Robust and Easy-to-Use One-Pot Workflow for Label-Free Single-Cell Proteomics.

The analysis of ultralow input samples or even individual cells is essential to answering a multitude of biomedical questions, but current proteomic workflows are limited in their sensitivity and reproducibility. Here, we report a comprehensive workflow that includes improved strategies for all steps, from cell lysis to data analysis. Thanks to convenient-to-handle 1 µL sample volume and standardized 384-well plates, the workflow is easy for even novice users to implement. At the same time, it can be performed semi-automatized using CellenONE, which allows for the highest reproducibility. To achieve high throughput, ultrashort gradient lengths down to 5 min were tested using advanced µ-pillar columns. Data-dependent acquisition (DDA), wide-window acquisition (WWA), data-independent acquisition (DIA), and commonly used advanced data analysis algorithms were benchmarked. Using DDA, 1790 proteins covering a dynamic range of four orders of magnitude were identified in a single cell. Using DIA, proteome coverage increased to more than 2200 proteins identified from single-cell level input in a 20 min active gradient. The workflow enabled differentiation of two cell lines, demonstrating its suitability to cellular heterogeneity determination.

Correction to: The urinary microbiome shows different bacterial genera in renal transplant recipients and non-transplant patients at time of acute kidney injury - a pilot study.

An amendment to this paper has been published and can be accessed via the original article.

The urinary microbiome shows different bacterial genera in renal transplant recipients and non-transplant patients at time of acute kidney injury - a pilot study.

BACKGROUND: In the past urine was considered sterile. Through the introduction of next generation sequencing, it has become clear that a urinary microbiome exists. Acute kidney injury (AKI) represents a major threat to kidney transplant recipients. Remarkable changes in the urinary metabolome occur during AKI, which may influence the urinary microbiome. To our knowledge, this is the first study that examines the urinary microbiome in renal transplant recipients (RTX) and non-transplant recipients (nRTX) at time of AKI. METHODS: In this cross-sectional pilot-study the urinary microbiome of 21 RTX and 9 nRTX with AKI was examined. Clean catch morning urine samples were obtained from all patients on the first day of AKI diagnosis. AKI was defined according to KDIGO guidelines. Urinary microbiota and the urinary metabolome during AKI were assessed in one patient. 16S rRNA sequencing was performed. Sequences were processed using UPARSE-pipeline for operational taxonomic units (OTU) and taxon finding. RESULTS: We successfully extracted and sequenced bacterial DNA from 100% of the urine samples. All 30 patients revealed at least 106,138 reads. 319 OTU and 211 different genera were identified. The microbiotic diversity richness in the RTX group was no different from the nRTX group. Eighteen genera were solely present in nRTX and 7 in RTX. CONCLUSIONS: The urinary microbiome at time of AKI showed different bacterial genera in RTX compared to nRTX. The nRTX group exhibited no different diversity to the RTX group. Irrespective of the status of a previous renal transplantation, the urinary microbiome comprised > 210 different genera. An intraindividual change in microbiota diversity and richness was observed in one study patient during recovery from AKI.

Global typology of urban energy use and potentials for an urbanization mitigation wedge.

The aggregate potential for urban mitigation of global climate change is insufficiently understood. Our analysis, using a dataset of 274 cities representing all city sizes and regions worldwide, demonstrates that economic activity, transport costs, geographic factors, and urban form explain 37% of urban direct energy use and 88% of urban transport energy use. If current trends in urban expansion continue, urban energy use will increase more than threefold, from 240 EJ in 2005 to 730 EJ in 2050. Our model shows that urban planning and transport policies can limit the future increase in urban energy use to 540 EJ in 2050 and contribute to mitigating climate change. However, effective policies for reducing urban greenhouse gas emissions differ with city type. The results show that, for affluent and mature cities, higher gasoline prices combined with compact urban form can result in savings in both residential and transport energy use. In contrast, for developing-country cities with emerging or nascent infrastructures, compact urban form, and transport planning can encourage higher population densities and subsequently avoid lock-in of high carbon emission patterns for travel. The results underscore a significant potential urbanization wedge for reducing energy use in rapidly urbanizing Asia, Africa, and the Middle East.

Quantitative phosphoproteomics of the ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-mutated and rad3-related (ATR) dependent DNA damage response in Arabidopsis thaliana.

