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Identifying precise molecular forms of proteins can improve our understanding of function Proteoforms—the different forms of proteins produced from the genome with a variety of sequence variations, splice isoforms, and myriad posttranslational modifications ( 1 )—are critical elements in all biological systems (see the figure, left). Yang et al. ( 2 ) recently showed that the functions of proteins produced from splice variants from a given gene—different proteoforms—can be as different as those for proteins encoded by entirely different genes. Li et al. ( 3 ) showed that splice variants play a central role in modulating complex traits. However, the standard paradigm of proteomic analysis, the “bottom-up” strategy pioneered by Eng and Yates some 20 years ago ( 4 ), does not directly identify proteoforms. We argue that proteomic analysis needs to provide the identities and abundances of the proteoforms themselves, rather than just their peptide surrogates. Developing new proteome-wide strategies to accomplish this goal presents a formidable but not insurmountable technological challenge that will benefit the biomedical community.

Proteomics has progressed radically in the last 5 years and is now on par with most genomic technologies in throughput and comprehensiveness. Analyzing peptide mixtures by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS) has emerged as the main technology for in-depth proteome analysis whereas two-dimensional gel electrophoresis, low-resolution MALDI, and protein arrays are playing niche roles. MS-based proteomics is rapidly becoming quantitative through both label-free and stable isotope labeling technologies. The latest generation of mass spectrometers combines extremely high resolving power, mass accuracy, and very high sequencing speed in routine proteomic applications. Peptide fragmentation is mostly performed in low-resolution but very sensitive and fast linear ion traps. However, alternative fragmentation methods and high-resolution fragment analysis are becoming much more practical. Recent advances in computational proteomics are removing the data analysis bottleneck. Thus, in a few specialized laboratories, \"precision proteomics\" can now identify and quantify almost all fragmented peptide peaks. Huge challenges and opportunities remain in technology development for proteomics; thus, this is not \"the beginning of the end\" but surely \"the end of the beginning.\"

Genome mining has become a key technology to exploit natural product diversity. Although 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. In the present study, a streamlined computational workflow is provided, consisting of two new software tools: the ‘biosynthetic gene similarity clustering and prospecting engine’ (BiG-SCAPE), which facilitates fast and interactive sequence similarity network analysis of biosynthetic gene clusters and gene cluster families; and the ‘core analysis of syntenic orthologues to prioritize natural product gene clusters’ (CORASON), which elucidates phylogenetic relationships within and across these families. BiG-SCAPE is validated by correlating its output to metabolomic data across 363 actinobacterial strains and the discovery potential of CORASON is demonstrated by comprehensively mapping biosynthetic diversity across a range of detoxin/rimosamide-related gene cluster families, culminating in the characterization of seven detoxin analogues. Two bioinformatic tools, BiG-SCAPE and CORASON, enable sequence similarity network and phylogenetic analysis of gene clusters and their families across hundreds of strains and in large datasets, leading to the discovery of new natural products.

Top-down mass spectrometry is an emerging technology which strives to preserve the post-translationally modified forms of proteins present in vivo by measuring them intact, rather than measuring peptides produced from them by proteolysis. The top-down technology is beginning to capture the interest of biologists and mass spectrometrists alike, with a main goal of deciphering interaction networks operative in cellular pathways. Here we outline recent approaches and applications of top-down mass spectrometry as well as an outlook for its future.

