Protein-protein interactions are essential for protein complex formation and function. Affinity purification coupled with mass spectrometry (AP-MS) is the method of choice for studying protein-protein interactions at the systems level under different physiological conditions. Although effective in capturing stable protein interactions, transient, weak, and/or dynamic interactors are often lost due to extended procedures during conventional AP-MS experiments. To circumvent this problem, we have recently developed XAP (in vivo cross-linking (X)-assisted affinity purification)-MS strategy to better preserve dynamic protein complexes under native lysis conditions. In addition, we have developed XBAP (in vivo cross-linking (X)-assisted bimolecular tandem affinity purification)-MS method by incorporating XAP with bimolecular affinity purification to define dynamic and heterogeneous protein subcomplexes. Here we describe general experimental protocols of XAP- and XBAP-MS to study dynamic protein complexes and their subcomplexes, respectively. Specifically, we present their applications in capturing and identifying proteasome dynamic interactors and ubiquitin receptor (UbR)-proteasome subcomplexes.

Protein-protein interactions are essential to various physiological processes in living cells. A full characterization of protein interactions is critical to our understanding of their roles in the regulation of protein functions. Affinity purification coupled with mass spectrometry (AP-MS) has become one of the most effective approaches to systematically study protein-protein interactions. In combination with quantitative mass spectrometry, specific interacting proteins can be efficiently distinguished from nonspecific background proteins. Based on interaction affinity and kinetics, protein interactions can be classified into different categories such as stable and dynamic interactions. Standard biochemical methods are effective in capturing and identifying stable protein interactions but are not sufficient enough to identify dynamic interactors. In this chapter, we describe integrated strategies to allow the identification of dynamic interactors of protein complexes by incorporating new sample preparation methods with SILAC-based quantitation.

High-temperature superconducting REBCO coated conductor is one of the main candidates for next-generation high field magnets in fusion reactors and particle accelerators owing to their high current-carrying capability. Although these materials can operate at higher temperatures and generate higher magnetic fields than their counterparts with lower critical temperatures, protecting the REBCO magnet against quench is challenging. A variety of candidate technologies that may be able to enable self-protection, including no-insulation technology and insulative coatings with temperature-dependent resistance, are in development. In order to understand current sharing and thermal processes during a quench, we model a REBCO pancake coil as an electrical circuit, considering power generation and heat transfer along conductor turns, and study the current distribution around a local defect with lower critical current. The magnetic field and coil terminal voltage predicted by the simulation was compared to published experimental results. Our results provide useful insights into how current sharing occurs around defects.

To enable the physics research that continues to deepen our understanding of the Universe, future circular colliders will require a critical and unique instrument—magnets that can generate a dipole field of 20 T and above. However, today’s maturing magnet technology for low-temperature superconductors (Nb-Ti and Nb3 Sn) can lead to a maximum dipole field of around 16 T. High-temperature superconductors such as REBCO can, in principle, generate higher dipole fields but significant challenges exist for both conductor and magnet technology. To address these challenges, several critical research needs, including direct needs on instrumentation and measurements, are identified to push for the maximum dipole fields a REBCO accelerator magnet can generate. We discuss the research needs by reviewing the current results and outlining the perspectives for future technology development, followed by a brief update on the status of the technology development at Lawrence Berkeley National Laboratory. We present a roadmap for the next decade to develop 20 T-class REBCO accelerator magnets as an enabling instrument for future energy-frontier accelerator complex.

In recent years, cross-linking mass spectrometry (XL-MS) has made enormous strides as a technology for probing protein-protein interactions (PPIs) and elucidating architectures of multisubunit assemblies. To define conformational and interaction dynamics of protein complexes under different physiological conditions, various quantitative cross-linking mass spectrometry (QXL-MS) strategies based on stable isotope labeling have been developed. These QXL-MS approaches have effectively allowed comparative analysis of cross-links to determine their relative abundance changes at global scales. Although successful, it remains challenging to consistently obtain quantitative measurements on low-abundant cross-links. Therefore, targeted QXL-MS is needed to enable MS "Western" analysis of cross-links to enhance sensitivity and reliability in quantitation. To this end, we have established a robust parallel reaction monitoring (PRM)-based targeted QXL-MS platform using sulfoxide-containing MS-cleavable cross-linker disuccinimidyl sulfoxide (DSSO), permitting label-free comparative analysis of selected cross-links across multiple samples. In addition, we have applied this methodology to study phosphorylation-dependent conformational dynamics of the human 26S proteasome. The PRM-based targeted QXL-MS analytical platform described here is applicable for all sulfoxide-containing MS-cleavable cross-linkers and can be directly adopted for comparative studies of protein-protein interactions in various cellular contexts.

In this paper, we consider the advantages of an alternative design concept for HTS accelerator magnets operating at 20 K or above. The idea is primarily built on using REBCO tape as the main conductor, but may be applicable to other HTS. The key concepts are to align REBCO tapes in the most favourable field orientation and to make joints for every turn such that the tapes will not have to be wound over the saddle ends. We argue that such a concept involving resistive joints is viable at 20 K or above due to an increased cryogenic efficiency, and has multiple advantages that would more than compensate for the resistive heating cost penalty. First, the favourable tape orientation can allow a much higher current carrying capability. Second, the short unit length of tapes equal to the length of the magnet will be much more economical and can be specified at a higher performance than a long continuous piece equal to the number of turns multiplied by the length of the magnet. Third, any defective conductor can be replaced easily and at a much lower cost than an entire coil. Fourth, with each tape separately sourced and connected, efficient grading with stress management can be achieved. Fifth, the straight section of the magnet would be modular and easily scalable for production in industry. Correspondingly, the most challenging part is the end cap design and joint technology, whose geometrical constraints are well within national laboratories' capabilities, making the R&D and prototyping phases much more affordable, with a turnover time much quicker than testing full size magnets. Additional attractive potentials include conductor development (e.g., double-sided extra thick REBCO), novel diagnostics (e.g., individual tape quench detection and protection), synergy with fusion devices research (e.g., demountable joints), and other possibilities.

