Database search engines for bottom-up proteomics largely ignore peptide fragment ion intensities during the automated scoring of tandem mass spectra against protein databases. Recent advances in deep ...learning allow the accurate prediction of peptide fragment ion intensities. Using these predictions to calculate additional intensity-based scores helps to overcome this drawback. Here, we describe a processing workflow termed INFERYS™ rescoring for the intensity-based rescoring of Sequest HT search engine results in Thermo Scientific™ Proteome Discoverer™ 2.5 software. The workflow is based on the deep learning platform INFERYS capable of predicting fragment ion intensities, which runs on personal computers without the need for graphics processing units. This workflow calculates intensity-based scores comparing peptide spectrum matches from Sequest HT and predicted spectra. Resulting scores are combined with classical search engine scores for input to the false discovery rate estimation tool Percolator. We demonstrate the merits of this approach by analyzing a classical HeLa standard sample and exemplify how this workflow leads to a better separation of target and decoy identifications, in turn resulting in increased peptide spectrum match, peptide and protein identification numbers. On an immunopeptidome dataset, this workflow leads to a 50% increase in identified peptides, emphasizing the advantage of intensity-based scores when analyzing low-intensity spectra or analytes with very similar physicochemical properties that require vast search spaces. Overall, the end-to-end integration of INFERYS rescoring enables simple and easy access to a powerful enhancement to classical database search engines, promising a deeper, more confident and more comprehensive analysis of proteomic data from any organism by unlocking the intensity dimension of tandem mass spectra for identification and more confident scoring.
Medicinal chemistry has discovered thousands of potent protein and lipid kinase inhibitors. These may be developed into therapeutic drugs or chemical probes to study kinase biology. Because of ...polypharmacology, a large part of the human kinome currently lacks selective chemical probes. To discover such probes, we profiled 1,183 compounds from drug discovery projects in lysates of cancer cell lines using Kinobeads. The resulting 500,000 compound-target interactions are available in ProteomicsDB and we exemplify how this molecular resource may be used. For instance, the data revealed several hundred reasonably selective compounds for 72 kinases. Cellular assays validated GSK986310C as a candidate SYK (spleen tyrosine kinase) probe and X-ray crystallography uncovered the structural basis for the observed selectivity of the CK2 inhibitor GW869516X. Compounds targeting PKN3 were discovered and phosphoproteomics identified substrates that indicate target engagement in cells. We anticipate that this molecular resource will aid research in drug discovery and chemical biology.
Despite the increasing use of high-throughput experiments in molecular biology, methods for evaluating and classifying the acquired results have not kept pace, requiring significant manual efforts to ...do so. Here, we present CiRCus, a framework to generate custom machine learning models to classify results from high-throughput proteomics binding experiments. We show the experimental procedure that guided us to the layout of this framework as well as the usage of the framework on an example data set consisting of 557 166 protein/drug binding curves achieving an AUC of 0.9987. By applying our classifier to the data, only 6% of the data might require manual investigation. CiRCus bundles two applications, a minimal interface to label a training data set (CindeR) and an interface for the generation of random forest classifiers with optional optimization of pretrained models (CurveClassification). CiRCus is available on https://github.com/kusterlab accompanied by an in-depth user manual and video tutorial.
Pregnancy and lactation-associated osteoporosis (PLO) is a rare skeletal disorder characterized by early-onset osteoporosis typically manifestating with vertebral compression fractures or transient ...osteoporosis of the hip. We hypothesized that genetic variants may play a role in the development of PLO. This study aimed to analyze the presence of genetic variants and a potential association with the clinical presentation in PLO. 42 women with PLO were included from 2013 to 2019 in a multicenter study in Germany. All cases underwent comprehensive genetic analysis based on a custom-designed gene panel including genes relevant for skeletal disorders. The skeletal status was assessed using dual-energy X-ray absorptiometry (DXA). Subgroups were further analyzed by serum bone turnover markers (n = 31) and high-resolution peripheral computed tomography (HR-pQCT; n = 23). We detected relevant genetic variants in 21 women (50%), with LRP5, WNT1 and COL1A1/A2 being the most commonly involved genes. The mean number of vertebral compression fractures was 3.3 ± 3.4 per case with a significantly higher occurrence in the subgroup with genetic variants (4.8 ± 3.7 vs. 1.8 ± 2.3, p = 0.02). Among the total cohort, DXA Z-scores were significantly lower at the lumbar spine compared to the femoral neck (p = 0.002). HR-pQCT revealed a pronounced reduction of trabecular and cortical thickness, while trabecular number was within the reference range. Eighteen women (43%) received a bone-specific therapy (primarily teriparatide). Overall, a steep increase in bone mass (+37.7%) was observed after 3 years. In conclusion, pregnancy and lactation represent skeletal risk factors, which may unmask hereditary bone disorders leading to PLO. These cases were affected more severely. Nevertheless, a timely diagnosis and adequate treatment can ensure a substantial recovery potential even without specific therapy. Patients with genetically induced low bone turnover (e.g.; LRP5, WNT1) may especially benefit from osteo-anabolic medication.
