MetaNovo: An open-source pipeline for probabilistic peptide discovery in complex metaproteomic datasets.

BACKGROUND: Microbiome research is providing important new insights into the metabolic interactions of complex microbial ecosystems involved in fields as diverse as the pathogenesis of human diseases, agriculture and climate change. Poor correlations typically observed between RNA and protein expression datasets make it hard to accurately infer microbial protein synthesis from metagenomic data. Additionally, mass spectrometry-based metaproteomic analyses typically rely on focused search sequence databases based on prior knowledge for protein identification that may not represent all the proteins present in a set of samples. Metagenomic 16S rRNA sequencing only targets the bacterial component, while whole genome sequencing is at best an indirect measure of expressed proteomes. Here we describe a novel approach, MetaNovo, that combines existing open-source software tools to perform scalable de novo sequence tag matching with a novel algorithm for probabilistic optimization of the entire UniProt knowledgebase to create tailored sequence databases for target-decoy searches directly at the proteome level, enabling metaproteomic analyses without prior expectation of sample composition or metagenomic data generation and compatible with standard downstream analysis pipelines. RESULTS: We compared MetaNovo to published results from the MetaPro-IQ pipeline on 8 human mucosal-luminal interface samples, with comparable numbers of peptide and protein identifications, many shared peptide sequences and a similar bacterial taxonomic distribution compared to that found using a matched metagenome sequence database-but simultaneously identified many more non-bacterial peptides than the previous approaches. MetaNovo was also benchmarked on samples of known microbial composition against matched metagenomic and whole genomic sequence database workflows, yielding many more MS/MS identifications for the expected taxa, with improved taxonomic representation, while also highlighting previously described genome sequencing quality concerns for one of the organisms, and identifying an experimental sample contaminant without prior expectation. CONCLUSIONS: By estimating taxonomic and peptide level information directly on microbiome samples from tandem mass spectrometry data, MetaNovo enables the simultaneous identification of peptides from all domains of life in metaproteome samples, bypassing the need for curated sequence databases to search. We show that the MetaNovo approach to mass spectrometry metaproteomics is more accurate than current gold standard approaches of tailored or matched genomic sequence database searches, can identify sample contaminants without prior expectation and yields insights into previously unidentified metaproteomic signals, building on the potential for complex mass spectrometry metaproteomic data to speak for itself.

Proteogenomic Analysis of Mycobacterium smegmatis Using High Resolution Mass Spectrometry.

Biochemical evidence is vital for accurate genome annotation. The integration of experimental data collected at the proteome level using high resolution mass spectrometry allows for improvements in genome annotation by providing evidence for novel gene models, while validating or modifying others. Here, we report the results of a proteogenomic analysis of a reference strain of Mycobacterium smegmatis (mc(2)155), a fast growing model organism for the pathogenic Mycobacterium tuberculosis-the causative agent for Tuberculosis. By integrating high throughput LC/MS/MS proteomic data with genomic six frame translation and ab initio gene prediction databases, a total of 2887 ORFs were identified, including 2810 ORFs annotated to a Reference protein, and 63 ORFs not previously annotated to a Reference protein. Further, the translational start site (TSS) was validated for 558 Reference proteome gene models, while upstream translational evidence was identified for 81. In addition, N-terminus derived peptide identifications allowed for downstream TSS modification of a further 24 gene models. We validated the existence of six previously described interrupted coding sequences at the peptide level, and provide evidence for four novel frameshift positions. Analysis of peptide posterior error probability (PEP) scores indicates high-confidence novel peptide identifications and shows that the genome of M. smegmatis mc(2)155 is not yet fully annotated. Data are available via ProteomeXchange with identifier PXD003500.

Predictors of clinical and radiological outcome in patients with fractures of the acetabulum and concomitant posterior dislocation of the hip.

We aimed to identify variables associated with clinical and radiological outcome following fractures of the acetabulum associated with posterior dislocation of the hip. Using a prospective database of 1076 such fractures, we identified 109 patients with this combined injury managed operatively within three weeks and followed up for two or more years. The patients had a mean age of 42 years (15 to 79), 78 (72%) were male, and 84 (77%) had been involved in motor vehicle accidents. Using multivariate analysis the quality of reduction of the fracture was identified as the only significant predictor of radiological grade, clinical function and the development of post-traumatic arthritis (p < 0.001). All patients lacking anatomical reduction developed arthritis whereas only 25.5% (24 patients) with an anatomical reduction did so (p = 0.05). The quality of the reduction of the fracture is the most important variable in forecasting the outcome for patients with this injury. The interval to reduction of the dislocation of the hip may be less important than previously described.

Pediatric ankle fractures: evaluation and treatment.

Pediatric ankle fractures account for approximately 5% of pediatric fractures and 15% of physeal injuries. The biomechanical differences between mature and immature bones, as well as the differing forces applied to those bones, help explain the differences between adult and pediatric fractures. The potential complications associated with pediatric ankle fractures include those seen with adult fractures (such as posttraumatic arthritis, stiffness, and reflex sympathetic dystrophy) as well as those that result from physeal damage (including leg-length discrepancy, angular deformity, or a combination thereof). The goals of treatment are to achieve and maintain a satisfactory reduction and to avoid physeal arrest. A knowledge of common pediatric ankle fracture patterns and the pitfalls associated with their evaluation and treatment will aid the clinician in the effective management of these injuries.

Heuristic statistical analysis of fluorescence fluctuation data with bright spikes: application to ligand binding to the human serotonin receptor expressed in Escherichia coli cells.

A statistical method for the analysis of fluorescence fluctuation amplitudes including bright spikes is presented. This situation arises e. g. when fluorescent ligands interact with receptors carrying multiple binding sites. The technique gives information on the amount of bound ligand in solution, making it a complementary technique to fluorescence correlation spectroscopy analysis, which cannot be applied in this situation. Two simple statistical tests are proposed that can discriminate between fluorescence intensities originating from free ligands or complexes. The performance of the two tests is evaluated and compared on mixtures of a fluorophore and fluorophore-coated beads that mimic the behaviour of multi-liganded complexes. An application to ligand binding to the serotonin receptor, expressed on Escherichia coli cells, is also provided. Specific binding of a fluorophore to this receptor, as well as competition with several ligands, is assessed.

Single-stage talectomy and tibiocalcaneal arthrodesis as a salvage of severe, rigid equinovarus deformity.

Seven adult males with rigid, severe equinovarus deformities underwent single-stage corrective surgery. The procedure included extensive soft tissue release, talectomy and tibiocalcaneal, calcaneocuboid, and tibionavicular fusion. All seven patients fused successfully at an average of 3.5 months with a plantigrade foot. All feet were stiff and had an average of 3.5 cm limb length discrepancy. All of the patients were satisfied with the surgery and would choose to have the procedure again. This small series represents one method of achieving a plantigrade foot in patients with severe, rigid equinovarus deformities.