23 research outputs found
Proteomic Interrogation of Human Chromatin
Chromatin proteins provide a scaffold for DNA packaging and a basis for epigenetic regulation and genomic maintenance. Despite understanding its functional roles, mapping the chromatin proteome (i.e. the “Chromatome”) is still a continuing process. Here, we assess the biological specificity and proteomic extent of three distinct chromatin preparations by identifying proteins in selected chromatin-enriched fractions using mass spectrometry-based proteomics. These experiments allowed us to produce a chromatin catalog, including several proteins ranging from highly abundant histone proteins to less abundant members of different chromatin machinery complexes. Using a Normalized Spectral Abundance Factor approach, we quantified relative abundances of the proteins across the chromatin enriched fractions giving a glimpse into their chromosomal abundance. The large-scale data sets also allowed for the discovery of a variety of novel post-translational modifications on the identified chromatin proteins. With these comparisons, we find one of the probed methods to be qualitatively superior in specificity for chromatin proteins, but inferior in proteomic extent, evidencing a compromise that must be made between biological specificity and broadness of characterization. Additionally, we attempt to identify proteins in eu- and heterochromatin, verifying the enrichments by characterizing the post-translational modifications detected on histone proteins from these chromatin regions. In summary, our results provide insights into the value of different methods to extract chromatin-associated proteins and provide starting points to study the factors that may be involved in directing gene expression and other chromatin-related processes
Collective mass spectrometry approaches reveal broad and combinatorial modification of high mobility group protein A1a
H3K4 demethylation by Jarid1a and Jarid1b contributes to retinoblastoma-mediated gene silencing during cellular senescence
BclAF1 restriction factor is neutralized by proteasomal degradation and microRNA repression during human cytomegalovirus infection
Using Glycinylation, a Chemical Derivatization Technique, for the Quantitation of Ubiquitinated Proteins
A practical guide for analysis of histone post-translational modifications by mass spectrometry: Best practices and pitfalls
The histone H4 proteoform dynamics in response to SUV4-20 inhibition reveals single molecule mechanisms of inhibitor resistance
Analysis of Histone Posttranslational Modifications from Nucleolus-Associated Chromatin by Mass Spectrometry
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PILOT_PROTEIN: Identification of Unmodified and Modified Proteins via High-Resolution Mass Spectrometry and Mixed-Integer Linear Optimization
A novel protein identification framework, PILOT_PROTEIN, has been developed to construct a comprehensive list of all unmodified proteins that are present in a living sample. It uses the peptide identification results from the PILOT_SEQUEL algorithm to initially determine all unmodified proteins within the sample. Using a rigorous biclustering approach that groups incorrect peptide sequences with other homologous sequences, the number of false positives reported is minimized. A sequence tag procedure is then incorporated along with the untargeted PTM identification algorithm, PILOT_PTM, to determine a list of all modification types and sites for each protein. The unmodified protein identification algorithm, PILOT_PROTEIN, is compared to the methods SEQUEST, InsPecT, X!Tandem, VEMS, and ProteinProspector using both prepared protein samples and a more complex chromatin digest. The algorithm demonstrates superior protein identification accuracy with a lower false positive rate. All materials are freely available to the scientific community at http://pumpd.princeton.edu