Constructing Human Proteoform Families Using Intact-Mass and Top-Down Proteomics with a Multi-Protease Global Post-Translational Modification Discovery Database.

Complex human biomolecular processes are made possible by the diversity of human proteoforms. Constructing proteoform families, groups of proteoforms derived from the same gene, is one way to represent this diversity. Comprehensive, high-confidence identification of human proteoforms remains a central challenge in mass spectrometry-based proteomics. We have previously reported a strategy for proteoform identification using intact-mass measurements, and we have since improved that strategy by mass calibration based on search results, the use of a global post-translational modification discovery database, and the integration of top-down proteomics results with intact-mass analysis. In the present study, we combine these strategies for enhanced proteoform identification in total cell lysate from the Jurkat human T lymphocyte cell line. We collected, processed, and integrated three types of proteomics data (NeuCode-labeled intact-mass, label-free top-down, and multi-protease bottom-up) to maximize the number of confident proteoform identifications. The integrated analysis revealed 5950 unique experimentally observed proteoforms, which were assembled into 848 proteoform families. Twenty percent of the observed proteoforms were confidently identified at a 3.9% false discovery rate, representing 1207 unique proteoforms derived from 484 genes.

HyPR-MS for Multiplexed Discovery of MALAT1, NEAT1, and NORAD lncRNA Protein Interactomes.

RNA-protein interactions are integral to the regulation of gene expression. RNAs have diverse functions and the protein interactomes of individual RNAs vary temporally, spatially, and with physiological context. These factors make the global acquisition of individual RNA-protein interactomes an essential endeavor. Although techniques have been reported for discovery of the protein interactomes of specific RNAs they are largely laborious, costly, and accomplished singly in individual experiments. We developed HyPR-MS for the discovery and analysis of the protein interactomes of multiple RNAs in a single experiment while also reducing design time and improving efficiencies. Presented here is the application of HyPR-MS to simultaneously and selectively isolate the interactomes of lncRNAs MALAT1, NEAT1, and NORAD. Our analysis features the proteins that potentially contribute to both known and previously undiscovered roles of each lncRNA. This platform provides a powerful new multiplexing tool for the efficient and cost-effective elucidation of specific RNA-protein interactomes.

Elucidating Protein-DNA Interactions in Human Alphoid Chromatin via Hybridization Capture and Mass Spectrometry.

The centromere is the chromosomal locus where the kinetochore forms and is critical for ensuring proper segregation of sister chromatids during cell division. A substantial amount of effort has been devoted to understanding the characteristic features and roles of the centromere, yet some fundamental aspects of the centromere, such as the complete list of elements that define it, remain obscure. It is well-known that human centromeres include a highly repetitive class of DNA known as alpha satellite, or alphoid, DNA. We present here the first DNA-centric examination of human protein-alpha satellite interactions, employing an approach known as HyCCAPP (hybridization capture of chromatin-associated proteins for proteomics) to identify the protein components of alphoid chromatin in a human cell line. Using HyCCAPP, cross-linked alpha satellite chromatin was isolated from cell lysate, and captured proteins were analyzed via mass spectrometry. After being compared to proteins identified in control pulldown experiments, 90 proteins were identified as enriched at alphoid DNA. This list included many known centromere-binding proteins in addition to multiple novel alpha satellite-binding proteins, such as LRIF1, a heterochromatin-associated protein. The ability of HyCCAPP to reveal both known as well as novel alphoid DNA-interacting proteins highlights the validity and utility of this approach.

Investigating the impacts of DNA binding mode and sequence on thermodynamic quantities and water exchange values for two small molecule drugs.

Doxorubicin and nogalamycin are antitumor antibiotics that interact with DNA via intercalation and threading mechanisms, respectively. Because the importance of water, particularly its impact on entropy changes, has been established in other biological processes, we investigated the role of water in these two drug-DNA binding events. We used the osmotic stress method to calculate the number of water molecules exchanged (Δnwater), and isothermal titration calorimetry to measure Kbinding, ΔH, and ΔS for two synthetic DNAs, poly(dA·dT) and poly(dG·dC), and calf thymus DNA (CT DNA). For nogalamycin, Δnwater<0 for CT DNA and poly(dG·dC). For doxorubicin, Δnwater>0 for CT DNA and Δnwater<0 for poly(dG·dC). For poly(dA·dT), Δnwater~0 with both drugs. Net enthalpy changes were always negative, but net entropy changes depended on the drug. The effect of water exchange on the overall sign of entropy change appears to be smaller than other contributions.