Nucleosomes containing mutant K27M histones in diffuse intrinsic pediatric gliomas (DIPG) exclude PRC2 binding and recruit BET bromodomain proteins; however, residual PRC2-dependent repression of specific loci, is required for DIPG oncogenesis. These results provide a rationale for targeting these epigenetic regulators in patients. Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brainstem tumor characterized by rapid and uniform patient demise 1 . A heterozygous point mutation of histone H3 occurs in more than 80% of these tumors and results in a lysine-to-methionine substitution (H3K27M) 2 , 3 . Expression of this histone mutant is accompanied by a reduction in the levels of polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation (H3K27me3), and this is hypothesized to be a driving event of DIPG oncogenesis 4 , 5 . Despite a major loss of H3K27me3, PRC2 activity is still detected in DIPG cells positive for H3K27M 6 , 7 . To investigate the functional roles of H3K27M and PRC2 in DIPG pathogenesis, we profiled the epigenome of H3K27M-mutant DIPG cells and found that H3K27M associates with increased H3K27 acetylation (H3K27ac). In accordance with previous biochemical data 5 , the majority of the heterotypic H3K27M-K27ac nucleosomes colocalize with bromodomain proteins at the loci of actively transcribed genes, whereas PRC2 is excluded from these regions; this suggests that H3K27M does not sequester PRC2 on chromatin. Residual PRC2 activity is required to maintain DIPG proliferative potential, by repressing neuronal differentiation and function. Finally, to examine the therapeutic potential of blocking the recruitment of bromodomain proteins by heterotypic H3K27M-K27ac nucleosomes in DIPG cells, we performed treatments in vivo with BET bromodomain inhibitors and demonstrate that they efficiently inhibit tumor progression, thus identifying this class of compounds as potential therapeutics in DIPG.

Mutations in IDH1 and IDH2 (encoding isocitrate dehydrogenase 1 and 2) drive the development of gliomas and other human malignancies. Mutant IDH1 induces epigenetic changes that promote tumorigenesis, but the scale and reversibility of these changes are unknown. Here, using human astrocyte and glioma tumorsphere systems, we generate a large-scale atlas of mutant-IDH1-induced epigenomic reprogramming. We characterize the reversibility of the alterations in DNA methylation, the histone landscape, and transcriptional reprogramming that occur following IDH1 mutation. We discover genome-wide coordinate changes in the localization and intensity of multiple histone marks and chromatin states. Mutant IDH1 establishes a CD24 + population with a proliferative advantage and stem-like transcriptional features. Strikingly, prolonged exposure to mutant IDH1 results in irreversible genomic and epigenetic alterations. Together, these observations provide unprecedented high-resolution molecular portraits of mutant-IDH1-dependent epigenomic reprogramming. These findings have substantial implications for understanding of mutant IDH function and for optimizing therapeutic approaches to targeting IDH-mutant tumors. This study uses human astrocytes and glioma tumorspheres to generate an atlas of mutant-IDH1-induced epigenomic reprogramming. The findings have implications for understanding mutant IDH function and for optimizing approaches to target IDH-mutant tumors.

Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p -acetyl- l -phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries. Cell-free protein synthesis allows for producing proteins without the need of a host organism, thus sparing the researcher experimental hassle. Here, the authors developed a cell-free synthesis method that enables incorporating non-standard amino acids in the product.

Ali Shilatifard and colleagues generate Drosophila lines expressing catalytically deficient Trr, which normally deposits H3K4me1 at enhancers. Trr mutants undergo normal development and show minimal changes in gene expression. Histone H3 lysine 4 monomethylation (H3K4me1) is an evolutionarily conserved feature of enhancer chromatin catalyzed by the COMPASS-like methyltransferase family, which includes Trr in Drosophila melanogaster and MLL3 (encoded by KMT2C ) and MLL4 (encoded by KMT2D ) in mammals 1 , 2 , 3 . Here we demonstrate that Drosophila embryos expressing catalytically deficient Trr eclose and develop to productive adulthood. Parallel experiments with a trr allele that augments enzyme product specificity show that conversion of H3K4me1 at enhancers to H3K4me2 and H3K4me3 is also compatible with life and results in minimal changes in gene expression. Similarly, loss of the catalytic SET domains of MLL3 and MLL4 in mouse embryonic stem cells (mESCs) does not disrupt self-renewal. Drosophila embryos with trr alleles encoding catalytic mutants manifest subtle developmental abnormalities when subjected to temperature stress or altered cohesin levels. Collectively, our findings suggest that animal development can occur in the context of Trr or mammalian COMPASS-like proteins deficient in H3K4 monomethylation activity and point to a possible role for H3K4me1 on cis -regulatory elements in specific settings to fine-tune transcriptional regulation in response to environmental stress.

Non-standard amino acids are incorporated into proteins at large numbers of sites using evolved translation components in recoded bacteria. Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technology has been largely restricted to proteins containing a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein production for p -acetyl- L -phenylalanine and p -azido- L -phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled production of elastin-like-polypeptides with 30 nsAA residues at high yields (∼50 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.