Proteogenomics of Non-smoking Lung Cancer in East Asia Delineates Molecular Signatures of Pathogenesis and Progression.

Lung cancer in East Asia is characterized by a high percentage of never-smokers, early onset and predominant EGFR mutations. To illuminate the molecular phenotype of this demographically distinct disease, we performed a deep comprehensive proteogenomic study on a prospectively collected cohort in Taiwan, representing early stage, predominantly female, non-smoking lung adenocarcinoma. Integrated genomic, proteomic, and phosphoproteomic analysis delineated the demographically distinct molecular attributes and hallmarks of tumor progression. Mutational signature analysis revealed age- and gender-related mutagenesis mechanisms, characterized by high prevalence of APOBEC mutational signature in younger females and over-representation of environmental carcinogen-like mutational signatures in older females. A proteomics-informed classification distinguished the clinical characteristics of early stage patients with EGFR mutations. Furthermore, integrated protein network analysis revealed the cellular remodeling underpinning clinical trajectories and nominated candidate biomarkers for patient stratification and therapeutic intervention. This multi-omic molecular architecture may help develop strategies for management of early stage never-smoker lung adenocarcinoma.

A RIPK1-specific PROTAC degrader achieves potent antitumor activity by enhancing immunogenic cell death.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies.

PBAF loss leads to DNA damage-induced inflammatory signaling through defective G2/M checkpoint maintenance.

The PBRM1 subunit of the PBAF (SWI/SNF) chromatin remodeling complex is mutated in ∼40% of clear cell renal cancers. PBRM1 loss has been implicated in responses to immunotherapy in renal cancer, but the mechanism is unclear. DNA damage-induced inflammatory signaling is an important factor determining immunotherapy response. This response is kept in check by the G2/M checkpoint, which prevents progression through mitosis with unrepaired damage. We found that in the absence of PBRM1, p53-dependent p21 up-regulation is delayed after DNA damage, leading to defective transcriptional repression by the DREAM complex and premature entry into mitosis. Consequently, DNA damage-induced inflammatory signaling pathways are activated by cytosolic DNA. Notably, p53 is infrequently mutated in renal cancer, so PBRM1 mutational status is critical to G2/M checkpoint maintenance. Moreover, we found that the ability of PBRM1 deficiency to predict response to immunotherapy correlates with expression of the cytosolic DNA-sensing pathway in clinical samples. These findings have implications for therapeutic responses in renal cancer.

Spatial control of the APC/C ensures the rapid degradation of cyclin B1.

The proper control of mitosis depends on the ubiquitin-mediated degradation of the right mitotic regulator at the right time. This is effected by the Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase that is regulated by the Spindle Assembly Checkpoint (SAC). The SAC prevents the APC/C from recognising Cyclin B1, the essential anaphase and cytokinesis inhibitor, until all chromosomes are attached to the spindle. Once chromosomes are attached, Cyclin B1 is rapidly degraded to enable chromosome segregation and cytokinesis. We have a good understanding of how the SAC inhibits the APC/C, but relatively little is known about how the APC/C recognises Cyclin B1 as soon as the SAC is turned off. Here, by combining live-cell imaging, in vitro reconstitution biochemistry, and structural analysis by cryo-electron microscopy, we provide evidence that the rapid recognition of Cyclin B1 in metaphase requires spatial regulation of the APC/C. Using fluorescence cross-correlation spectroscopy, we find that Cyclin B1 and the APC/C primarily interact at the mitotic apparatus. We show that this is because Cyclin B1, like the APC/C, binds to nucleosomes, and identify an 'arginine-anchor' in the N-terminus as necessary and sufficient for binding to the nucleosome. Mutating the arginine anchor on Cyclin B1 reduces its interaction with the APC/C and delays its degradation: cells with the mutant, non-nucleosome-binding Cyclin B1 become aneuploid, demonstrating the physiological relevance of our findings. Together, our data demonstrate that mitotic chromosomes promote the efficient interaction between Cyclin B1 and the APC/C to ensure the timely degradation of Cyclin B1 and genomic stability.

PhoXplex: Combining Phospho-enrichable Cross-Linking with Isobaric Labeling for Quantitative Proteome-Wide Mapping of Protein Interfaces.

