NEW YORK (GenomeWeb) – Researchers from the University of Virginia and University of Pennsylvania have devised a new workflow for top-down analysis of histone proteoforms.
Detailed in a paper published last month in Molecular & Cellular Proteomics, the workflow combines several recently developed mass spec methods for improved analysis of intact proteins, which the researchers applied to identify several histone proteoforms from HeLa cells with up to 81 percent sequence coverage.
Additionally, they showed their approach was able to distinguish between separate proteoforms with overlapping m/z that were not separated by chromatography, as well as characterize with sequence coverage of up to 93 percent truncated forms of histone H2A and H2B they identified within the fraction they analyzed.
The effort, which was led by UVA researcher Donald Hunt and Penn researcher Benjamin Garcia, utilized a number of mass spec techniques, several of them worked out in Hunt's lab, to improve histone analysis.
Histones have become a particular area of focus for top-down proteomics as their post-translational modifications are key to their function. Because top-down analyses look at undigested proteins, researchers are able profile the intact molecule with all its modifications rather than tying that information together from collections of identified peptides. And while this is valuable in a variety of contexts, it is especially so for histones where such methods have the potential to unravel what is often called the "histone code" — the different histone PTM combinations thought to play a significant role in DNA transcription.
The complexity of these molecules, however, presents significant challenges to their analysis.
Using a Thermo Fisher Scientific Orbitrap Velos Pro/Elite instrument, the researchers brought several methods to bear on the problem. The instrument was modified to add a glow discharge ionization source at the front of the machine, with which they ionized ETD and ionized ion/ion proton transfer (IIPT) reagents for ion/ion reactions. By placing this source at the front of the instrument, they were able to collect ions from multiple iterations of these ion/ion reactions, which allows for analysis of significantly more target ions, improving signal-to-noise.
Use of IIPT in combination with ETD simplifies the ETD spectra by dispersing fragment ions over the observed m/z range, the authors noted.
The researchers also used parallel ion parking, in which selected ions in the ion trap are excited to reduce their reactivity, which allows for the use of IIPT without reacting with precursors outside the targeted m/z range. This, they noted, allowed them to achieve better separation of highly similar proteoforms that would otherwise be difficult to separate via chromatography and which might be isolated and fragmented together leading to hard-to-interpret MS/MS spectra.
The MCP study was the first time such "ion parking has been demonstrated on a commercially available linear ion trap using ion cloud densities that lend themselves to high-resolution MS/MS analyses," the authors said, adding that the believed the technique would in the future become a powerful tool for identification of closely related proteoforms.