Heat 'n Beat: A Universal High-Throughput End-to-End Proteomics Sample Processing Platform in under an Hour.

Proteomic analysis by mass spectrometry of small (≤2 mg) solid tissue samples from diverse formats requires high throughput and comprehensive proteome coverage. We developed a nearly universal, rapid, and robust protocol for sample preparation, suitable for high-throughput projects that encompass most cell or tissue types. This end-to-end workflow extends from original sample to loading the mass spectrometer and is centered on a one-tube homogenization and digestion method called Heat 'n Beat (HnB). It is applicable to most tissues, regardless of how they were fixed or embedded. Sample preparation was divided into separate challenges. The initial sample washing and final peptide cleanup steps were adapted to three tissue sources: fresh frozen (FF), optimal cutting temperature (OCT) compound embedded (FF-OCT), and formalin-fixed paraffin embedded (FFPE). Third, for core processing, tissue disruption and lysis were decreased to a 7 min heat and homogenization treatment, and reduction, alkylation, and proteolysis were optimized into a single step. The refinements produced near doubled peptide yield when compared to our earlier method ABLE delivered a consistently high digestion efficiency of 85-90%, reported by ProteinPilot, and required only 38 min for core processing in a single tube, with the total processing time being 53-63 min. The robustness of HnB was demonstrated on six organ types, a cell line, and a cancer biopsy. Its suitability for high-throughput applications was demonstrated on a set of 1171 FF-OCT human cancer biopsies, which were processed for end-to-end completion in 92 h, producing highly consistent peptide yield and quality for over 3513 MS runs.

Pan-cancer proteomic map of 949 human cell lines.

The proteome provides unique insights into disease biology beyond the genome and transcriptome. A lack of large proteomic datasets has restricted the identification of new cancer biomarkers. Here, proteomes of 949 cancer cell lines across 28 tissue types are analyzed by mass spectrometry. Deploying a workflow to quantify 8,498 proteins, these data capture evidence of cell-type and post-transcriptional modifications. Integrating multi-omics, drug response, and CRISPR-Cas9 gene essentiality screens with a deep learning-based pipeline reveals thousands of protein biomarkers of cancer vulnerabilities that are not significant at the transcript level. The power of the proteome to predict drug response is very similar to that of the transcriptome. Further, random downsampling to only 1,500 proteins has limited impact on predictive power, consistent with protein networks being highly connected and co-regulated. This pan-cancer proteomic map (ProCan-DepMapSanger) is a comprehensive resource available at https://cellmodelpassports.sanger.ac.uk.

Strategies to enable large-scale proteomics for reproducible research.

Reproducible research is the bedrock of experimental science. To enable the deployment of large-scale proteomics, we assess the reproducibility of mass spectrometry (MS) over time and across instruments and develop computational methods for improving quantitative accuracy. We perform 1560 data independent acquisition (DIA)-MS runs of eight samples containing known proportions of ovarian and prostate cancer tissue and yeast, or control HEK293T cells. Replicates are run on six mass spectrometers operating continuously with varying maintenance schedules over four months, interspersed with ~5000 other runs. We utilise negative controls and replicates to remove unwanted variation and enhance biological signal, outperforming existing methods. We also design a method for reducing missing values. Integrating these computational modules into a pipeline (ProNorM), we mitigate variation among instruments over time and accurately predict tissue proportions. We demonstrate how to improve the quantitative analysis of large-scale DIA-MS data, providing a pathway toward clinical proteomics.

Partitioning global change: Assessing the relative importance of changes in climate and land cover for changes in avian distribution.

Understanding the relative impact of climate change and land cover change on changes in avian distribution has implications for the future course of avian distributions and appropriate management strategies. Due to the dynamic nature of climate change, our goal was to investigate the processes that shape species distributions, rather than the current distributional patterns. To this end, we analyzed changes in the distribution of Eastern Wood Pewees (Contopus virens) and Red-eyed Vireos (Vireo olivaceus) from 1997 to 2012 using Breeding Bird Survey data and dynamic correlated-detection occupancy models. We estimated the local colonization and extinction rates of these species in relation to changes in climate (hours of extreme temperature) and changes in land cover (amount of nesting habitat). We fit six nested models to partition the deviance explained by spatial and temporal components of land cover and climate. We isolated the temporal components of environmental variables because this is the essence of global change. For both species, model fit was significantly improved when we modeled vital rates as a function of spatial variation in climate and land cover. Model fit improved only marginally when we added temporal variation in climate and land cover to the model. Temporal variation in climate explained more deviance than temporal variation in land cover, although both combined only explained 20% (Eastern Wood Pewee) and 6% (Red-eyed Vireo) of temporal variation in vital rates. Our results showing a significant correlation between initial occupancy and environmental covariates are consistent with biological expectation and previous studies. The weak correlation between vital rates and temporal changes in covariates indicated that we have yet to identify the most relevant components of global change influencing the distributions of these species and, more importantly, that spatially significant covariates are not necessarily driving temporal shifts in avian distributions.