Integrating cross-linking mass spectrometry (XL-MS) into structural biology workflows provides valuable information about the spatial arrangement of amino acid stretches, which can guide elucidation of protein assembly architecture. Additionally, the combination of XL-MS with peptide quantitation techniques is a powerful approach to delineate protein interface dynamics across diverse conditions. While XL-MS is increasingly effective with isolated proteins or small complexes, its application to whole-cell samples poses technical challenges related to analysis depth and throughput. The use of enrichable cross-linkers has greatly improved the detectability of protein interfaces in a proteome-wide scale, facilitating global protein-protein interaction mapping. Therefore, bringing together enrichable cross-linking and multiplexed peptide quantification is an appealing approach to enable comparative characterization of structural attributes of proteins and protein interactions. Here, we combined phospho-enrichable cross-linking with TMT labeling to develop a streamline workflow (PhoXplex) for the detection of differential structural features across a panel of cell lines in a global scale. We achieved deep coverage with quantification of over 9000 cross-links and long loop-links in total including potentially novel interactions. Overlaying AlphaFold predictions and disorder protein annotations enables exploration of the quantitative cross-linking data set, to reveal possible associations between mutations and protein structures. Lastly, we discuss current shortcomings and perspectives for deep whole-cell profiling of protein interfaces at large-scale.

Secretome of Cancer-Associated Fibroblasts (CAFs) Influences Drug Sensitivity in Cancer Cells.

Resistance is a major problem with effective cancer treatment and the stroma forms a significant portion of the tumor mass but traditional drug screens involve cancer cells alone. Cancer-associated fibroblasts (CAFs) are a major tumor stroma component and its secreted proteins may influence the function of cancer cells. The majority of secretome studies compare different cancer or CAF cell lines exclusively. Here, we present the direct characterization of the secreted protein profiles between CAFs and KRAS mutant-cancer cell lines from colorectal, lung, and pancreatic tissues using multiplexed mass spectrometry. 2573 secreted proteins were annotated, and differential analysis highlighted understudied CAF-enriched secreted proteins, including Wnt family member 5B (WNT5B), in addition to established CAF markers, such as collagens. The functional role of CAF secreted proteins was explored by assessing its effect on the response to 97 anticancer drugs since stromal cells may cause a differing cancer drug response, which may be missed on routine drug screening using cancer cells alone. CAF secreted proteins caused specific effects on each of the cancer cell lines, which highlights the complexity and challenges in cancer treatment and so the importance to consider stromal elements.

EPEC-induced activation of the Ca2+ transporter TRPV2 leads to pyroptotic cell death.

The enteropathogenic Escherichia coli (EPEC) type III secretion system effector Tir, which mediates intimate bacterial attachment to epithelial cells, also triggers Ca2+ influx followed by LPS entry and caspase-4-dependent pyroptosis, which could be antagonized by the effector NleF. Here we reveal the mechanism by which EPEC induces Ca2+ influx. We show that in the intestinal epithelial cell line SNU-C5, Tir activates the mechano/osmosensitive cation channel TRPV2 which triggers extracellular Ca2+ influx. Tir-induced Ca2+ influx could be blocked by siRNA silencing of TRPV2, pre-treatment with the TRPV2 inhibitor SET2 or by growing cells in low osmolality medium. Pharmacological activation of TRPV2 in the absence of Tir failed to initiate caspase-4-dependent cell death, confirming the necessity of Tir. Consistent with the model implicating activation on translocation of TRPV2 from the ER to plasma membrane, inhibition of protein trafficking by either brefeldin A or the effector NleA prevented TRPV2 activation and cell death. While infection with EPECΔnleA triggered pyroptotic cell death, this could be prevented by NleF. Taken together this study shows that while integration of Tir into the plasma membrane activates TRPV2, EPEC uses NleA to inhibit TRPV2 trafficking and NleF to inhibit caspase-4 and pyroptosis.

Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx-dependent pyroptosis in intestinal epithelial cells.

