Searle, B. | bioRxiv Science

bioRxiv2018

Incorporating in-source fragment information improves metabolite identification accuracy in untargeted LC-MS datasets

[ABSTRACT]In-source fragmentation occurs as a byproduct of electrospray ionization. We find that ions produced as a result of in-source fragmentation often match fragment ions produced during MS/MS fragmentation and we take advantage of this phenomenon in a novel algorithm to analyze LC-MS metabolomics datasets. Our approach organizes co-eluting MS1 features into a single peak group and then identifies in-source fragments among co-eluting features using MS/MS spectral libraries. We tested our approach using previously published data of verified metabolites, and compared the results to features detected by other mainstream metabolomics tools. Our results indicate that considering in-source fragment information as a part of the identification process increases annotation quality, allowing us to leverage MS/MS data in spectrum libraries even if MS/MS scans were not collected.

bioinformatics↗

bioRxiv2018

Quantification of nuclear protein dynamics reveals chromatin remodeling during acute protein degradation

Sequencing-based technologies cannot measure post-transcriptional dynamics of the nuclear proteome, but unbiased mass-spectrometry measurements of chromatin-associated proteins remain difficult. In this work, we have combined facile nuclear sub-fractionation approaches with data-independent acquisition mass spectrometry to improve detection and quantification of nuclear proteins in human cells and tissues. Nuclei are isolated and subjected to a series of extraction conditions that enrich for nucleoplasm, euchromatin, heterochromatin and nuclear-membrane associated proteins. Using this approach, we can measure peptides from over 70% of the expressed nuclear proteome. As we are physically separating chromatin compartments prior to analysis, proteins can be assigned into functional chromatin environments to illuminate systems-wide nuclear protein dynamics. The integrity of nuclear sub-compartments were validated with immunofluorescence, which confirms the presence of key markers during chromatin extraction. We then apply this method to study the nuclear proteome-wide response to pharmacological degradation of the BET bromodomain proteins. BET degradation leads to widespread changes in chromatin composition, and we discover global HDAC1/2-mediated remodeling of chromatin previously bound by BET bromodomains. In summary, we have developed a technology for reproducible, comprehensive characterization of the nuclear proteome to observe the systems-wide nuclear protein dynamics.

systems biology↗