Proteomics research routinely involves identifying peptides and proteins via MS/MS sequence database search. Thus the database search engine is an integral tool in many proteomics research groups. ...Here, we introduce the Comet search engine to the existing landscape of commercial and open‐source database search tools. Comet is open source, freely available, and based on one of the original sequence database search tools that has been widely used for many years.
Protein chemical cross-linking and mass spectrometry enable the analysis of protein–protein interactions and protein topologies; however, complicated cross-linked peptide spectra require specialized ...algorithms to identify interacting sites. The Kojak cross-linking software application is a new, efficient approach to identify cross-linked peptides, enabling large-scale analysis of protein–protein interactions by chemical cross-linking techniques. The algorithm integrates spectral processing and scoring schemes adopted from traditional database search algorithms and can identify cross-linked peptides using many different chemical cross-linkers with or without heavy isotope labels. Kojak was used to analyze both novel and existing data sets and was compared to existing cross-linking algorithms. The algorithm provided increased cross-link identifications over existing algorithms and, equally importantly, the results in a fraction of computational time. The Kojak algorithm is open-source, cross-platform, and freely available. This software provides both existing and new cross-linking researchers alike an effective way to derive additional cross-link identifications from new or existing data sets. For new users, it provides a simple analytical resource resulting in more cross-link identifications than other methods.
The ability to reliably and reproducibly measure any protein of the human proteome in any tissue or cell type would be transformative for understanding systems-level properties as well as specific ...pathways in physiology and disease. Here, we describe the generation and verification of a compendium of highly specific assays that enable quantification of 99.7% of the 20,277 annotated human proteins by the widely accessible, sensitive, and robust targeted mass spectrometric method selected reaction monitoring, SRM. This human SRMAtlas provides definitive coordinates that conclusively identify the respective peptide in biological samples. We report data on 166,174 proteotypic peptides providing multiple, independent assays to quantify any human protein and numerous spliced variants, non-synonymous mutations, and post-translational modifications. The data are freely accessible as a resource at http://www.srmatlas.org/, and we demonstrate its utility by examining the network response to inhibition of cholesterol synthesis in liver cells and to docetaxel in prostate cancer lines.
Display omitted
•Human SRMAtlas: 166,174 proteotypic peptides representing the human proteome•Resource of verified high-resolution spectra and multiplexed SRM assays•Supports proteome-scale quantification as well as hypothesis-driven research•Web database with free unlimited access
This resource enables the accurate detection and quantification of any known or predicted human protein from complex biological samples.
Abstract
The development of reliable, mixed-culture biotechnological processes hinges on understanding how microbial ecosystems respond to disturbances. Here we reveal extensive phenotypic plasticity ...and niche complementarity in oleaginous microbial populations from a biological wastewater treatment plant. We perform meta-omics analyses (metagenomics, metatranscriptomics, metaproteomics and metabolomics) on in situ samples over 14 months at weekly intervals. Based on 1,364 de novo metagenome-assembled genomes, we uncover four distinct fundamental niche types. Throughout the time-series, we observe a major, transient shift in community structure, coinciding with substrate availability changes. Functional omics data reveals extensive variation in gene expression and substrate usage amongst community members. Ex situ bioreactor experiments confirm that responses occur within five hours of a pulse disturbance, demonstrating rapid adaptation by specific populations. Our results show that community resistance and resilience are a function of phenotypic plasticity and niche complementarity, and set the foundation for future ecological engineering efforts.
Spectral matching sequence database search engines commonly used on mass spectrometry-based proteomics experiments excel at identifying peptide sequence ions, and in addition, possible sequence ions ...carrying post-translational modifications (PTMs), but most do not provide confidence metrics for the exact localization of those PTMs when several possible sites are available. Localization is absolutely required for downstream molecular cell biology analysis of PTM function in vitro and in vivo. Therefore, we developed PTMProphet, a free and open-source software tool integrated into the Trans-Proteomic Pipeline, which reanalyzes identified spectra from any search engine for which pepXML output is available to provide localization confidence to enable appropriate further characterization of biologic events. Localization of any type of mass modification (e.g., phosphorylation) is supported. PTMProphet applies Bayesian mixture models to compute probabilities for each site/peptide spectrum match where a PTM has been identified. These probabilities can be combined to compute a global false localization rate at any threshold to guide downstream analysis. We describe the PTMProphet tool, its underlying algorithms, and demonstrate its performance on ground-truth synthetic peptide reference data sets, one previously published small data set, one new larger data set, and also on a previously published phosphoenriched data set where the correct sites of modification are unknown. Data have been deposited to ProteomeXchange with identifier PXD013210.