Clustering of the enteropathogenic Escherichia coli (EPEC) type III secretion system (T3SS) effector translocated intimin receptor (Tir) by intimin leads to actin polymerisation and pyroptotic cell death in macrophages. The effect of Tir clustering on the viability of EPEC-infected intestinal epithelial cells (IECs) is unknown. We show that EPEC induces pyroptosis in IECs in a Tir-dependent but actin polymerisation-independent manner, which was enhanced by priming with interferon gamma (IFNγ). Mechanistically, Tir clustering triggers rapid Ca2+ influx, which induces lipopolysaccharide (LPS) internalisation, followed by activation of caspase-4 and pyroptosis. Knockdown of caspase-4 or gasdermin D (GSDMD), translocation of NleF, which blocks caspase-4 or chelation of extracellular Ca2+, inhibited EPEC-induced cell death. IEC lines with low endogenous abundance of GSDMD were resistant to Tir-induced cell death. Conversely, ATP-induced extracellular Ca2+ influx enhanced cell death, which confirmed the key regulatory role of Ca2+ in EPEC-induced pyroptosis. We reveal a novel mechanism through which infection with an extracellular pathogen leads to pyroptosis in IECs.

GENCODE 2021.

The GENCODE project annotates human and mouse genes and transcripts supported by experimental data with high accuracy, providing a foundational resource that supports genome biology and clinical genomics. GENCODE annotation processes make use of primary data and bioinformatic tools and analysis generated both within the consortium and externally to support the creation of transcript structures and the determination of their function. Here, we present improvements to our annotation infrastructure, bioinformatics tools, and analysis, and the advances they support in the annotation of the human and mouse genomes including: the completion of first pass manual annotation for the mouse reference genome; targeted improvements to the annotation of genes associated with SARS-CoV-2 infection; collaborative projects to achieve convergence across reference annotation databases for the annotation of human and mouse protein-coding genes; and the first GENCODE manually supervised automated annotation of lncRNAs. Our annotation is accessible via Ensembl, the UCSC Genome Browser and https://www.gencodegenes.org.

SimPLIT: Simplified Sample Preparation for Large-Scale Isobaric Tagging Proteomics.

Large scale proteomic profiling of cell lines can reveal molecular signatures attributed to variable genotypes or induced perturbations, enabling proteogenomic associations and elucidation of pharmacological mechanisms of action. Although isobaric labeling has increased the throughput of proteomic analysis, the commonly used sample preparation workflows often require time-consuming steps and costly consumables, limiting their suitability for large scale studies. Here, we present a simplified and cost-effective one-pot reaction workflow in a 96-well plate format (SimPLIT) that minimizes processing steps and demonstrates improved reproducibility compared to alternative approaches. The workflow is based on a sodium deoxycholate lysis buffer and a single detergent cleanup step after peptide labeling, followed by quick off-line fractionation and MS2 analysis. We showcase the applicability of the workflow in a panel of colorectal cancer cell lines and by performing target discovery for a set of molecular glue degraders in different cell lines, in a 96-sample assay. Using this workflow, we report frequently dysregulated proteins in colorectal cancer cells and uncover cell-dependent protein degradation profiles of seven cereblon E3 ligase modulators (CRL4CRBN). Overall, SimPLIT is a robust method that can be easily implemented in any proteomics laboratory for medium-to-large scale TMT-based studies for deep profiling of cell lines.

Discovery of high-confidence human protein-coding genes and exons by whole-genome PhyloCSF helps elucidate 118 GWAS loci.

The most widely appreciated role of DNA is to encode protein, yet the exact portion of the human genome that is translated remains to be ascertained. We previously developed PhyloCSF, a widely used tool to identify evolutionary signatures of protein-coding regions using multispecies genome alignments. Here, we present the first whole-genome PhyloCSF prediction tracks for human, mouse, chicken, fly, worm, and mosquito. We develop a workflow that uses machine learning to predict novel conserved protein-coding regions and efficiently guide their manual curation. We analyze more than 1000 high-scoring human PhyloCSF regions and confidently add 144 conserved protein-coding genes to the GENCODE gene set, as well as additional coding regions within 236 previously annotated protein-coding genes, and 169 pseudogenes, most of them disabled after primates diverged. The majority of these represent new discoveries, including 70 previously undetected protein-coding genes. The novel coding genes are additionally supported by single-nucleotide variant evidence indicative of continued purifying selection in the human lineage, coding-exon splicing evidence from new GENCODE transcripts using next-generation transcriptomic data sets, and mass spectrometry evidence of translation for several new genes. Our discoveries required simultaneous comparative annotation of other vertebrate genomes, which we show is essential to remove spurious ORFs and to distinguish coding from pseudogene regions. Our new coding regions help elucidate disease-associated regions by revealing that 118 GWAS variants previously thought to be noncoding are in fact protein altering. Altogether, our PhyloCSF data sets and algorithms will help researchers seeking to interpret these genomes, while our new annotations present exciting loci for further experimental characterization.