Tuning recombinant protein expression to match secretion capacity.

BACKGROUND: The secretion of recombinant disulfide-bond containing proteins into the periplasm of Gram-negative bacterial hosts, such as E. coli, has many advantages that can facilitate product isolation, quality and activity. However, the secretion machinery of E. coli has a limited capacity and can become overloaded, leading to cytoplasmic retention of product; which can negatively impact cell viability and biomass accumulation. Fine control over recombinant gene expression offers the potential to avoid this overload by matching expression levels to the host secretion capacity. RESULTS: Here we report the application of the RiboTite gene expression control system to achieve this by finely controlling cellular expression levels. The level of control afforded by this system allows cell viability to be maintained, permitting production of high-quality, active product with enhanced volumetric titres. CONCLUSIONS: The methods and systems reported expand the tools available for the production of disulfide-bond containing proteins, including antibody fragments, in bacterial hosts.

E. coli strain engineering for the production of advanced biopharmaceutical products.

Since the emergence of the biopharmaceutical industry in the 1980's, Escherichia coli, has played an important role in the industrial production of recombinant proteins and plasmid DNA for therapeutic use. Currently, advanced biopharmaceutical products, including rationally designed recombinant proteins and viral-vector gene therapies, offer unprecedented promise for the long-term management, and even cure of disease. As such, E. coli remains an important production host for the biopharmaceutical industry. This review provides insight into the industrially relevant strain engineering approaches used to enhance both the quantity and quality of these therapeutic products.

Development of a generic ß-lactamase screening system for improved signal peptides for periplasmic targeting of recombinant proteins in Escherichia coli.

Targeting of recombinant proteins to the Escherichia coli periplasm is a desirable industrial processing tool to allow formation of disulphide bonds, aid folding and simplify recovery. Proteins are targeted across the inner membrane to the periplasm by an N-terminal signal peptide. The sequence of the signal peptide determines its functionality, but there is no method to predict signal peptide function for specific recombinant proteins, so multiple signal peptides must be screened for their ability to translocate each recombinant protein, limiting throughput. We present a screening system for optimising signal peptides for translocation of a single chain variable (scFv) antibody fragment employing TEM1 ß-lactamase (Bla) as a C-terminal reporter of periplasmic localisation. The Pectobacterium carotovorum PelB signal peptide was selected as the starting point for a mutagenic screen. ß-lactamase was fused to the C-terminal of scFv and ß-lactamase activity was correlated against scFv translocation. Signal peptide libraries were generated and screened for ß-lactamase activity, which correlated well to scFv::Bla production, although only some high activity clones had improved periplasmic translocation of scFv::Bla. Selected signal peptides were investigated in fed-batch fermentations for production and translocation of scFv::Bla and scFv without the Bla fusion. Improved signal peptides increased periplasmic scFv activity by ~40%.

Optimizing host cell physiology and stress avoidance for the production of recombinant human tumour necrosis factor α in Escherichia coli.

As high-level recombinant protein production (RPP) exerts a massive stress on the production host, an extensive literature on RPP optimization focuses on separating the growth phase from RPP production once sufficient biomass has been obtained. The aim of the current investigation was to optimize the benefits of the relatively neglected alternative strategy to achieve high-level RPP during growth by minimizing stress on the host. High yields of the biopharmaceutical recombinant human tumour necrosis factor alpha (rhTNFα) were obtained by fed-batch fermentation relevant to industrial production based upon parameters that most severely affected RPP in preliminary laboratory scale batch cultures. Decreasing the inducer concentration and growth temperature, but increasing the production period, were far more effective for increasing RPP yields than changing the growth phase at which production was induced. High yields of up to 5 g l-1 of rhTNFα were obtained with minimal plasmid loss, even in synthetic media that lack animal-derived components and are therefore fully compliant with regulatory requirements. Most of the product was soluble and biologically active. In summary, stress minimization was shown to be an effective way to optimize the production of rhTNFα. Data generated in shake-flask experiments allowed the design of intensified bioreactor cultures in which RPP and growth could be balanced, leading to higher yield of both rhTNFα and biomass than with previous fermentations. An additional benefit of this approach is avoidance of lysis during harvesting and downstream processing.

