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Intact proteins yield to proteomics Conventional 'bottom-up' proteomics, in which mass spectrometry is used to analyse peptide mixtures made by tryptic digestion of target proteins, is a powerful way of characterizing complex proteomes. However, the technique has limitations when considering different protein isoforms and combinations of post-translational modifications. The 'top-down' approach is generally thought to be impractical because of the limitations of mass spectrometry and difficulties with automation. A new top-down system presented here avoids these problems by using a four-dimensional separation system that achieves greater proteome coverage than conventional methods. A proof-of-principle experiment shows that the method is capable of identifying previously undetected isoforms and isoform-specific post-translational modifications caused by cellular senescence. 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 analysis 1 has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry 2 . This ‘bottom-up’ process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous 2 characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species 3 . ‘Top-down’ interrogation of whole proteins can overcome these problems for individual proteins 4 , 5 , but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here we show, using a new four-dimensional separation system, identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kDa 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 ageing (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database 6 , 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 research 7 .

We have developed a targeted method to quantify all combinations of methylation on an H3 peptide containing lysines 27 and 36 (H3K27-K36). By using stable isotopes that separately label the histone backbone and its methylations, we tracked the rates of methylation and demethylation in myeloma cells expressing high vs. low levels of the methyltransferase MMSET/WHSC1/NSD2. Following quantification of 99 labeled H3K27-K36 methylation states across time, a kinetic model converged to yield 44 effective rate constants qualifying each methylation and demethylation step as a function of the methylation state on the neighboring lysine. We call this approach MS-based measurement and modeling of histone methylation kinetics (M4K). M4K revealed that, when dimethylation states are reached on H3K27 or H3K36, rates of further methylation on the other site are reduced as much as 100-fold. Overall, cells with high MMSET have as much as 33-fold increases in the effective rate constants for formation of H3K36 mono- and dimethylation. At H3K27, cells with high MMSET have elevated formation of K27me1, but even higher increases in the effective rate constants for its reversal by demethylation. These quantitative studies lay bare a bidirectional antagonism between H3K27 and H3K36 that controls the writing and erasing of these methylation marks. Additionally, the integrated kinetic model was used to correctly predict observed abundances of H3K27-K36 methylation states within 5% of that actually established in perturbed cells. Such predictive power for how histone methylations are established should have major value as this family of methyltransferases matures as drug targets.

PARP3 is a member of the ADP-ribosyl transferase superfamily that we show accelerates the repair of chromosomal DNA single-strand breaks in avian DT40 cells. Two-dimensional nuclear magnetic resonance experiments reveal that PARP3 employs a conserved DNA-binding interface to detect and stably bind DNA breaks and to accumulate at sites of chromosome damage. PARP3 preferentially binds to and is activated by mononucleosomes containing nicked DNA and which target PARP3 trans-ribosylation activity to a single-histone substrate. Although nicks in naked DNA stimulate PARP3 autoribosylation, nicks in mononucleosomes promote the trans-ribosylation of histone H2B specifically at Glu2. These data identify PARP3 as a molecular sensor of nicked nucleosomes and demonstrate, for the first time, the ribosylation of chromatin at a site-specific DNA single-strand break. Chromosomal single-strand DNA breaks occur frequently and require repair to avoid disease outcomes. Here, the authors show that in bird cells, PARP3 accelerates this repair, and use structural biology and cell biology techniques to reveal details of the mechanism of action.

The transmembrane receptor 'ROR2' resembles members of the receptor tyrosine kinase family of signalling receptors in sequence but its' signal transduction mechanisms remain enigmatic. This problem has particular importance because mutations in ROR2 are associated with two human skeletal dysmorphology syndromes, recessive Robinow Syndrome (RS) and dominant acting Brachydactyly type B (BDB). Here we show, using a constitutive dimerisation approach, that ROR2 exhibits dimerisation-induced tyrosine kinase activity and the ROR2 C-terminal domain, which is deleted in BDB, is required for recruitment and activation of the non-receptor tyrosine kinase Src. Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity. Eight sites of Src-mediated ROR2 phosphorylation have been identified by mass spectrometry. Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes. These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src.

The Cardiomyopathy–associated gene 5 ( Cmya5 ) encodes myospryn, a large tripartite motif (TRIM)-related protein found predominantly in cardiac and skeletal muscle. Cmya5 is an expression biomarker for a number of diseases affecting striated muscle and may also be a schizophrenia risk gene. To further understand the function of myospryn in striated muscle, we searched for additional myospryn paralogs. Here we identify a novel muscle-expressed TRIM-related protein minispryn, encoded by Fsd2 , that has extensive sequence similarity with the C-terminus of myospryn. Cmya5 and Fsd2 appear to have originated by a chromosomal duplication and are found within evolutionarily-conserved gene clusters on different chromosomes. Using immunoaffinity purification and mass spectrometry we show that minispryn co-purifies with myospryn and the major cardiac ryanodine receptor (RyR2) from heart. Accordingly, myospryn, minispryn and RyR2 co-localise at the junctional sarcoplasmic reticulum of isolated cardiomyocytes. Myospryn redistributes RyR2 into clusters when co-expressed in heterologous cells whereas minispryn lacks this activity. Together these data suggest a novel role for the myospryn complex in the assembly of ryanodine receptor clusters in striated muscle.

