Large-Scale Quantitative Cross-Linking and Mass Spectrometry Provides New Insight on Protein Conformational Plasticity within Organelles, Cells, and Tissues.

Many proteins can exist in multiple conformational states to achieve distinct functional roles. These states include alternative conformations, variable PTMs, and association with interacting protein, nucleotide, and ligand partners. Quantitative chemical cross-linking of live cells, organelles, or tissues together with mass spectrometry provides the relative abundance of cross-link levels formed in two or more compared samples, which depends both on the relative levels of existent protein conformational states in the compared samples as well as the relative likelihood of the cross-link originating from each.

Exploring clinician perspectives of systems-based practice: A physician training challenge.

Introduction: Systems-based practice (SBP) has been a competency in US graduate medical education for over 20 years, but it is not well implemented. SBP is loosely defined as physician's responsiveness to the larger system of healthcare. The aim of this study was to describe healthcare professionals' perspectives regarding: (1) their knowledge and beliefs about SBP and (2) their beliefs regarding factors in clinical learning environments (CLE) that facilitate or hinder operationalisation and learning of SBP.

Combining Quantitative Proteomics and Interactomics for a Deeper Insight into Molecular Differences between Human Cell Lines.

In modern biomedical research, cultivable cell lines are an indispensable tool, and the selection of cell lines that exhibit specific functional profiles is often critical to success. Cellular functional pathways have evolved through the selection of protein intra- and intermolecular interactions collectively referred to as the interactome. In the present work, quantitative in vivo protein cross-linking and mass spectrometry were used to probe large-scale protein interactome differences among three commonly employed human cell lines, namely, HEK293, MCF-7, and HeLa cells.

Role of RNA structural plasticity in modulating HIV-1 genome packaging and translation.

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5' polyA element, resulting in formation of an extended 5' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs.

Chemical cross-linking and mass spectrometry enabled systems-level structural biology.

Structural information on protein-protein interactions (PPIs) is essential for improved understanding of regulatory interactome networks that confer various physiological and pathological responses. Additionally, maladaptive PPIs constitute desirable therapeutic targets due to inherently high disease state specificity. Recent advances in chemical cross-linking strategies coupled with mass spectrometry (XL-MS) have positioned XL-MS as a promising technology to not only elucidate the molecular architecture of individual protein assemblies, but also to characterize proteome-wide PPI networks.

Combining quantitative proteomics and interactomics for a deeper insight into molecular differences between human cell lines.

Cellular functional pathways have evolved through selection based on fitness benefits conferred through protein intra- and inter-molecular interactions that comprise all protein conformational features and protein-protein interactions, collectively referred to as the interactome. While the interactome is regulated by proteome levels, it is also regulated independently by, post translational modification, co-factor, and ligand levels, as well as local protein environmental factors, such as osmolyte concentration, pH, ionic strength, temperature and others. In modern biomedical research, cultivatable cell lines have become an indispensable tool, with selection of optimal cell lines that exhibit specific functional profiles being critical for success in many cases.

Interactome dynamics during heat stress signal transmission and reception.

Among evolved molecular mechanisms, cellular stress response to altered environmental conditions to promote survival is among the most fundamental. The presence of stress-induced unfolded or misfolded proteins and molecular registration of these events constitute early steps in cellular stress response. However, what stress-induced changes in protein conformations and protein-protein interactions within cells initiate stress response and how these features are recognized by cellular systems are questions that have remained difficult to answer, requiring new approaches.

Multidimensional Cross-Linking and Real-Time Informatics for Multiprotein Interaction Studies.

Chemical cross-linking combined with mass spectrometry is a technique used to study protein structures and identify protein complexes. Traditionally, chemical cross-linkers contain two reactive groups, allowing them to covalently bond a pair of proximal residues, either within a protein or between two proteins. The output of a cross-linking experiment is a list of interacting site pairs that provide structural constraints for modeling of new structures and complexes.

Exploiting a rodent cell block for intrinsic resistance to HIV-1 gene expression in human T cells.

Unlike humans, mice are unable to support HIV-1 infection. This is due, in part, to a constellation of defined minor, species-specific differences in conserved host proteins needed for viral gene expression. Here, we used precision CRISPR/Cas9 gene editing to engineer a "mousified" version of one such host protein, cyclin T1 (CCNT1), in human T cells.

A planar quadrupole device for transmitting and trapping ions in high vacuum.

Rationale: Hybrid mass spectrometers combine multiple mass analyzers to achieve optimal performance in terms of tandem mass spectrometry, high mass resolving power, and mass measurement accuracy for studying highly complex samples. As a result, the need for transport, trapping, and control of ion kinetic energies is critical for the successful integration of multiple mass analyzers and hybrid instrument operation. In addition, transportation of ion populations between two physically distinct locations can result in time-of-flight (TOF) discrimination against ions with widely disparate m/z values, compromising full mass spectral performance.

Integrated Analysis of Cross-Links and Dead-End Peptides for Enhanced Interpretation of Quantitative XL-MS.

Chemical cross-linking with mass spectrometry provides low-resolution structural information on proteins in cells and tissues. Combined with quantitation, it can identify changes in the interactome between samples, for example, control and drug-treated cells or young and old mice. A difference can originate from protein conformational changes that alter the solvent-accessible distance separating the cross-linked residues.

HIV-1 virological synapse formation enhances infection spread by dysregulating Aurora Kinase B.

