The in silico human surfaceome Bausch-Fluck, Damaris; Goldmann, Ulrich; Müller, Sebastian ...
Proceedings of the National Academy of Sciences - PNAS,
11/2018, Letnik:
115, Številka:
46
Journal Article
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Cell-surface proteins are of great biomedical importance, as demonstrated by the fact that 66% of approved human drugs listed in the DrugBank database target a cell-surface protein. Despite this ...biomedical relevance, there has been no comprehensive assessment of the human surfaceome, and only a fraction of the predicted 5,000 human transmembrane proteins have been shown to be located at the plasma membrane. To enable analysis of the human surfaceome, we developed the surfaceome predictor SURFY, based on machine learning. As a training set, we used experimentally verified high-confidence cell-surface proteins from the Cell Surface Protein Atlas (CSPA) and trained a random forest classifier on 131 features per protein and, specifically, per topological domain. SURFY was used to predict a human surfaceome of 2,886 proteins with an accuracy of 93.5%, which shows excellent overlap with known cell-surface protein classes (i.e., receptors). In deposited mRNA data, we found that between 543 and 1,100 surfaceome genes were expressed in cancer cell lines and maximally 1,700 surfaceome genes were expressed in embryonic stem cells and derivative lines. Thus, the surfaceome diversity depends on cell type and appears to be more dynamic than the nonsurface proteome. To make the predicted surfaceome readily accessible to the research community, we provide visualization tools for intuitive interrogation (wlab.ethz.ch/surfaceome). The in silico surfaceome enables the filtering of data generated by multiomics screens and supports the elucidation of the surfaceome nanoscale organization.
In this Perspective, we discuss developments in mass-spectrometry-based proteomic technology over the past decade from the viewpoint of our laboratory. We also reflect on existing challenges and ...limitations, and explore the current and future roles of quantitative proteomics in molecular systems biology, clinical research and personalized medicine.
Deciphering physiological changes that mediate transition of Mycobacterium tuberculosis between replicating and nonreplicating states is essential to understanding how the pathogen can persist in an ...individual host for decades. We have combined RNA sequencing (RNA-seq) of 5′ triphosphate-enriched libraries with regular RNA-seq to characterize the architecture and expression of M. tuberculosis promoters. We identified over 4,000 transcriptional start sites (TSSs). Strikingly, for 26% of the genes with a primary TSS, the site of transcriptional initiation overlapped with the annotated start codon, generating leaderless transcripts lacking a 5′ UTR and, hence, the Shine-Dalgarno sequence commonly used to initiate ribosomal engagement in eubacteria. Genes encoding proteins with active growth functions were markedly depleted from the leaderless transcriptome, and there was a significant increase in the overall representation of leaderless mRNAs in a starvation model of growth arrest. The high percentage of leaderless genes may have particular importance in the physiology of nonreplicating M. tuberculosis.
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•A resource for the identification of in vitro active promoters in M. tuberculosis•A quarter of all genes in M. tuberculosis are expressed as leaderless mRNAs•Leaderless mRNAs are differentially associated with toxin-antitoxin modules•Abundance of leaderless mRNAs increases during starvation-induced growth arrest
In this study, Cortes, Young, and colleagues report genome-wide mapping of transcriptional start sites combined with RNA sequencing and shotgun proteomics in the human pathogen Mycobacterium tuberculosis. A striking finding is a high proportion of genes expressed in the form of leaderless transcripts lacking the Shine-Dalgarno sequence conventionally required for translation. The distribution of functional gene classes between leaderless and Shine-Dalgarno transcriptomes suggests that changes in the specificity of translation may play a role in bacterial adaptation during infection.
Targeted proteomics by selected/multiple reaction monitoring (S/MRM) or, on a larger scale, by SWATH (sequential window acquisition of all theoretical spectra) MS (mass spectrometry) typically relies ...on spectral reference libraries for peptide identification. Quality and coverage of these libraries are therefore of crucial importance for the performance of the methods. Here we present a detailed protocol that has been successfully used to build high-quality, extensive reference libraries supporting targeted proteomics by SWATH MS. We describe each step of the process, including data acquisition by discovery proteomics, assertion of peptide-spectrum matches (PSMs), generation of consensus spectra and compilation of MS coordinates that uniquely define each targeted peptide. Crucial steps such as false discovery rate (FDR) control, retention time normalization and handling of post-translationally modified peptides are detailed. Finally, we show how to use the library to extract SWATH data with the open-source software Skyline. The protocol takes 2-3 d to complete, depending on the extent of the library and the computational resources available.
Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts ...for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts. P. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive. Our results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.
Accurate measurements of cellular protein concentrations are invaluable to quantitative studies of gene expression and physiology in living cells. Here, we developed a versatile mass spectrometric ...workflow based on data‐independent acquisition proteomics (DIA/SWATH) together with a novel protein inference algorithm (xTop). We used this workflow to accurately quantify absolute protein abundances in Escherichia coli for > 2,000 proteins over > 60 growth conditions, including nutrient limitations, non‐metabolic stresses, and non‐planktonic states. The resulting high‐quality dataset of protein mass fractions allowed us to characterize proteome responses from a coarse (groups of related proteins) to a fine (individual) protein level. Hereby, a plethora of novel biological findings could be elucidated, including the generic upregulation of low‐abundant proteins under various metabolic limitations, the non‐specificity of catabolic enzymes upregulated under carbon limitation, the lack of large‐scale proteome reallocation under stress compared to nutrient limitations, as well as surprising strain‐dependent effects important for biofilm formation. These results present valuable resources for the systems biology community and can be used for future multi‐omics studies of gene regulation and metabolic control in E. coli.
Synopsis
Accurate proteomic measurements of absolute protein mass fractions in Escherichia coli allowed the characterization of proteome responses under > 60 diverse growth conditions from a coarse (groups of related proteins) to a fine (individual) protein level.
The study presents a mass spectrometric workflow based on data‐independent acquisition proteomics and a novel protein inference algorithm (xTop) optimized for absolute protein quantification.
The mass spectrometric data was benchmarked and calibrated with absolute protein mass fractions obtained by ribosome profiling.
A plethora of novel biological findings are presented, including lack of large‐scale proteome reallocation under stress compared to nutrient limitations, regulation of outer membrane proteins, and effects important for motility and biofilm formation.
Accurate proteomic measurements of absolute protein mass fractions in Escherichia coli allowed the characterization of proteome responses under > 60 diverse growth conditions from a coarse (groups of related proteins) to a fine (individual) protein level.
Mycobacterium tuberculosis remains a health concern due to its ability to enter a non-replicative dormant state linked to drug resistance. Understanding transitions into and out of dormancy will ...inform therapeutic strategies. We implemented a universally applicable, label-free approach to estimate absolute cellular protein concentrations on a proteome-wide scale based on SWATH mass spectrometry. We applied this approach to examine proteomic reorganization of M. tuberculosis during exponential growth, hypoxia-induced dormancy, and resuscitation. The resulting data set covering >2,000 proteins reveals how protein biomass is distributed among cellular functions during these states. The stress-induced DosR regulon contributes 20% to cellular protein content during dormancy, whereas ribosomal proteins remain largely unchanged at 5%–7%. Absolute protein concentrations furthermore allow protein alterations to be translated into changes in maximal enzymatic reaction velocities, enhancing understanding of metabolic adaptations. Thus, global absolute protein measurements provide a quantitative description of microbial states, which can support the development of therapeutic interventions.
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•A SWATH MS-based strategy can estimate genome-wide absolute protein concentrations•This technique revealed absolute protein abundances in Mtb during growth and dormancy•The DosR regulon makes up 20% of the total protein biomass during dormancy•Absolute protein abundances reveal maximal enzymatic reaction velocities in metabolism
Schubert et al. develop a strategy to estimate absolute cellular protein concentrations on a global scale based on the proteomic technique SWATH MS. They use this strategy to generate a resource of absolute protein concentrations in Mycobacterium tuberculosis during exponential growth, induction of dormancy, and resuscitation.
