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For proteins of < 20 kDa, this new radical site dissociation method cleaves different and many more backbone bonds than the conventional MS/MS methods (e.g., collisionally activated dissociation, CAD) that add energy directly to the even-electron ions. A minimum kinetic energy difference between the electron and ion maximizes capture; a 1 eV difference reduces capture by 10(3). Thus, in an FTMS ion cell with added electron trapping electrodes, capture appears to be achieved best at the boundary between the potential wells that trap the electrons and ions, now providing 80 +/- 15% precursor ion conversion efficiency. Capture cross section is dependent on the ionic charge squared (z2), minimizing the secondary dissociation of lower charge fragment ions. Electron capture is postulated to occur initially at a protonated site to release an energetic (approximately 6 eV) H. atom that is captured at a high-affinity site such as -S-S- or backbone amide to cause nonergodic (before energy randomization) dissociation. Cleavages between every pair of amino acids in mellitin (2.8 kDa) and ubiquitin (8.6 kDa) are represented in their ECD and CAD spectra, providing complete data for their de novo sequencing. Because posttranslational modifications such as carboxylation, glycosylation, and sulfation are less easily lost in ECD than in CAD, ECD assignments of their sequence positions are far more specific.

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The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B-cells and targeted by somatic mutations in B-cell lymphomas. Here we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions in mice. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B-cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets in B-cells, and in human B-cell lymphomas. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GCB-type DLBCLs are mostly addicted to EZH2, regardless of mutation status, but not the more differentiated ABC-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting.

A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large scale proteome analysis1 has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry (MS)2. This “bottom up” process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous2 characterization of alternative splice forms, diverse modifications (e.g., acetylation and methylation), and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species3. “Top down” interrogation of whole proteins can overcome these problems for individual proteins4,5, but has not been achieved on a proteome scale due to the lack of intact protein fractionation methods that are well integrated with tandem MS. Here we show, using a new four dimensional (4D) separation system, identification of 1,043 gene products from human cells that are dispersed into >3,000 protein species created by post-translational modification, RNA splicing, and proteolysis. The overall system produced >20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kilodaltons and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply-modified species in response to accelerated cellular aging (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database6, the data provide precise correlations to individual genes and proof-of-concept for large scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research7.

Characterization of larger proteins by mass spectrometry (MS) is especially promising because the information complements that of classical techniques and can be obtained on as little as 10-17 mol of protein. Using MS to localize errors in the DNA-derived sequence or modifications (posttranslational, derivatized active sites, etc.) usually involves extensive proteolysis to yield peptides of <3 kDa, with separation and MS/MS to compare their sequences to those expected (the “bottom up” approach). In contrast, an alternative “top down” approach limits the dissociation (proteolysis or MS/MS) to yield larger products from which a small set of complementary peptides can be found whose masses sum to those of the molecule. Thus a disagreement with the predicted molecular mass can be localized to a fragment(s) without examining all others, with further dissociation of the fragments in the same way providing further localization. Using carbonic anhydrase (29 kDa) as an example, Fourier transform mass spectrometry is unusually effective for the bottom up approach, in that a single spectrum of an extensive chymotryptic digest identifies 64 expected peptides, but these only cover 95% of the sequence; 20 fragment masses are unassigned so that any set whose masses sum to that of the molecule would be misleading. Extensive Lys-C dissociation yields 17 peptides, 23 unassigned masses, and 96% coverage. In the contrasting “top down” approach, less extensive initial dissociation by Lys-C, MS/MS, or CNBr in each case provides 100% coverage, so that modified protein fragment(s) could easily be recognized among the complementary sets. MS/MS of such a fragment or more extensive proteolysis provide further localization of the modification. The combined methods cleaved 137 of the 258 amide bonds between residues.

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 fragmentation of proteins up to 29 kDa is achieved with UVPD including the unambiguous localization of a single residue mutation and several protein modifications on Pin1 (Q13526), a protein implicated in the development of Alzheimer’s disease and in cancer pathogenesis. The 5 nanosecond, high-energy activation afforded by UVPD exhibits far less precursor ion-charge state dependence than conventional collision-based and electron-based dissociation methods.

Genome mining has become a key technology to exploit natural product diversity. While initially performed on a single-genome basis, the process is now being scaled up to mine entire genera, strain collections and microbiomes. However, no bioinformatic framework is currently available for effectively analyzing datasets of this size and complexity. Here, we provide a streamlined computational workflow consisting of two new software tools: The 'Biosynthetic Gene Similarity Clustering And Prospecting Engine' (BiG-SCAPE) facilitates fast and interactive sequence similarity network analysis of biosynthetic gene clusters and gene cluster families. 'CORe Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: