large highly charged UVPD product ions with reagent anions produces charge-reduced fragment ions and disperses them across a larger m/z domain, thus decreasing the overlap in the isotopic distributions. 33,37 This process also increases the S/N ratios of large PTM-localizing fragment ions, thus improving their deconvolution and identification, hence increasing the sequence coverage of larger proteins. 33,37 Here, we capitalize on the attributes of UVPD to generate informative PTM-localizing fragment ions and PTCR to decongest the MS/MS spectra to aid in the characterization of six unbranched ubiquitin tetramers linked at Lys6, Lys11, Lys29, Lys33, Lys48, and Lys63, respectively.…”

“…33,37 This process also increases the S/N ratios of large PTM-localizing fragment ions, thus improving their deconvolution and identification, hence increasing the sequence coverage of larger proteins. 33,37 Here, we capitalize on the attributes of UVPD to generate informative PTM-localizing fragment ions and PTCR to decongest the MS/MS spectra to aid in the characterization of six unbranched ubiquitin tetramers linked at Lys6, Lys11, Lys29, Lys33, Lys48, and Lys63, respectively. We specifically aim to increase the identification of PTM-containing fragment ions larger than the size of a Ub monomer (8.6 kDa) and up to the size of a Ub tetramer (34 kDa).…”

“…Ion activation methods like electron transfer dissociation supplemented with higher energy collision dissociation (known as EThcD) , and ultraviolet photodissociation (UVPD) facilitate the analysis of larger molecules by producing rich fragmentation patterns . UVPD has been used to characterize combinatorial patterns of modifications and has shown promising results for the analysis of intact proteins. − UVPD and electron-based methods have been used to elucidate the linkage patterns of intact ubiquitin polymers. − , ETD was used to reveal the linkage and branching patterns of intact Ub-trimers . Electron transfer dissociation supplemented with collisional activation (known as ETciD) was used to elucidate the structure of Ub-tetramers, and EThcD was used to access the linkage sites in two-component branched proteins .…”