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Top-down proteomics is emerging as a viable method for the routine identification of hundreds to thousands of proteins. In this work we report the largest top-down study to date, with the identification of 1,220 proteins from the transformed human cell line H1299 at a false discovery rate of 1%. Multiple separation strategies were utilized, including the(More)
With the prospect of resolving whole protein molecules into their myriad proteoforms on a proteomic scale, the question of their quantitative analysis in discovery mode comes to the fore. Here, we demonstrate a robust pipeline for the identification and stringent scoring of abundance changes of whole protein forms <30 kDa in a complex system. The input is(More)
Pilot Project #1--the identification and characterization of human histone H4 proteoforms by top-down MS--is the first project launched by the Consortium for Top-Down Proteomics (CTDP) to refine and validate top-down MS. Within the initial results from seven participating laboratories, all reported the probability-based identification of human histone H4(More)
The top-down approach to proteomics offers compelling advantages due to the potential to provide complete characterization of protein sequence and post-translational modifications. Here we describe the implementation of 193 nm ultraviolet photodissociation (UVPD) in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete(More)
Many top-down proteomics experiments focus on identifying and localizing PTMs and other potential sources of “mass shift” on a known protein sequence. A simple application to match ion masses and facilitate the iterative hypothesis testing of PTM presence and location would assist with the data analysis in these experiments. ProSight Lite is a free software(More)
UNLABELLED Measuring post-translational modifications on transcription factors by targeted mass spectrometry is hampered by low protein abundance and inefficient isolation. Here, we utilized HaloTag technology to overcome these limitations and evaluate various top down mass spectrometry approaches for measuring NF-κB p65 proteoforms isolated from human(More)
Histones, and their modifications, are critical components of cellular programming and epigenetic inheritance. Recently, cancer genome sequencing has uncovered driver mutations in chromatin modifying enzymes spurring high interest how such mutations change histone modification patterns. Here, we applied Top-Down mass spectrometry for the characterization of(More)
Efforts to map the human protein interactome have resulted in information about thousands of multi-protein assemblies housed in public repositories, but the molecular characterization and stoichiometry of their protein subunits remains largely unknown. Here, we report a computational search strategy that supports hierarchical top-down analysis for precise(More)
The automated processing of data generated by top down proteomics would benefit from improved scoring for protein identification and characterization of highly related protein forms (proteoforms). Here we propose the "C-score" (short for Characterization Score), a Bayesian approach to the proteoform identification and characterization problem, implemented(More)
Top-down proteomics is capable of identifying and quantitating unique proteoforms through the analysis of intact proteins. We extended the coverage of the label-free technique, achieving differential analysis of whole proteins <30 kDa from the proteomes of growing and senescent human fibroblasts. By integrating improved control software with more instrument(More)