Eps8 is involved in both cell signalling and receptor trafficking. It is a known phosphorylation substrate for two proteins involved in the fibroblast growth factor receptor (FGFR) signalling pathway: the receptor itself and Src. Here we report a differential proteomic analysis of Eps8 aimed to identify specific FGFR and Src family kinase dependent phosphosites and co-associated phosphodependent binding partners. This study reveals a total of 22 Eps8 pTyr and pSer/Thr phosphorylation sites, including those that are dependent on Src family and FGFR kinase activity. Peptide affinity purification of proteins that bind to a selection of the pTyr phosphosites has identified a range of novel Eps8 binding partners including members of the intracellular vesicle trafficking machinery (clathrin and AP-2), proteins which have been shown to regulate activated receptor trafficking (NBR1 and Vav2), and proteins involved in receptor signalling (IRS4 and Shp2). Collectively this study significantly extends the understanding of Eps8 post-translational modification by regulated phosphorylation, identifies novel Eps8 binding partners implicated in receptor trafficking and signalling, and confirms the functions of Eps8 at the nexus of receptor signalling and vesicular trafficking.

Prostate cancer is generally considered an immunologically \"cold\" tumor type that is insensitive to immunotherapy. Targeting surface antigens on tumors through cellular therapy can induce a potent antitumor immune response to \"heat up\" the tumor microenvironment. However, many antigens expressed on prostate tumor cells are also found on normal tissues, potentially causing on-target, off-tumor toxicities and a suboptimal therapeutic index. Our studies revealed that six-transmembrane epithelial antigen of prostate-2 (STEAP2) was a prevalent prostate cancer antigen that displayed high, homogeneous cell surface expression across all stages of disease with limited distal normal tissue expression, making it ideal for therapeutic targeting. A multifaceted lead generation approach enabled development of an armored STEAP2 chimeric antigen receptor T cell (CAR-T) therapeutic candidate, AZD0754. This CAR-T product was armored with a dominant-negative TGF-β type II receptor, bolstering its activity in the TGF-β-rich immunosuppressive environment of prostate cancer. AZD0754 demonstrated potent and specific cytotoxicity against antigen-expressing cells in vitro despite TGF-β-rich conditions. Further, AZD0754 enforced robust, dose-dependent in vivo efficacy in STEAP2-expressing cancer cell line-derived and patient-derived xenograft mouse models, and exhibited encouraging preclinical safety. Together, these data underscore the therapeutic tractability of STEAP2 in prostate cancer as well as build confidence in the specificity, potency, and tolerability of this potentially first-in-class CAR-T therapy.

Deletions and chromosome re-arrangements are common features of cancer cells. We have established a new two-component system reporting on epigenetic silencing or deletion of an actively transcribed gene adjacent to a double-strand break (DSB). Unexpectedly, we find that a targeted DSB results in a minority (<10%) misrepair event of kilobase deletions encompassing the DSB site and transcribed gene. Deletions are reduced upon RNaseH1 over-expression and increased after knockdown of the DNA:RNA helicase Senataxin, implicating a role for DNA:RNA hybrids. We further demonstrate that the majority of these large deletions are dependent on the 3′ flap endonuclease XPF. DNA:RNA hybrids were detected by DNA:RNA immunoprecipitation in our system after DSB generation. These hybrids were reduced by RNaseH1 over-expression and increased by Senataxin knock-down, consistent with a role in deletions. Overall, these data are consistent with DNA:RNA hybrid generation at the site of a DSB, mis-processing of which results in genome instability in the form of large deletions.

Prostate cancer is generally considered an immunologically \"cold\" tumor type that is insensitive to immunotherapy. Targeting surface antigens on tumors through cellulartherapy can induce a potent antitumor immune response to \"heat up\" the tumor microenvironment. However, many antigens expressed on prostate tumor cells are also found on normal tissues, potentially causing on-target, off-tumortoxicities and a suboptimal therapeutic index. Our studies revealed that six-transmembrane epithelial antigen of prostate-2 (STEAP2) was a prevalent prostate cancer antigen that displayed high, homogeneous cell surface expression across all stages of disease with limited distal normal tissue expression, making it ideal for therapeutic targeting. A multifaceted lead generation approach enabled development of an armored STEAP2 chimeric antigen receptor T cell (CAR-T) therapeutic candidate, AZD0754. This CAR-T product was armored with a dominant-negative TGF-[beta] type II receptor, bolstering its activity in the TGF-[beta]-rich immunosuppressive environment of prostate cancer. AZD0754 demonstrated potent and specific cytotoxicity against antigen-expressing cells in vitro despite TGF-[beta]-rich conditions. Further, AZD0754 enforced robust, dose-dependent in vivo efficacy in STEAP2- expressing cancer cell line-derived and patient-derived xenograft mouse models, and exhibited encouraging preclinical safety. Together, these data underscore the therapeutic tractability of STEAP2 in prostate cancer as well as build confidence in the specificity, potency, and tolerability of this potentially first-in-class CAR-T therapy.

The working model to describe the mechanisms used to replicate the cancer-associated virus Epstein-Barr virus (EBV) is partly derived from comparisons with other members of the Herpes virus family. Many genes within the EBV genome are homologous across the herpes virus family. Published transcriptome data for the EBV genome during its lytic replication cycle show extensive transcription, but the identification of the proteins is limited. We have taken a global proteomics approach to identify viral proteins that are expressed during the EBV lytic replication cycle. We combined an enrichment method to isolate cells undergoing EBV lytic replication with SILAC-labeling coupled to mass-spectrometry and identified viral and host proteins expressed during the OPEN ACCESS Pathogens 2015, 4 740 EBV lytic replication cycle. Amongst the most frequently identified viral proteins are two components of the DNA replication machinery, the single strand DNA binding protein BALF2, DNA polymerase accessory protein BMRF1 and both subunits of the viral ribonucleoside-diphosphate reductase enzyme (BORF2 and BaRF1). An additional 42 EBV lytic cycle proteins were also detected. This provides proteomic identification for many EBV lytic replication cycle proteins and also identifies post-translational modifications.