HIV-1 spreads efficiently through direct cell-to-cell transmission at virological synapses (VSs) formed by interactions between HIV-1 envelope proteins (Env) on the surface of infected cells and CD4 receptors on uninfected target cells. Env-CD4 interactions bring the infected and uninfected cellular membranes into close proximity and induce transport of viral and cellular factors to the VS for efficient virion assembly and HIV-1 transmission. Using novel, cell-specific stable isotope labeling and quantitative mass spectrometric proteomics, we identified extensive changes in the levels and phosphorylation states of proteins in HIV-1 infected producer cells upon mixing with CD4+ target cells under conditions inducing VS formation.

Integrated Analysis of Cross-Links and Dead-End Peptides for Enhanced Interpretation of Quantitative XL-MS.

XL-MS provides low-resolution structural information of proteins in cells and tissues. Combined with quantitation, it can identify changes in the interactome between samples, for example, control and drug-treated cells, or young and old mice. A difference can originate from protein conformational changes altering the solvent-accessible distance separating the cross-linked residues.

Studying Protein-Ligand Interactions by Protein Denaturation and Quantitative Cross-Linking Mass Spectrometry.

Recently, several mass spectrometry methods have utilized protein structural stability for the quantitative study of protein-ligand engagement. These protein-denaturation approaches, which include thermal proteome profiling (TPP) and stability of proteins from rates of oxidation (SPROX), evaluate ligand-induced denaturation susceptibility changes with a MS-based readout. The different techniques of bottom-up protein-denaturation methods each have their own advantages and challenges.

Skeletal muscle mitochondrial interactome remodeling is linked to functional decline in aged female mice.

Genomic, transcriptomic and proteomic approaches have been used to gain insight into molecular underpinnings of aging in laboratory animals and in humans. However, protein function in biological systems is under complex regulation and includes factors besides abundance levels, such as modifications, localization, conformation and protein-protein interactions. By making use of quantitative chemical cross-linking technologies, we show that changes in the muscle mitochondrial interactome contribute to mitochondrial functional decline in aging in female mice.

Therapy professionals in critical care: A UK wide workforce survey.

Introduction: National guidelines suggest recommended staffing levels for therapies. The aim of this study was to capture information on existing staffing levels, roles and responsibilities and service structures.

Methods: An observational study using online surveys distributed to 245 critical care units across the United Kingdom (UK).

Towards a structurally resolved human protein interaction network.

Cellular functions are governed by molecular machines that assemble through protein-protein interactions. Their atomic details are critical to studying their molecular mechanisms. However, fewer than 5% of hundreds of thousands of human protein interactions have been structurally characterized.

Mitochondrial interactome quantitation reveals structural changes in metabolic machinery in the failing murine heart.

Advancements in cross-linking mass spectrometry (XL-MS) bridge the gap between purified systems and native tissue environments, allowing the detection of protein structural interactions in their native state. Here we use isobaric quantitative protein interaction reporter technology (iqPIR) to compare the mitochondria protein interactomes in healthy and hypertrophic murine hearts, 4 weeks post-transaortic constriction. The failing heart interactome includes 588 statistically significant cross-linked peptide pairs altered in the disease condition.

Differential Proteome and Interactome Analysis Reveal the Basis of Pleiotropy Associated With the Histidine Methyltransferase Hpm1p.

The methylation of histidine is a post-translational modification whose function is poorly understood. Methyltransferase histidine protein methyltransferase 1 (Hpm1p) monomethylates H243 in the ribosomal protein Rpl3p and represents the only known histidine methyltransferase in Saccharomyces cerevisiae. Interestingly, the hpm1 deletion strain is highly pleiotropic, with many extraribosomal phenotypes including improved growth rates in alternative carbon sources.

Improved Interpretation of Protein Conformational Differences and Ligand Occupancy in Large-Scale Cross-Link Data.

Chemical cross-linking of proteins in complex samples, cells, or even tissues is emerging to provide unique structural information on proteins and complexes that exist within native or nativelike environments. The public database XLinkDB automatically maps cross-links to available structures based on sequence homology. Structures most likely to reflect protein conformations in the cross-linked sample are routinely identified by having cross-linked residues separated by Euclidean distances within the maximum span of the applied cross-linker.

Upregulation of mitochondrial ATPase inhibitory factor 1 (ATPIF1) mediates increased glycolysis in mouse hearts.

In hypertrophied and failing hearts, fuel metabolism is reprogrammed to increase glucose metabolism, especially glycolysis. This metabolic shift favors biosynthetic function at the expense of ATP production. Mechanisms responsible for the switch are poorly understood.

Removal of stormwater dissolved organic nitrogen through biotransformation using activated carbon.

Conventional bioretention systems are not effectively designed to remove stormwater dissolved organic nitrogen (DON). Biotransformation study on five organic nitrogenous compounds with different values for adsorption on coal activated carbon (AC) and bioavailability revealed that adsorption is a greater controlling factor for ammonification than bioavailability. This study also showed three apparent benefits: enhancement of the ammonification rate, ammonification of the bio-recalcitrant organic nitrogenous compounds, for example, pyrrole, and bio-regeneration of the adsorbent (coal AC).

CsrA-Controlled Proteins Reveal New Dimensions of Acinetobacter baumannii Desiccation Tolerance.

Hospital environments are excellent reservoirs for the opportunistic pathogen Acinetobacter baumannii in part because it is exceptionally tolerant to desiccation. We found that relative to other A. baumannii strains, the virulent strain AB5075 was strikingly desiccation resistant at 2% relative humidity (RH), suggesting that it is a good model for studies of the functional basis of this trait.

Investigation into reversed-phase chromatography peptide separation systems part V: Establishment of a screening strategy for development of methods for assessment of pharmaceutical peptides' purity.

The paper describes a simple and rapid reversed-phase UHPLC method development screening strategy for the purity determination of peptide-based pharmaceuticals. The protocol utilises five disparate column and six volatile or non-volatile mobile phases (i.e.