The 26S proteasome is a mega-dalton protein complex responsible for intracellular degradation in eukaryotes. It is composed of two subcomplexes: the 20S core particle and the 19S regulatory particle, which form compositionally and structurally heterogeneous proteasome complexes in cells. To fully characterize the 26S proteasome, it is necessary to understand its structural and functional diversities. Multiple mass spectrometry (MS) methodologies have been developed in recent years for the study of proteasome structural dynamics in which biochemically isolated complexes are subjected to analysis. Due to the inherent heterogeneity of proteasome complexes, single-bait affinity purification typically results in a mixture of compositionally heterogeneous complexes regardless of the baits, making accurate assessment of complex-specific conformations and functions challenging. To facilitate complex-centric analysis, we have adopted a bimolecular affinity purification method utilizing a dual-bait cell line expressing tagged 19S and tagged 20S subunits to improve the homogeneity of the resulting 26S holocomplexes. To establish the method, four types of purifications were performed and the resulting samples were extensively examined by biochemical analysis and two label-free quantitative MS methods. Our results have demonstrated the effectiveness of this purification strategy in improving the complex homogeneity for downstream biochemical and MS characterizations. This strategy will be valuable for facilitating detailed quantitative assessments of complex-specific molecular details under different conditions and can be directly adopted for studying other complexes.

Detecting local heat-dissipating zones in high-temperature superconductor (HTS) magnets is a challenging task due to slow propagation of such zones in HTS conductors. For long conductor lengths, voltage-based methods may not provide a sufficient sensitivity or redundancy, and therefore nonvoltage-based detection alternatives are being sought. One of those is the recently proposed method of Eigen Frequency Thermometry (EFT), which is an active acoustic technique for a fast and nonintrusive detection of 'hot spots,' utilizing temperature dependence of the conductor elastic moduli. In this work, we demonstrate the efficiency of EFT for detecting localized heating in a 1.2-m-long sample of REBCO tape immersed in liquid nitrogen, and benchmark sensitivity of the acoustic detection with respect to voltage, hot spot temperature, and power dissipation in the conductor. Modifying the original technique for differential mode of operation enables a much improved sensitivity, and adds a hot spot localization capability. Furthermore, we adapt this technique to subscale coils wound with REBCO CORC conductor built in the framework of U.S. Magnet Development Program. A successful thermal-based detection of dissipation onset at the critical current for a two-layer canted CORC dipole assembly is discussed.

Abstract Background Restrictive patterns of pulmonary function abnormalities associated with asbestos exposure are well described. Studies are less consistent, however, regarding the association of asbestos inhalation with airway dysfunction and obstructive impairment. Methods We compared pulmonary function test results between 277 chrysotile exposed workers (22% non-smokers) and 177 unexposed controls (50.3% non-smokers). Information on exposure and smoking were collected using a standardized questionnaire. Standardized spirometric and DCLO Measurement methods were utilized. CXRs were read based on ILO pneumoconiosis guidelines. Results Asbestos exposed subjects had significantly reduced FVC, FEV1, FEV1/FVC and DLCO. Restricting the analysis to non-smokers, asbestos workers still had about 3% lower FEV1/FVC ratio than controls, but this difference did not reach statistical significance. Among exposed workers, the presence of radiographic evidence of asbestosis further lowered FVC and DLCO but not FEV1/FVC compared to asbestos exposure without radiographic asbestosis. Additionally, smoking asbestos workers had significantly lower DLCO compared to non-smoking workers. Conclusion Asbestos exposure, especially when radiographic evidence of interstitial fibrosis from asbestosis is present, leads to significant decreases in FVC, FEV1 and the DLCO. However, asbestos exposure alone is not significantly associated with a reduction of the FEV1/FVC. Smoking-asbestos workers had significantly lower DLCO than their non-smoking counterparts. Whether asbestos interacts with smoking additively or synergistically on DLCO needs further investigation. Similarly, further studies are needed to assess the progression and clinical significance of asbestos induced airway dysfunction.

Work in yeast models has benefitted tremendously from the insertion of epitope or fluorescence tags at the native gene locus to study protein function and behavior under physiological conditions. In contrast, work in mammalian cells largely relies on overexpression of tagged proteins because high-quality antibodies are only available for a fraction of the mammalian proteome. CRISPR/Cas9-mediated genome editing has recently emerged as a powerful genome-modifying tool that can also be exploited to insert various tags and fluorophores at gene loci to study the physiological behavior of proteins in most organisms, including mammals. Here we describe a versatile toolset for rapid tagging of endogenous proteins. The strategy utilizes CRISPR/Cas9 and microhomology-mediated end joining repair for efficient tagging. We provide tools to insert 3×HA, His₆FLAG, His₆-Biotin-TEV-RGSHis₆, mCherry, GFP, and the auxin-inducible degron tag for compound-induced protein depletion. This approach and the developed tools should greatly facilitate functional analysis of proteins in their native environment.