Research advancing our understanding of Mycobacterium tuberculosis (Mtb) biology and complex host-Mtb interactions requires consistent and precise quantitative measurements of Mtb proteins. We ...describe the generation and validation of a compendium of assays to quantify 97% of the 4,012 annotated Mtb proteins by the targeted mass spectrometric method selected reaction monitoring (SRM). Furthermore, we estimate the absolute abundance for 55% of all Mtb proteins, revealing a dynamic range within the Mtb proteome of over four orders of magnitude, and identify previously unannotated proteins. As an example of the assay library utility, we monitored the entire Mtb dormancy survival regulon (DosR), which is linked to anaerobic survival and Mtb persistence, and show its dynamic protein-level regulation during hypoxia. In conclusion, we present a publicly available research resource that supports the sensitive, precise, and reproducible quantification of virtually any Mtb protein by a robust and widely accessible mass spectrometric method.
•A resource of quantitative assays for 97% of the annotated Mtb proteins was developed•In unfractionated lysates, 72% of the Mtb proteome is detectable using these assays•Absolute protein concentrations were estimated for 55% of the Mtb proteome•DosR regulon proteins are dynamically regulated in an in vitro model of Mtb dormancy
Accurate classification of breast tumors is vital for patient management decisions and enables more precise cancer treatment. Here, we present a quantitative proteotyping approach based on sequential ...windowed acquisition of all theoretical fragment ion spectra (SWATH) mass spectrometry and establish key proteins for breast tumor classification. The study is based on 96 tissue samples representing five conventional breast cancer subtypes. SWATH proteotype patterns largely recapitulate these subtypes; however, they also reveal varying heterogeneity within the conventional subtypes, with triple negative tumors being the most heterogeneous. Proteins that contribute most strongly to the proteotype-based classification include INPP4B, CDK1, and ERBB2 and are associated with estrogen receptor (ER) status, tumor grade status, and HER2 status. Although these three key proteins exhibit high levels of correlation with transcript levels (R > 0.67), general correlation did not exceed R = 0.29, indicating the value of protein-level measurements of disease-regulated genes. Overall, this study highlights how cancer tissue proteotyping can lead to more accurate patient stratification.
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•Proteotyping of 96 breast tumors by SWATH mass spectrometry•Three key proteins for breast tumor classification•Varying degrees of heterogeneity within conventional breast cancer subtypes•Generally modest correlation between protein and transcript levels in tumor tissue
Bouchal et al. explore and confirm the suitability of SWATH-MS for proteotyping of human tumor samples at relatively high throughput. Results indicate that proteotype-based classification resolves more variability than is apparent from conventional subtyping and potentially improves current classification.
Task demands modulate tactile localization in sighted humans, presumably through weight adjustments in the spatial integration of anatomical, skin-based, and external, posture-based information. In ...contrast, previous studies have suggested that congenitally blind humans, by default, refrain from automatic spatial integration and localize touch using only skin-based information. Here, sighted and congenitally blind participants localized tactile targets on the palm or back of one hand, while ignoring simultaneous tactile distractors at congruent or incongruent locations on the other hand. We probed the interplay of anatomical and external location codes for spatial congruency effects by varying hand posture: the palms either both faced down, or one faced down and one up. In the latter posture, externally congruent target and distractor locations were anatomically incongruent and vice versa. Target locations had to be reported either anatomically ("palm" or "back" of the hand), or externally ("up" or "down" in space). Under anatomical instructions, performance was more accurate for anatomically congruent than incongruent target-distractor pairs. In contrast, under external instructions, performance was more accurate for externally congruent than incongruent pairs. These modulations were evident in sighted and blind individuals. Notably, distractor effects were overall far smaller in blind than in sighted participants, despite comparable target-distractor identification performance. Thus, the absence of developmental vision seems to be associated with an increased ability to focus tactile attention towards a non-spatially defined target. Nevertheless, that blind individuals exhibited effects of hand posture and task instructions in their congruency effects suggests that, like the sighted, they automatically integrate anatomical and external information during tactile localization. Moreover, spatial integration in tactile processing is, thus, flexibly adapted by top-down information-here, task instruction-even in the absence of developmental vision.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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