David Butcher

Shared Research Resources (SRRs), also known as Core Facilities or Research Service Centers are essential for advancing scientific research in academic, government, industry, and non-profit institutions since they provide access to advanced resources like innovative research technologies, latest equipment in variety of platforms, and expert personnel with skills to solve complex problems as well as providing training and pursue technology development. Traditionally, SRRs are thought of as confined to academic R1 universities and non-profit organizations. However, many regional centers and national laboratories also feature core facilities that cater to local, regional, national, and international users. Like traditional SRRs, these facilities provide user training, in-person access, and remote access to instruments and services. In this section, we will discuss some of the opportunities these cores provide to scientists worldwide. We will also discuss some of the challenges these facilities face in running and maintaining such a large and continuous operation with many users.

This protocol describes the culture of E. coli MG1655 from a glycerol stock solution and subsequent lysis. It is intended for generating small quantities of whole cell lysate from cells grown in nutrient-rich (e.g. LB or TB medium) and/or nutrient-poor (e.g. M9 minimal) media for proteomics analysis. Culture and lysis of cells in a single Bio-Reaction tube minimizes the possibility of contamination. All cell culture steps should be carried out using sterile materials and proper aseptic technique. This procedure is intended to be carried out in a biosafety cabinet, but it can also easily be performed on a bench top with a Bunsen burner. Applications of this protocol at the National MagLab have utilized E. coli stocks from the Coli Genetic Stock Center at Yale University. The final lysate typically contains 300 - 500 μg/mL of protein for M9 minimal medium cultures and 750 - 1000 μg/mL for LB medium cultures, as determined by BCA assay. The cell lysis protocol is adapted from Preparing human WCL for analysis by qualitative TDMS by Caroline DeHart, with a few changes.

Human biology is tightly linked to proteins, yet most measurements do not precisely determine alternatively spliced sequences or posttranslational modifications. Here, we present the primary structures of ~30,000 unique proteoforms, nearly 10 times more than in previous studies, expressed from 1690 human genes across 21 cell types and plasma from human blood and bone marrow. The results, compiled in the Blood Proteoform Atlas (BPA), indicate that proteoforms better describe protein-level biology and are more specific indicators of differentiation than their corresponding proteins, which are more broadly expressed across cell types. We demonstrate the potential for clinical application, by interrogating the BPA in the context of liver transplantation and identifying cell and proteoform signatures that distinguish normal graft function from acute rejection and other causes of graft dysfunction.

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS (“PEPPI-MS”), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.

Flavohemoglobins (fHbs) are heme proteins found in prokaryotic and eukaryotic microbes. They are involved in NO detoxification through an NO˙ dioxygenase mechanism. The N-terminal heme globin domain allows for binding of gaseous ligands whereas a C-terminal NADH/FADH binding domain facilitates association of redox cofactors necessary for ligand reduction. The NO˙ dioxygenase function is important in facilitating immune resistance by protecting the cell from nitrosative stress brought about by a host organism; as a result, bacterial flavoHbs have recently been considered as targets for the development of new antibiotics. Here, photoacoustic calorimetry and transient absorption spectroscopy have been used to characterize energetics, structural dynamics, and kinetics of CO migration within bacterial flavoHbs from Ralstonia eutropha (FHP) and Staphylococcus aureus (HMP_Sa) in the presence and absence of antibiotic azole compounds. In FHP, the ligand photo-release is associated with ΔH = 26.2 ± 7.0 kcal mol⁻¹ and ΔV = 25.0 ± 1.5 mL mol⁻¹ while in HMP_Sa, ΔH = 34.7 ± 8.0 kcal mol⁻¹ and ΔV = 28.6 ± 17 mL mol⁻¹ were observed, suggesting distinct structural changes associated with ligand escape from FHP and HMP_Sa. In the presence of ketoconazole, the CO escape leads to a more negative enthalpy change and volume change whereas association of miconazole to FHP or HMP_Sa does not impact the reaction volume. These data are in agreement with the computational results that propose distinct binding sites for ketoconazole and miconazole on CO bound FHP. Miconazole or ketoconazole binding to either protein has only a negligible impact on the CO association rates, indicating that azole drugs do not impact flavoHbs interactions with gaseous ligands but may inhibit the NOD activity through preventing the electron transfer between FAD and heme cofactors.