The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis).

qcML: an exchange format for quality control metrics from mass spectrometry experiments.

Quality control is increasingly recognized as a crucial aspect of mass spectrometry based proteomics. Several recent papers discuss relevant parameters for quality control and present applications to extract these from the instrumental raw data. What has been missing, however, is a standard data exchange format for reporting these performance metrics. We therefore developed the qcML format, an XML-based standard that follows the design principles of the related mzML, mzIdentML, mzQuantML, and TraML standards from the HUPO-PSI (Proteomics Standards Initiative). In addition to the XML format, we also provide tools for the calculation of a wide range of quality metrics as well as a database format and interconversion tools, so that existing LIMS systems can easily add relational storage of the quality control data to their existing schema. We here describe the qcML specification, along with possible use cases and an illustrative example of the subsequent analysis possibilities. All information about qcML is available at http://code.google.com/p/qcml.

Investigation on the performance of dedicated radiotherapy positioning devices for MR scanning for prostate planning.

The purpose of this study was to investigate performance of the couch and coil mounts designed for MR-simulation prostate scanning using data from ten volunteers. Volunteers were scanned using the standard MR scanning protocol with the MR coil directly strapped on the external body and the volunteer lying on the original scanner table. They also were scanned using a MR-simulation table top and pelvic coil mounts. MR images from both setups were compared in terms of body contour variation and image quality effects within particular organs of interest. Six-field conformal plans were generated on the two images with assigned bulk density for dose calculation. With the MR-simulation devices, the anterior skin deformation was reduced by up to 1.7 cm. The hard tabletop minimizes the posterior body deformation which can be up to 2.3 cm on the standard table, depending on the weight of volunteer. The image signal-to-noise ratio reduced by 14% and 25% on large field of view (FOV) and small FOV images, respectively, after using the coil mount; the prostate volume contoured on two images showed difference of 1.05 ± 0.66 cm3. The external body deformation caused a mean dose reduction of 0.6 ± 0.3 Gy, while the coverage reduced by 22% ± 13% and 27% ± 6% in V98 and V100, respectively. A dedicated MR simulation setup for prostate radiotherapy is essential to ensure the agreement between planning anatomy and treatment anatomy. The image signal was reduced after applying the coil mount, but no significant effect was found on prostate contouring.

Development and performance evaluation of an ultralow flow nanoliquid chromatography-tandem mass spectrometry set-up.

LC-MS/MS is the most commonly used technique for the identification and characterization of proteins. The efficiency of the electrospray process is a critical factor in LC-MS/MS. Despite the benefits associated with very low flow rates for the ionization efficiency, most LC-MS/MS platforms are operated at relatively high flow rates. The purpose of this work was to develop a nano LC system operable at a flow rate of 20 nL/min, applicable for routine analysis in proteomics laboratories. Peptide separation was performed with an analytical column packed with 2 µm porous chromatographic beads, a length of 25 cm and an inner diameter (i.d.) of 25 µm. Practical usability, reproducibility, and overall performance of the system were evaluated with a tryptic peptide mixture generated from HeLa cells. Using 100 ng of sample, we identified on average 3721 protein groups based on 25,699 peptides. We demonstrate that the number of peptides identified with this system increases with decreasing flow rates. Probing the sensitivity of the set-up we analyzed only 10 ng of the sample, identifying an average number of 2042 protein groups based on 11 424 peptides. All MS data have been deposited in the ProteomeXchange with identifier PXD000396 (http://proteomecentral.proteomexchange.org/dataset/PXD000396).

Deep and precise quantification of the mouse synaptosomal proteome reveals substantial remodeling during postnatal maturation.