The type III secretion system effector EspO of enterohaemorrhagic Escherichia coli inhibits apoptosis through an interaction with HAX-1.

Many enteric pathogens employ a type III secretion system (T3SS) to translocate effector proteins directly into the host cell cytoplasm, where they subvert signalling pathways of the intestinal epithelium. Here, we report that the anti-apoptotic regulator HS1-associated protein X1 (HAX-1) is an interaction partner of the T3SS effectors EspO of enterohaemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, OspE of Shigella flexneri and Osp1STYM of Salmonella enterica serovar Typhimurium. EspO, OspE and Osp1STYM have previously been reported to interact with the focal adhesions protein integrin linked kinase (ILK). We found that EspO localizes both to the focal adhesions (ILK localisation) and mitochondria (HAX-1 localisation), and that increased expression of HAX-1 leads to enhanced mitochondrial localisation of EspO. Ectopic expression of EspO, OspE and Osp1STYM protects cells from apoptosis induced by staurosporine and tunicamycin. Depleting cells of HAX-1 indicates that the anti-apoptotic activity of EspO is HAX-1 dependent. Both HAX-1 and ILK were further confirmed as EspO1-interacting proteins during infection using T3SS-delivered EspO1. Using cell detachment as a proxy for cell death we confirmed that T3SS-delivered EspO1 could inhibit cell death induced during EPEC infection, to a similar extent as the anti-apoptotic effector NleH, or treatment with the pan caspase inhibitor z-VAD. In contrast, in cells lacking HAX-1, EspO1 was no longer able to protect against cell detachment, while NleH1 and z-VAD maintained their protective activity. Therefore, during both infection and ectopic expression EspO protects cells from cell death by interacting with HAX-1. These results suggest that despite the differences between EHEC, C. rodentium, Shigella and S. typhimurium infections, hijacking HAX-1 anti-apoptotic signalling is a common strategy to maintain the viability of infected cells. TAKE AWAY: EspO homologues are found in EHEC, Shigella, S. typhimurium and some EPEC. EspO homologues interact with HAX-1. EspO protects infected cells from apoptosis. EspO joins a growing list of T3SS effectors that manipulate cell death pathways.

Citrobacter rodentium induces rapid and unique metabolic and inflammatory responses in mice suffering from severe disease.

The mouse pathogen Citrobacter rodentium is used to model infections with enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC). Pathogenesis is commonly modelled in mice developing mild disease (e.g., C57BL/6). However, little is known about host responses in mice exhibiting severe colitis (e.g., C3H/HeN), which arguably provide a more clinically relevant model for human paediatric enteric infection. Infection of C3H/HeN mice with C. rodentium results in rapid colonic colonisation, coinciding with induction of key inflammatory signatures and colonic crypt hyperplasia. Infection also induces dramatic changes to bioenergetics in intestinal epithelial cells, with transition from oxidative phosphorylation (OXPHOS) to aerobic glycolysis and higher abundance of SGLT4, LDHA, and MCT4. Concomitantly, mitochondrial proteins involved in the TCA cycle and OXPHOS were in lower abundance. Similar to observations in C57BL/6 mice, we detected simultaneous activation of cholesterol biogenesis, import, and efflux. Distinctly, however, the pattern recognition receptors NLRP3 and ALPK1 were specifically induced in C3H/HeN. Using cell-based assays revealed that C. rodentium activates the ALPK1/TIFA axis, which is dependent on the ADP-heptose biosynthesis pathway but independent of the Type III secretion system. This study reveals for the first time the unfolding intestinal epithelial cells' responses during severe infectious colitis, which resemble EPEC human infections.

GENCODE reference annotation for the human and mouse genomes.

The accurate identification and description of the genes in the human and mouse genomes is a fundamental requirement for high quality analysis of data informing both genome biology and clinical genomics. Over the last 15 years, the GENCODE consortium has been producing reference quality gene annotations to provide this foundational resource. The GENCODE consortium includes both experimental and computational biology groups who work together to improve and extend the GENCODE gene annotation. Specifically, we generate primary data, create bioinformatics tools and provide analysis to support the work of expert manual gene annotators and automated gene annotation pipelines. In addition, manual and computational annotation workflows use any and all publicly available data and analysis, along with the research literature to identify and characterise gene loci to the highest standard. GENCODE gene annotations are accessible via the Ensembl and UCSC Genome Browsers, the Ensembl FTP site, Ensembl Biomart, Ensembl Perl and REST APIs as well as https://www.gencodegenes.org.