Rationale: The molecular environment is known to impact the secondary and tertiary structures of biomolecules both in solution and in the gas phase, shifting the equilibrium between different conformational and oligomerization states. However, there is a lack of studies monitoring the impacts of solution additives and gas‐phase modifiers on biomolecules characterized using ion mobility techniques. Methods: The effect of solution additives and gas‐phase modifiers on the molecular environment of two common heme proteins, bovine cytochrome c and equine myoglobin, is investigated as a function of the time after desolvation (e.g., 100–500 ms) using nanoelectrospray ionization coupled to trapped ion mobility spectrometry with detection by time‐of‐flight mass spectrometry. Organic compounds used as additives/modifiers (methanol, acetonitrile, acetone) were either added to the aqueous protein solution before ionization or added to the ion mobility bath gas by nebulization. Results: Changes in the mobility profiles are observed depending on the starting solution composition (i.e., in aqueous solution at neutral pH or in the presence of organic content: methanol, acetone, or acetonitrile) and the protein. In the presence of gas‐phase modifiers (i.e., N ₂ doped with methanol, acetone, or acetonitrile), a shift in the mobility profiles driven by the gas‐modifier mass and size and changes in the relative abundances and number of IMS bands are observed. Conclusions: We attribute the observed changes in the mobility profiles in the presence of gas‐phase modifiers to a clustering/declustering mechanism by which organic molecules adsorb to the protein ion surface and lower energetic barriers for interconversion between conformational states, thus redefining the free energy landscape and equilibria between conformers. These structural biology experiments open new avenues for manipulation and interrogation of biomolecules in the gas phase with the potential to emulate a large suite of solution conditions, ultimately including conditions that more accurately reflect a variety of intracellular environments.

Deoxyribonucleic acids can form a wide variety of structural motifs which differ greatly from the typical antiparallel duplex stabilized by Watson-Crick base pairing. Many of these structures are thought to occur in vivo and may have essential roles in the biology of the cell. Among these is the parallel-stranded duplex – a structural motif in which DNA strands associate in a head-to-head fashion with the 5’ ends at the same end of the duplex which is stabilized by reverse Watson-Crick base pairing. In this study, parallel- and antiparallel- stranded DNA duplexes formed from two different 12-mer oligonucleotides were studied using native electrospray ionization combined with trapped ion mobility spectrometry and mass spectrometry (TIMS-MS). The DNA duplex charge plays an important role in the gas-phase mobility profile, with a more compact form in negative mode than in positive mode ( ~100 Å2 between -4 and +4). Despite sequence mismatches, homo- and hetero- DNA duplexes were formed in solution and transfer to the gas-phase, where a more compact structure was observed for the parallel compared to the antiparallel duplexes ( ~50 Å2), in good agreement with theoretical calculations. Theoretical studies suggest that a reduction (or compaction) along the helical axis of the parallel- and anti-parallel DNA duplexes is observed upon transfer to the gas-phase.

Type 1 nonsymbiotic hemoglobins are found in a wide variety of land plants and exhibit very high affinities for exogenous gaseous ligands. These proteins are presumed to have a role in protecting plant cells from oxidative stress under etiolated/hypoxic conditions through NO dioxygenase activity. In this study we have employed photoacoustic calorimetry, time-resolved absorption spectroscopy, and classical molecular dynamics simulations in order to elucidate thermodynamics, kinetics, and ligand migration pathways upon CO photodissociation from WT and a H73L mutant of type 1 nonsymbiotic hemoglobin from Oryza sativa (rice). We observe a temperature dependence of the resolved thermodynamic parameters for CO photodissociation from CO-rHb1 which we attribute to temperature dependent formation of a network of electrostatic interactions in the vicinity of the heme propionate groups. We also observe slower ligand escape from the protein matrix under mildly acidic conditions in both the WT and H73L mutant (τ = 134 ± 19 and 90 ± 15 ns). Visualization of transient hydrophobic channels within our classical molecular dynamics trajectories allows us to attribute this phenomenon to a change in the ligand migration pathway which occurs upon protonation of the distal His73, His117, and His152. Protonation of these residues may be relevant to the functioning of the protein in vivo given that etiolation/hypoxia can cause a decrease in intracellular pH in plant cells.