During postnatal murine maturation, behavioral patterns emerge and become shaped by experience-dependent adaptations. During the same period, the morphology of dendritic spines, the morphological correlates of excitatory synapses, is known to change, and there is evidence of concurrent alterations of the synaptosomal protein machinery. To obtain comprehensive and quantitative insights in the developmental regulation of the proteome of synapses, we prepared cortical synaptosomal fractions from a total of 16 individual juvenile and adult mouse brains (age 3 or 8 weeks, respectively). We then applied peptide-based iTRAQ labeling (four pools of 4 animals) and high-resolution two-dimensional peptide fractionation (99 SCX fractions and 3 h reversed-phase gradients) using a hybrid CID-HCD acquisition method on a Velos Orbitrap mass spectrometer to identify a comprehensive set of synaptic proteins and to quantify changes in protein expression. We obtained a data set tracking expression levels of 3500 proteins mapping to 3427 NCBI GeneIDs during development with complete quantification data available for 3422 GeneIDs, which, to the best of our knowledge, constitutes the deepest coverage of the synaptosome proteome to date. The inclusion of biological replicates in a single mass spectrometry analysis demonstrated both high reproducibility of our synaptosome preparation method as well as high precision of our quantitative data (correlation coefficient R = 0.87 for the biological replicates). To evaluate the validity of our data, the developmental regulation of eight proteins identified in our analysis was confirmed independently using western blotting. A gene ontology analysis confirmed the synaptosomal nature of a large fraction of identified proteins. Of note, the set of the most strongly regulated proteins revealed candidates involved in neurological processes in health and disease states. This highlights the fact that developmentally regulated proteins can play additional roles in neurological disease processes. All data have been deposited to the ProteomeXchange with identifier PXD000552.

MS Amanda, a universal identification algorithm optimized for high accuracy tandem mass spectra.

Today's highly accurate spectra provided by modern tandem mass spectrometers offer considerable advantages for the analysis of proteomic samples of increased complexity. Among other factors, the quantity of reliably identified peptides is considerably influenced by the peptide identification algorithm. While most widely used search engines were developed when high-resolution mass spectrometry data were not readily available for fragment ion masses, we have designed a scoring algorithm particularly suitable for high mass accuracy. Our algorithm, MS Amanda, is generally applicable to HCD, ETD, and CID fragmentation type data. The algorithm confidently explains more spectra at the same false discovery rate than Mascot or SEQUEST on examined high mass accuracy data sets, with excellent overlap and identical peptide sequence identification for most spectra also explained by Mascot or SEQUEST. MS Amanda, available at http://ms.imp.ac.at/?goto=msamanda , is provided free of charge both as standalone version for integration into custom workflows and as a plugin for the Proteome Discoverer platform.

Extensive thoracoabdominal aortic electro septotomy: A case report of a novel approach in the treatment of chronic aortic dissections.

Treatment of post-dissection arch and thoracoabdominal aortic aneurysms presents significant therapeutic challenges. True lumen collapse or take off of aortic branches from the false lumen makes endograft alignment difficult, if not impossible. We present herein the first successful case of an extensive thoracoabdominal electro aortic septotomy of the entire dissection membrane from the aortic arch down to the aortic bifurcation during an open redo aortic arch replacement employing the frozen elephant trunk technique. The procedure was performed on a 59 years old female patient presenting with a progressive post-dissection aortic aneurysm during follow-up with a maximum diameter of 6 cm 11 years after operating on an acute type A aortic dissection. Due to the extensive longitudinal aortic electric septotomy, we created a new "common lumen" for subsequent endovascular completion of the repair.

Breakthrough Infections in SARS-CoV-2-Vaccinated Multiple Myeloma Patients Improve Cross-Protection against Omicron Variants.

Patients with multiple myeloma (MM) are a heterogenous, immunocompromised group with increased risk for COVID-19 morbidity and mortality but impaired responses to primary mRNA SARS-CoV-2 vaccination. The effects of booster vaccinations and breakthrough infections (BTIs) on antibody (Ab) levels and cross-protection to variants of concern (VOCs) are, however, not sufficiently evaluated. Therefore, we analysed humoral and cellular vaccine responses in MM patients stratified according to disease stage/treatment into group (1) monoclonal gammopathy of undetermined significance, (2) after stem cell transplant (SCT) without immunotherapy (IT), (3) after SCT with IT, and (4) progressed MM, and in healthy subjects (prospective cohort study). In contrast to SARS-CoV-2 hu-1-specific Ab levels, Omicron-specific Abs and their cross-neutralisation capacity remained low even after three booster doses in a majority of MM patients. In particular, progressed MM patients receiving anti-CD38 mAb and those after SCT with IT were Ab low responders and showed delayed formation of spike-specific B memory cells. However, MM patients with hybrid immunity (i.e., vaccination and breakthrough infection) had improved cross-neutralisation capacity against VOCs, yet in the absence of severe COVID-19 disease. Our results indicate that MM patients require frequent variant-adapted booster vaccinations and/or changes to other vaccine formulations/platforms, which might have similar immunological effects as BTIs.

