Thermal Profiling of Automotive Turbochargers in Durability Tests

A major portion of the development of an automotive powertrain system is devoted to robustness and durability testing to ascertain the viability of the design. For turbochargers, thermo-mechanical fatigue is often considered as life limiting failure mechanism for the turbine section, therefore, these tests involve repeated and continuous cycling of the turbocharger for hundreds of hours. The Thermal History Coatings (THC) can offer a new and unique solution. THCs are applied to the surface of a component and, when heated, the coating permanently changes according to the maximum temperature of exposure. The technique has been used in several turbomachinery, and other applications to capture the spatial temperature distribution of critical components. However, the turbocharger durability test presents new challenges for the technique. It has not been tested in this type of application and repeated cycling operation can test the response of the coating on the temperature measurements. In this paper, the capability of the THC for this application was investigated. For the first time, the effect of cyclic operation on the THC is reported. The measurement capability was demonstrated on two turbine housings tested on a gas stand, one for a single cycle, another for 10 cycles. The results show that the surface temperature profile of the two turbine housings can be accurately recorded and the results are validated against the installed thermocouples. The demonstration indicates that the THC can be used to acquire accurate and detailed spatial temperature distributions. This information improves the interpretation of a durability test.

C25-modified rifamycin derivatives with improved activity against Mycobacterium abscessus

Infections caused by Mycobacterium abscessus are difficult to treat due to its intrinsic resistance to most antibiotics. Formation of biofilms and the capacity of M. abscessus to survive inside host phagocytes further complicate eradication. Herein, we explored whether addition of a carbamate-linked group at the C25 position of rifamycin SV blocks enzymatic inactivation by ArrMab, an ADP-ribosyltransferase conferring resistance to rifampicin. Unlike rifampicin, 5j, a benzyl piperidine rifamycin derivative with a morpholino substituted C3 position, is not modified by purified ArrMab. Additionally, we show that the ArrMab D82 residue is essential for catalytic activity. Thermal profiling of ArrMab in the presence of 5j, rifampicin or rifabutin shows that 5j does not bind to ArrMab. We found that the activity of 5j is comparable to amikacin against M. abscessus planktonic cultures and pellicles. Critically, 5j also exerts potent antimicrobial activity against M. abscessus in human macrophages and shows synergistic activity with amikacin and azithromycin.

Diverse Hotspot Thermal Profiling Methods Detect Phosphorylation-Dependent Changes in Protein Stability

Hotspot thermal profiling (HTP) methods utilize modified-peptide level information in order to interrogate proteoform-specific stability inside of live cells. The first demonstration of HTP involved the integration of phosphopeptide enrichment into a TMT-based, single-LC separation thermal profiling workflow1. Here we present a new "label-fractionate-enrich" (LFE)-HTP method that involves high-pH reverse phase fractionation of TMT-labeled peptides prior to phosphopeptide enrichment, followed by peptide detection and quantitation using multi-notch LC-MS3. We find that LFE-HTP, while more resource intensive, improves the depth and precision of (phospho)proteoform coverage relative to the initial published HTP workflow. The fraction of detected phosphorylation sites that are significantly perturbed in this new dataset are consistent with those seen in our previous study, as well as those published by others, when compared head-to-head with the same analysis pipelines. Likewise, many hotspot phosphorylation sites identified in our paper are consistently reproduced by LFE-HTP as well as other modified HTP methods. The LFE-HTP dataset contains many novel hotspot phosphorylation sites that regulate the stability of diverse proteins, including phosphosites in the central glycolytic enzyme Aldolase A that are associated with monomer-to-oligomer formation, enzymatic activity and metabolic regulation in cancer cells. Our comparative analyses confirm that several variants of the HTP method can track modified proteoforms in live cells to detect and prioritize PTM-dependent changes in protein stability that may be associated with function.

Evaluation and Selection of Video Stabilization Techniques for UAV-Based Active Infrared Thermography Application

Unmanned Aerial Vehicles (UAVs) that can fly around an aircraft carrying several sensors, e.g., thermal and optical cameras, to inspect the parts of interest without removing them can have significant impact in reducing inspection time and cost. One of the main challenges in the UAV based active InfraRed Thermography (IRT) inspection is the UAV’s unexpected motions. Since active thermography is mainly concerned with the analysis of thermal sequences, unexpected motions can disturb the thermal profiling and cause data misinterpretation especially for providing an automated process pipeline of such inspections. Additionally, in the scenarios where post-analysis is intended to be applied by an inspector, the UAV’s unexpected motions can increase the risk of human error, data misinterpretation, and incorrect characterization of possible defects. Therefore, post-processing is required to minimize/eliminate such undesired motions using digital video stabilization techniques. There are number of video stabilization algorithms that are readily available; however, selecting the best suited one is also challenging. Therefore, this paper evaluates video stabilization algorithms to minimize/mitigate undesired UAV motion and proposes a simple method to find the best suited stabilization algorithm as a fundamental first step towards a fully operational UAV-IRT inspection system.