Data-independent acquisition (DIA) mass spectrometry, also known as Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH), is a popular label-free proteomics strategy to ...comprehensively quantify peptides/proteins utilizing mass spectral libraries to decipher inherently multiplexed spectra collected linearly across a mass range. Although there are many spectral libraries produced worldwide, the quality control of these libraries is lacking. We present the DIALib-QC (DIA library quality control) software tool for the systematic evaluation of a library's characteristics, completeness and correctness across 62 parameters of compliance, and further provide the option to improve its quality. We demonstrate its utility in assessing and repairing spectral libraries for correctness, accuracy and sensitivity.
The stomach bacterium Helicobacter pylori is one of the most prevalent human pathogens. It has dispersed globally with its human host, resulting in a distinct phylogeographic pattern that can be used ...to reconstruct both recent and ancient human migrations. The extant European population of H. pylori is known to be a hybrid between Asian and African bacteria, but there exist different hypotheses about when and where the hybridization took place, reflecting the complex demographic history of Europeans. Here, we present a 5300-year-old H. pylori genome from a European Copper Age glacier mummy. The "Iceman" H. pylori is a nearly pure representative of the bacterial population of Asian origin that existed in Europe before hybridization, suggesting that the African population arrived in Europe within the past few thousand years.
Abstract
Aims
Heart failure invariably affects patients with various forms of muscular dystrophy (MD), but the onset and molecular sequelae of altered structure and function resulting from ...full-length dystrophin (Dp427) deficiency in MD heart tissue are poorly understood. To better understand the role of dystrophin in cardiomyocyte development and the earliest phase of Duchenne muscular dystrophy (DMD) cardiomyopathy, we studied human cardiomyocytes differentiated from induced pluripotent stem cells (hiPSC-CMs) obtained from the urine of a DMD patient.
Methods and results
The contractile properties of patient-specific hiPSC-CMs, with no detectable dystrophin (DMD-CMs with a deletion of exon 50), were compared to CMs containing a CRISPR-Cas9 mediated deletion of a single G base at position 263 of the dystrophin gene (c.263delG-CMs) isogenic to the parental line of hiPSC-CMs from a healthy individual. We hypothesized that the absence of a dystrophin-actin linkage would adversely affect myofibril and cardiomyocyte structure and function. Cardiomyocyte maturation was driven by culturing long-term (80–100 days) on a nanopatterned surface, which resulted in hiPSC-CMs with adult-like dimensions and aligned myofibrils.
Conclusions
Our data demonstrate that lack of Dp427 results in reduced myofibril contractile tension, slower relaxation kinetics, and to Ca2+ handling abnormalities, similar to DMD cells, suggesting either retarded or altered maturation of cardiomyocyte structures associated with these functions. This study offers new insights into the functional consequences of Dp427 deficiency at an early stage of cardiomyocyte development in both patient-derived and CRISPR-generated models of dystrophin deficiency.
Cross-linking mass spectrometry (MS) has substantially matured as a method over the past 2 decades through parallel development in multiple labs, demonstrating its applicability to protein structure ...determination, conformation analysis, and mapping protein interactions in complex mixtures. Cross-linking MS has become a much-appreciated and routinely applied tool, especially in structural biology. Therefore, it is timely that the community commits to the development of methodological and reporting standards. This white paper builds on an open process comprising a number of events at community conferences since 2015 and identifies aspects of Cross-linking MS for which guidelines should be developed as part of a Cross-linking MS standards initiative.
Leitner et al. propose steps toward the development of best practices in Cross-linking mass spectrometry in this white paper. This community-driven effort represents a consensus of approximately 30 groups from academia and industry and, if successfully implemented, will increase transparency and facilitate data reuse.
PDF