Optimized nonlinear gradients for reversed-phase liquid chromatography in shotgun proteomics.

Reversed-phase liquid chromatography has become the preferred method for separating peptides in most of the mass spectrometry-based proteomics workflows of today. In the way the technique is typically applied, the peptides are released from the chromatography column by the gradual addition of an organic buffer according to a linear function. However, when applied to complex peptide mixtures, this approach leads to unequal spreads of the peptides over the chromatography time. To address this, we investigated the use of nonlinear gradients, customized for each setup at hand. We developed an algorithm to generate optimized gradient functions for shotgun proteomics experiments and evaluated it for two data sets consisting each of four replicate runs of a human complex sample. Our results show that the optimized gradients produce a more even spread of the peptides over the chromatography run, while leading to increased numbers of confident peptide identifications. In addition, the list of peptides identified using nonlinear gradients differed considerably from those found with the linear ones, suggesting that such gradients can be a valuable tool for increasing the proteome coverage of mass spectrometry-based experiments.

Quantitative phospho-proteomics to investigate the polo-like kinase 1-dependent phospho-proteome.

Polo-like kinase 1 (PLK1) is a key regulator of mitotic progression and cell division, and small molecule inhibitors of PLK1 are undergoing clinical trials to evaluate their utility in cancer therapy. Despite this importance, current knowledge about the identity of PLK1 substrates is limited. Here we present the results of a proteome-wide analysis of PLK1-regulated phosphorylation sites in mitotic human cells. We compared phosphorylation sites in HeLa cells that were or were not treated with the PLK1-inhibitor BI 4834, by labeling peptides via methyl esterification, fractionation of peptides by strong cation exchange chromatography, and phosphopeptide enrichment via immobilized metal affinity chromatography. Analysis by quantitative mass spectrometry identified 4070 unique mitotic phosphorylation sites on 2069 proteins. Of these, 401 proteins contained one or multiple phosphorylation sites whose abundance was decreased by PLK1 inhibition. These include proteins implicated in PLK1-regulated processes such as DNA damage, mitotic spindle formation, spindle assembly checkpoint signaling, and chromosome segregation, but also numerous proteins that were not suspected to be regulated by PLK1. Analysis of amino acid sequence motifs among phosphorylation sites down-regulated under PLK1 inhibition in this data set identified two potential novel variants of the PLK1 consensus motif.

EUBUCCO v0.1: European building stock characteristics in a common and open database for 200+ million individual buildings.

Building stock management is becoming a global societal and political issue, inter alia because of growing sustainability concerns. Comprehensive and openly accessible building stock data can enable impactful research exploring the most effective policy options. In Europe, efforts from citizen and governments generated numerous relevant datasets but these are fragmented and heterogeneous, thus hindering their usability. Here, we present EUBUCCO v0.1, a database of individual building footprints for ~202 million buildings across the 27 European Union countries and Switzerland. Three main attributes - building height, construction year and type - are included for respectively 73%, 24% and 46% of the buildings. We identify, collect and harmonize 50 open government datasets and OpenStreetMap, and perform extensive validation analyses to assess the quality, consistency and completeness of the data in every country. EUBUCCO v0.1 provides the basis for high-resolution urban sustainability studies across scales - continental, comparative or local studies - using a centralized source and is relevant for a variety of use cases, e.g., for energy system analysis or natural hazard risk assessments.

Validation of regulated protein phosphorylation events in yeast by quantitative mass spectrometry analysis of purified proteins.

Global phosphoproteomic studies based on MS have generated qualitative and quantitative data describing protein phosphorylation events in various biological systems. Since high-throughput data for protein modifications are inherently incomplete, we developed a strategy to extend and validate such primary datasets. We selected interesting protein candidates from a global screen in yeast and employed a modified histidine biotin tag that allows tandem affinity purifications of our targets under denaturing conditions. Products in question can be digested directly from affinity resins and phosphopeptides can be further enriched via TiO(2) before MS analysis. Our robust protocol can be amended for SILAC as well as iTRAQ quantifications or label-free approaches based on selective reaction monitoring, allowing completion of the phosphorylation pattern in a first step, followed by a detailed analysis of the phosphorylation kinetics. We exemplify the value of such a strategy by an in-depth analysis of Pan1, a highly phosphorylated factor involved in early steps of endocytosis. The study of Pan1 under osmotic stress conditions in different mutant backgrounds allowed us to differentiate between mitogen-activated protein kinase Hog1 driven and Hog1 independent stress responses.