Cell-type-specific visualisation and biochemical isolation of endogenous synaptic proteins in mice.

In recent years, the remarkable molecular complexity of synapses has been revealed, with over 1,000 proteins identified in the synapse proteome. Although it is known that different receptors and other synaptic proteins are present in different types of neurons, the extent of synapse diversity across the brain is largely unknown. This is mainly due to the limitations of current techniques. Here, we report an efficient method for the purification of synaptic protein complexes, fusing a high-affinity tag to endogenous PSD95 in specific cell types. We also developed a strategy, which enables the visualisation of endogenous PSD95 with fluorescent-protein tag in Cre-recombinase-expressing cells. We demonstrate the feasibility of proteomic analysis of synaptic protein complexes and visualisation of these in specific cell types. We find that the composition of PSD95 complexes purified from specific cell types differs from those extracted from tissues with diverse cellular composition. The results suggest that there might be differential interactions in the PSD95 complexes in different brain regions. We have detected differentially interacting proteins by comparing data sets from the whole hippocampus and the CA3 subfield of the hippocampus. Therefore, these novel conditional PSD95 tagging lines will not only serve as powerful tools for precisely dissecting synapse diversity in specific brain regions and subsets of neuronal cells, but also provide an opportunity to better understand brain region- and cell-type-specific alterations associated with various psychiatric/neurological diseases. These newly developed conditional gene tagging methods can be applied to many different synaptic proteins and will facilitate research on the molecular complexity of synapses.

The Citrobacter rodentium type III secretion system effector EspO affects mucosal damage repair and antimicrobial responses.

Infection with Citrobacter rodentium triggers robust tissue damage repair responses, manifested by secretion of IL-22, in the absence of which mice succumbed to the infection. Of the main hallmarks of C. rodentium infection are colonic crypt hyperplasia (CCH) and dysbiosis. In order to colonize the host and compete with the gut microbiota, C. rodentium employs a type III secretion system (T3SS) that injects effectors into colonic intestinal epithelial cells (IECs). Once injected, the effectors subvert processes involved in innate immune responses, cellular metabolism and oxygenation of the mucosa. Importantly, the identity of the effector/s triggering the tissue repair response is/are unknown. Here we report that the effector EspO ,an orthologue of OspE found in Shigella spp, affects proliferation of IECs 8 and 14 days post C. rodentium infection as well as secretion of IL-22 from colonic explants. While we observed no differences in the recruitment of group 3 innate lymphoid cells (ILC3s) and T cells, which are the main sources of IL-22 at the early and late stages of C. rodentium infection respectively, infection with ΔespO was characterized by diminished recruitment of sub-mucosal neutrophils, which coincided with lower abundance of Mmp9 and chemokines (e.g. S100a8/9) in IECs. Moreover, mice infected with ΔespO triggered significantly lesser nutritional immunity (e.g. calprotectin, Lcn2) and expression of antimicrobial peptides (Reg3ß, Reg3γ) compared to mice infected with WT C. rodentium. This overlapped with a decrease in STAT3 phosphorylation in IECs. Importantly, while the reduced CCH and abundance of antimicrobial proteins during ΔespO infection did not affect C. rodentium colonization or the composition of commensal Proteobacteria, they had a subtle consequence on Firmicutes subpopulations. EspO is the first bacterial virulence factor that affects neutrophil recruitment and secretion of IL-22, as well as expression of antimicrobial and nutritional immunity proteins in IECs.

Evidence for a novel overlapping coding sequence in POLG initiated at a CUG start codon.