TP-MAP - an Integrated Software Package for the Analysis of 1D and 2D Thermal Profiling Data

Thermal profiling (TP) has emerged as a promising experimental methodology for elucidating the molecular targets of drugs and metabolites on a proteome-wide scale. Here, we present the Thermal Profiling Meltome Analysis Program (TP-MAP) software package for the analysis and ranking of 1D and 2D thermal profiling datasets. TP-MAP provides a user-friendly interface to quickly identify hit candidates and further explore targets of interest via intersection and crosslinking to public databases.

N-glycosylation on Oryza Sativa Root Germin-like Protein 1 is conserved but not required for stability or activity

Germin and germin-like proteins (GLPs) are a broad family of extracellular glycoproteins ubiquitously distributed in plants. Overexpression of Oryza sativa root germin like protein 1 (OsRGLP1) enhances superoxide dismutase (SOD) activity in transgenic plants. Here, we report bioinformatic analysis and heterologous expression of OsRGLP1 to study the role of glycosylation on OsRGLP1 protein stability and activity. Sequence analysis of OsRGLP1 homologs identified diverse N-glycosylation sequons, one of which was highly conserved. We therefore expressed OsRGLP1 in glycosylation-competent Saccharomyces cerevisiae as a Maltose Binding Protein (MBP) fusion. Mass spectrometry analysis of purified OsRGLP1 showed it was expressed by S. cerevisiae in both N-glycosylated and unmodified forms. Glycoprotein thermal profiling showed little difference in the thermal stability of the glycosylated and unmodified protein forms. Circular Dichroism spectroscopy of MBP-OsRGLP1 and a N-Q glycosylation-deficient variant showed that both glycosylated and unmodified MBP-OsRGLP1 had similar secondary structure, and both forms had equivalent SOD activity. Together, we concluded that glycosylation was not critical for OsRGLP1 protein stability or activity, and it could therefore likely be produced in Escherichia coli without glycosylation. Indeed, we found that OsRGLP1 could be efficiently expressed and purified from K12 shuffle E. coli with a specific activity of 1251±70 Units/mg. In conclusion, we find that some highly conserved N-glycosylation sites are not necessarily required for protein stability or activity, and describe a suitable method for production of OsRGLP1 which paves the way for further characterization and use of this protein.

Boosting detection of low abundance proteins in thermal proteome profiling experiments by addition of an isobaric trigger channel to TMT multiplexes

ABSTRACTThe study of low abundance proteins is a challenge to discovery-based proteomics. Mass-spectrometry (MS) applications, such as thermal proteome profiling (TPP) face specific challenges in detection of the whole proteome as a consequence of the use of nondenaturing extraction buffers. TPP is a powerful method for the study of protein thermal stability, but quantitative accuracy is highly dependent on consistent detection. Therefore, TPP can be limited in its amenability to study low abundance proteins that tend to have stochastic or poor detection by MS. To address this challenge, we incorporated an affinity purified protein complex sample at submolar concentrations as an isobaric trigger channel into a mutant TPP (mTPP) workflow to provide reproducible detection and quantitation of the low abundance subunits of the Cleavage and Polyadenylation Factor (CPF) complex. The inclusion of an isobaric protein complex trigger channel increased detection an average of 40x for previously detected subunits and facilitated detection of CPF subunits that were previously below the limit of detection. Importantly, these gains in CPF detection did not cause large changes in melt temperature (Tm) calculations for other unrelated proteins in the samples, with a high positive correlation between Tm estimates in samples with and without isobaric trigger channel addition. Overall, the incorporation of affinity purified protein complex as an isobaric trigger channel within a TMT multiplex for mTPP experiments is an effective and reproducible way to gather thermal profiling data on proteins that are not readily detected using the original TPP or mTPP protocols.

A computational method for detection of ligand-binding proteins from dose range thermal proteome profiles

Detecting ligand-protein interactions in living cells is a fundamental challenge in molecular biology and drug research. Proteome-wide profiling of thermal stability as a function of ligand concentration promises to tackle this challenge. However, current data analysis strategies use preset thresholds that can lead to suboptimal sensitivity/specificity tradeoffs and limited comparability across datasets. Here, we present a method based on statistical hypothesis testing on curves, which provides control of the false discovery rate. We apply it to several datasets probing epigenetic drugs and a metabolite. This leads us to detect off-target drug engagement, including the finding that the HDAC8 inhibitor PCI-34051 and its analog BRD-3811 bind to and inhibit leucine aminopeptidase 3. An implementation is available as an R package from Bioconductor (https://bioconductor.org/packages/TPP2D). We hope that our method will facilitate prioritizing targets from thermal profiling experiments.