SIMPATIQCO: a server-based software suite which facilitates monitoring the time course of LC-MS performance metrics on Orbitrap instruments.

While the performance of liquid chromatography (LC) and mass spectrometry (MS) instrumentation continues to increase, applications such as analyses of complete or near-complete proteomes and quantitative studies require constant and optimal system performance. For this reason, research laboratories and core facilities alike are recommended to implement quality control (QC) measures as part of their routine workflows. Many laboratories perform sporadic quality control checks. However, successive and systematic longitudinal monitoring of system performance would be facilitated by dedicated automatic or semiautomatic software solutions that aid an effortless analysis and display of QC metrics over time. We present the software package SIMPATIQCO (SIMPle AuTomatIc Quality COntrol) designed for evaluation of data from LTQ Orbitrap, Q-Exactive, LTQ FT, and LTQ instruments. A centralized SIMPATIQCO server can process QC data from multiple instruments. The software calculates QC metrics supervising every step of data acquisition from LC and electrospray to MS. For each QC metric the software learns the range indicating adequate system performance from the uploaded data using robust statistics. Results are stored in a database and can be displayed in a comfortable manner from any computer in the laboratory via a web browser. QC data can be monitored for individual LC runs as well as plotted over time. SIMPATIQCO thus assists the longitudinal monitoring of important QC metrics such as peptide elution times, peak widths, intensities, total ion current (TIC) as well as sensitivity, and overall LC-MS system performance; in this way the software also helps identify potential problems. The SIMPATIQCO software package is available free of charge.

LivWell: a sub-national Dataset on the Living Conditions of Women and their Well-being for 52 Countries.

Data on women's living conditions and socio-economic development are important for understanding and addressing the pronounced challenges and inequalities faced by women worldwide. While such information is increasingly available at the national level, comparable data at the sub-national level are missing. We here present the LivWell global longitudinal dataset, which includes a set of key indicators on women's socio-economic status, health and well-being, access to basic services and demographic outcomes. It covers 447 regions in 52 countries and includes a total of 265 different indicators. The majority of these are based on 199 Demographic and Health Surveys (DHS) for the period 1990-2019 and are complemented by extensive information on socio-economic and climatic conditions in the respective regions. The resulting dataset offers various opportunities for policy-relevant research on gender inequality, inclusive development and demographic trends at the sub-national level.

Structural failure in bridging stentgrafts for branched endovascular aneurysm repair: a case-control study.

OBJECTIVE: To present a case series of spontaneous structural failure of bridging stentgrafts (BSGs) after branched endovascular aortic repair (bEVAR), as well as their failure types and their detection. While bEVAR is a safe and effective procedure, one main limitation is the reintervention rate associated with the BSGs. Structural failure of BSGs, defined as fabric disruption, stent fracture with leak or complete separation is a major cause for reinterventions and difficult to detect in computed tomography angiography (CTA). METHODS: From a multicenter bEVAR complication database, structural BSG failures were identified. Patient and BSG characteristics, detection mode, failure type, treatment and outcome were recorded and compared with bEVAR patients with intact BSGs. RESULTS: Twenty-three BSG failures were detected in 12 patients with only 43% directly identified in CTA, after a mean of 21.4 months after implantation. The BSGs were Advanta (n = 4), E-Ventus (n = 16) and BeGraft (n = 3) in 10 renal, 9 superior mesenteric, and 4 celiac branches. Religning with another BSG was successful in 20/22 cases, one BSG failure necessitated renal branch embolization (organ loss), and one mesenteric bypass surgery. Two reintervention-related mortalities occurred. CONCLUSION: Structural failure of BSGs is a serious limitation for bEVAR, which can result in high reintervention rates and serious complications. BSG failure typically occurs in single-layer types and events are clustered in patients. The necessary reinterventions carry serious morbidity and mortality. Since the use as BSG in bEVAR is off-label with all current BSG manufacturers, caution is advised regarding patient-informed consent.