BACKGROUND: POLG, located on nuclear chromosome 15, encodes the DNA polymerase γ(Pol γ). Pol γ is responsible for the replication and repair of mitochondrial DNA (mtDNA). Pol γ is the only DNA polymerase found in mitochondria for most animal cells. Mutations in POLG are the most common single-gene cause of diseases of mitochondria and have been mapped over the coding region of the POLG ORF. RESULTS: Using PhyloCSF to survey alternative reading frames, we found a conserved coding signature in an alternative frame in exons 2 and 3 of POLG, herein referred to as ORF-Y that arose de novo in placental mammals. Using the synplot2 program, synonymous site conservation was found among mammals in the region of the POLG ORF that is overlapped by ORF-Y. Ribosome profiling data revealed that ORF-Y is translated and that initiation likely occurs at a CUG codon. Inspection of an alignment of mammalian sequences containing ORF-Y revealed that the CUG codon has a strong initiation context and that a well-conserved predicted RNA stem-loop begins 14 nucleotides downstream. Such features are associated with enhanced initiation at near-cognate non-AUG codons. Reanalysis of the Kim et al. (2014) draft human proteome dataset yielded two unique peptides that map unambiguously to ORF-Y. An additional conserved uORF, herein referred to as ORF-Z, was also found in exon 2 of POLG. Lastly, we surveyed Clinvar variants that are synonymous with respect to the POLG ORF and found that most of these variants cause amino acid changes in ORF-Y or ORF-Z. CONCLUSIONS: We provide evidence for a novel coding sequence, ORF-Y, that overlaps the POLG ORF. Ribosome profiling and mass spectrometry data show that ORF-Y is expressed. PhyloCSF and synplot2 analysis show that ORF-Y is subject to strong purifying selection. An abundance of disease-correlated mutations that map to exons 2 and 3 of POLG but also affect ORF-Y provides potential clinical significance to this finding.

Proteomic navigation using proximity-labeling.

The identification of bona fide protein-protein interactions and the mapping of proteomes was greatly enhanced by protein tagging for generic affinity purification methods and analysis by mass spectrometry (AP-MS). The high quality of AP-MS data permitted the development of proteomic navigation by sequential tagging of identified interactions. However AP-MS is laborious and limited to relatively high affinity protein-protein interactions. Proximity labeling, first with the biotin ligase BirA, termed BioID, and then with ascorbate peroxidase, termed APEX, permits a greater reach into the proteome than AP-MS enabling both the identification of a wider field and weaker protein-protein interactions. This additional reach comes with the need for stringent controls. Proximity labeling also permits experiments in living cells allowing spatiotemporal investigations of the proteome. Here we discuss proximity labeling with accompanying methodological descriptions for E. coli and mammalian cells.

A polygenic burden of rare disruptive mutations in schizophrenia.

Schizophrenia is a common disease with a complex aetiology, probably involving multiple and heterogeneous genetic factors. Here, by analysing the exome sequences of 2,536 schizophrenia cases and 2,543 controls, we demonstrate a polygenic burden primarily arising from rare (less than 1 in 10,000), disruptive mutations distributed across many genes. Particularly enriched gene sets include the voltage-gated calcium ion channel and the signalling complex formed by the activity-regulated cytoskeleton-associated scaffold protein (ARC) of the postsynaptic density, sets previously implicated by genome-wide association and copy-number variation studies. Similar to reports in autism, targets of the fragile X mental retardation protein (FMRP, product of FMR1) are enriched for case mutations. No individual gene-based test achieves significance after correction for multiple testing and we do not detect any alleles of moderately low frequency (approximately 0.5 to 1 per cent) and moderately large effect. Taken together, these data suggest that population-based exome sequencing can discover risk alleles and complements established gene-mapping paradigms in neuropsychiatric disease.

Evaluation of a Dual Isolation Width Acquisition Method for Isobaric Labeling Ratio Decompression.

Isobaric labeling is a highly precise approach for protein quantification. However, due to the isolation interference problem, isobaric tagging suffers from ratio underestimation at the MS2 level. The use of narrow isolation widths is a rational approach to alleviate the interference problem; however, this approach compromises proteome coverage. We reasoned that although a very narrow isolation window will result in loss of peptide fragment ions, the reporter ion signals will be retained for a significant portion of the spectra. On the basis of this assumption, we have designed a dual isolation width acquisition (DIWA) method, in which each precursor is first fragmented with HCD using a standard isolation width for peptide identification and preliminary quantification, followed by a second MS2 HCD scan using a much narrower isolation width for the acquisition of quantitative spectra with reduced interference. We leverage the quantification obtained by the "narrow" scans to build linear regression models and apply these to decompress the fold-changes measured at the "standard" scans. We evaluate the DIWA approach using a nested two species/gene knockout TMT-6plex experimental design and discuss the perspectives of this approach.