•Genetic analysis of PLO cases was based on a custom-designed gene panel including genes relevant for skeletal disorders.•Relevant variants were found in 50% of the PLO cases.•PLO cases with relevant variants were affected more severely.•PLO cases showed a similar recovery independently of their genetic background.
Hypophosphatasia (HPP) is an inherited multisystem disorder predominantly affecting the mineralization of bones and teeth. HPP is caused by pathogenic variants in ALPL, which encodes tissue ...non-specific alkaline phosphatase (TNSALP). Variants of uncertain significance (VUS) cause diagnostic delay and uncertainty amongst patients and health care providers.
The ALPL gene variant database (https://alplmutationdatabase.jku.at/) is an open-access archive for interpretation of the clinical significance of variants reported in ALPL. The database contains coding and non-coding variants, including single nucleotide variants, insertions/deletions and structural variants affecting coding or non-coding sequences of ALPL. Each variant in the database is displayed with details explaining the corresponding pathogenicity, and all reported genotypes and phenotypes, including references. In 2021, the ALPL gene variant classification project was established to reclassify VUS and continuously assess and update genetic, phenotypic, and functional variant information in the database. For this purpose, the database provides a unique submission system for clinicians, geneticists, genetic counselors, and researchers to submit VUS within ALPL for classification. An international, multidisciplinary consortium of HPP experts has been established to reclassify the submitted VUS using a multi-step process adhering to the stringent ACMG/AMP variant classification guidelines. These steps include a clinical phenotype assessment, deep literature research including artificial intelligence technology, molecular genetic assessment, and in-vitro functional testing of variants in a co-transfection model to measure ALP residual activity.
This classification project and the ALPL gene variant database will serve the global medical community, widen the genotypic and phenotypic HPP spectrum by reporting and characterizing new ALPL variants based on ACMG/AMP criteria and thus facilitate improved genetic counseling and medical decision-making for affected patients and families. The project may also serve as a gold standard framework for multidisciplinary collaboration for variant interpretation in other rare diseases.
Many different inherited and acquired conditions can result in premature bone fragility/low bone mass disorders (LBMDs).
We aimed to elucidate the impact of genetic testing on differential diagnosis ...of adult LBMDs and at defining clinical criteria for predicting monogenic forms.
Four clinical centers broadly recruited a cohort of 394 unrelated adult women before menopause and men younger than 55 years with a bone mineral density (BMD) Z-score < -2.0 and/or pathological fractures. After exclusion of secondary causes or unequivocal clinical/biochemical hallmarks of monogenic LBMDs, all participants were genotyped by targeted next-generation sequencing.
In total, 20.8% of the participants carried rare disease-causing variants (DCVs) in genes known to cause osteogenesis imperfecta (COL1A1, COL1A2), hypophosphatasia (ALPL), and early-onset osteoporosis (LRP5, PLS3, and WNT1). In addition, we identified rare DCVs in ENPP1, LMNA, NOTCH2, and ZNF469. Three individuals had autosomal recessive, 75 autosomal dominant, and 4 X-linked disorders. A total of 9.7% of the participants harbored variants of unknown significance. A regression analysis revealed that the likelihood of detecting a DCV correlated with a positive family history of osteoporosis, peripheral fractures (> 2), and a high normal body mass index (BMI). In contrast, mutation frequencies did not correlate with age, prevalent vertebral fractures, BMD, or biochemical parameters. In individuals without monogenic disease-causing rare variants, common variants predisposing for low BMD (eg, in LRP5) were overrepresented.
The overlapping spectra of monogenic adult LBMD can be easily disentangled by genetic testing and the proposed clinical criteria can help to maximize the diagnostic yield.
Driven by electric mobility innovative winding methods increasing the copper fill ratio and thus the power density of electrical drives gain in importance. Compared to conventional windings, ...bar-wound windings based on rectangular cross sections besides optimized copper fill ratios also offer advantages in terms of automated production. Because of the high number of joints necessary for the manufacturing of the winding, contacting efforts increase significantly wherefore the process of laser welding, due to its short process times and high reproducibility shows potentials to realize the process step. This paper examines the most important interactions within the process of laser welding of insulated copper wires with rectangular cross sections in the context of electric drives production. Besides workpiece specific factors, the focus is set on process parameters, beam guidance strategies and specific quality requirements. For this purpose, a parameter study based on design of experiments is executed in which suitable measuring technologies are used to quantify the properties of the samples.
Selective mask and laser sintering are powder based, additive manufacturing technologies, which allow the production of complex parts layer-by-layer. Due to the high building chamber temperature ...during processing, a physical and chemical degradation of the used plastic powder occurs. To reuse the unmolten material and to realize reproducible part properties it is necessary to refresh the partcake powder and establish constant powder properties. In this publication PA12-powders are conditioned in a model experiment at building chamber temperatures for different periods. Process relevant material properties like phase transition temperatures or melt viscosity are analyzed. Also the influence of material pretreatment on resulting material and mechanical part properties by the use of degradated powder are investigated. Key words: degradation, aging, polyamide 12, mask sintering, laser sintering
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