Future land-use scenarios and the loss of wildlife habitats in the southeastern United States.

Land-use change is a major cause of wildlife habitat loss. Understanding how changes in land-use policies and economic factors can impact future trends in land use and wildlife habitat loss is therefore critical for conservation efforts. Our goal here was to evaluate the consequences of future land-use changes under different conservation policies and crop market conditions on habitat loss for wildlife species in the southeastern United States. We predicted the rates of habitat loss for 336 terrestrial vertebrate species by 2051. We focused on habitat loss due to the expansion of urban, crop, and pasture. Future land-use changes following business-as-usual conditions resulted in relatively low rates of wildlife habitat loss across the entire Southeast, but some ecoregions and species groups experienced much higher habitat loss than others. Increased crop commodity prices exacerbated wildlife habitat loss in most ecoregions, while the implementation of conservation policies (reduced urban sprawl, and payments for land conservation) reduced the projected habitat loss in some regions, to a certain degree. Overall, urban and crop expansion were the main drivers of habitat loss. Reptiles and wildlife species associated with open vegetation (grasslands, open woodlands) were the species groups most vulnerable to future land-use change. Effective conservation of wildlife habitat in the Southeast should give special consideration to future land-use changes, regional variations, and the forces that could shape land-use decisions.

An outer-membrane porin inducible by short-chain amides and urea in the methylotrophic bacterium Methylophilus methylotrophus.

The fmdA and fmdB genes encoding formamidase and a putative regulatory protein, respectively, from the methylotrophic bacterium Methylophilus methylotrophus were recloned with additional flanking DNA (pSW1). fmdC, encoding a weakly hydrophilic protein containing an N-terminal signal sequence, was identified upstream of fmdAB. The derived amino acid sequence of mature FmdC (M(r) 39204) showed that it was rich in beta-sheet and aromatic amino acids, and exhibited significant similarities to several outer-membrane porins from other bacteria. Cell fractionation studies showed that the protein was located in the outer membrane. Mature FmdC was purified and shown to consist of a single type of subunit (M(r) 40,000) with the predicted N-terminal amino acid sequence (GATISF-). SDS-PAGE and Western blotting of cells grown in continuous culture under various conditions showed that mature FmdC was induced by formamide, acetamide and urea, repressed by excess ammonia, and over-expressed during prolonged growth under formamide limitation. It is concluded that mature FmdC is a porin involved in the transport of short-chain amides and urea through the outer membrane of M. methylotrophus under conditions where these nitrogen sources are present at very low concentration.

Physiological and biochemical changes accompanying the loss of mucoidy by Pseudomonas aeruginosa.

Pseudomonas aeruginosa M60, a mucoid strain, was grown in continuous culture (D 0.05 h-1) under ammonia limitation with glucose as the carbon source. Steady-state alginate production occurred for only 1-2 d under these conditions [qalginate 0.097 g alginate h-1 (g dry wt cells)-1], after which time the percentage of mucoid cells and the alginate concentration in the culture decreased in parallel and approached zero after approximately 10 d. These changes were accompanied by similar decreases in the activities of the alginate biosynthetic enzymes (represented by phosphomannomutase and GDP-mannose dehydrogenase) and by a large increase in the activity of the first enzyme of the 'external' non-phosphorylative pathway of glucose metabolism, glucose dehydrogenase. In contrast, the activities of other enzymes associated with this pathway (gluconate dehydrogenase, 2-ketogluconate kinase plus 2-ketogluconate-6-phosphate reductase) or with the 'internal' phosphorylative pathway of glucose metabolism (glucokinase and glucose-6-phosphate dehydrogenase) remained essentially unchanged. The loss of mucoidy and alginate production was accompanied by the appearance of low concentrations of intracellular polyhydroxyalkanoate (PHA) and of extracellular gluconate and 2-ketogluconate (partly at the expense of alginate production and partly as a result of increased glucose consumption). It is suggested that ammonia-limited, glucose-excess cultures of P. aeruginosa growing at low dilution rate are unable fully to regulate the rate at which glucose and/or its 'external' pathway metabolites are taken up by the cell, and therefore form copious amounts of alginate in order both to overcome the potentially deleterious osmotic effects of accumulating surplus intracellular metabolites and to consume the surplus ATP generated by the further oxidation of these metabolites. The loss of mucoidy invokes the use of an alternative, but analogous, strategy via which non-mucoid cells produce an osmotically inactive intracellular product (PHA) plus increased amounts of the extracellular metabolites gluconate and 2-ketogluconate via the low-energy-yielding and, under these conditions, largely dead-end 'external' metabolic pathway.