Light-Mediated Directed Placement of Different DNA Sequences on Single Gold Nanoparticles.

This paper describes the light-directed functionalization of anisotropic gold nanoparticles with different thiolated-DNA oligomer (oligo) sequences. The starting nanoconstructs are gold nanostars (AuNS) uniformly grafted with one oligo sequence that are then exposed to fs-laser pulses at the plasmon resonance of the branches. The excitation selectively cleaves Au-S bonds at the tips of the branches to create vacant areas for functionalization with a different thiolated oligo sequence. Nanoconstructs synthesized by this approach present one oligo sequence on the AuNS body and branches and a different sequence at the tips. This process enables the formation of nanoparticle superstructures consisting of AuNS cores and small Au satellite nanoparticles at controlled locations after DNA hybridization. Our strategy enables selective oligo presentation at the single-particle level and opens prospects for sophisticated design of nanoscale assemblies that are important in a wide range of biological applications.

Label Free Particle-by-Particle Quantification of DNA Loading on Sorted Gold Nanostars.

This paper describes a label free technique for determining ligand loading on metal nanoparticles using a variant of secondary ion mass spectrometry. Au4004+ clusters bombard DNA-functionalized anisotropic gold nanostars and isotropic nanospheres with similar surface areas to determine ligand density. For each projectile impact, co-localized molecules within the emission area of a single impact (diameter of 10-15 nm) were examined for each particle. Individual nanoparticle analysis allows for determination of the relationship between particle geometry and DNA loading. We found that branched particles exhibited increased ligand density versus nanospheres and determined that positive and neutral curvature could facilitate additional loading. This methodology can be applied to optimize loading for any ligand-core interaction independent of nanoparticle core, ligand, or attachment chemistry.

The one hour yeast proteome.

We describe the comprehensive analysis of the yeast proteome in just over one hour of optimized analysis. We achieve this expedited proteome characterization with improved sample preparation, chromatographic separations, and by using a new Orbitrap hybrid mass spectrometer equipped with a mass filter, a collision cell, a high-field Orbitrap analyzer, and, finally, a dual cell linear ion trap analyzer (Q-OT-qIT, Orbitrap Fusion). This system offers high MS(2) acquisition speed of 20 Hz and detects up to 19 peptide sequences within a single second of operation. Over a 1.3 h chromatographic method, the Q-OT-qIT hybrid collected an average of 13,447 MS(1) and 80,460 MS(2) scans (per run) to produce 43,400 (x) peptide spectral matches and 34,255 (x) peptides with unique amino acid sequences (1% false discovery rate (FDR)). On average, each one hour analysis achieved detection of 3,977 proteins (1% FDR). We conclude that further improvements in mass spectrometer scan rate could render comprehensive analysis of the human proteome within a few hours.

Covalent modification of the NF-κB essential modulator (NEMO) by a chemical compound can regulate its ubiquitin binding properties in vitro.

Post-translational modification by ubiquitin plays important roles in multiple physiological and pathological processes. Ubiquitin-binding proteins play a critical role in recognizing and relaying polyubiquitin-based signaling. NEMO (NF-κB Essential Modulator) is a central player in canonical NF-κB signaling whose major function is to bind to Lys-63- and/or M1- (or linear) linked polyubiquitin chains generated in response to cell stimulation. Here we show that Withaferin A (WA), a steroidal lactone, causes a change in NEMO's interaction with specific types of polyubiquitin chains in vitro. WA induces full-length recombinant NEMO to bind to long Lys-48-linked polyubiquitin chains but not tetra-ubiquitin species. Significantly, the UBAN (ubiquitin binding in ABIN and NEMO) domain, essential for the ability of NEMO to bind M1/Lys-63-linked polyubiquitin, is dispensable for the WA-induced gain-of-function activity. Mass spectrometric analysis demonstrated that WA covalently modifies NEMO on a cysteine residue within the C-terminal zinc finger (ZF) domain. Point mutations to the ZF can reverse the WA-induced Lys-48-polyubiquitin binding phenotype. Our study demonstrates the feasibility of directly altering the ubiquitin interaction properties of an ubiquitin-binding protein by a chemical compound, thereby shedding light on a novel drug class to potentially alter polyubiquitin-based cellular processes.

Characterization of molecules binding to the 70K N-terminal region of fibronectin by IFAST purification coupled with mass spectrometry.

Fibronectin (Fn) is a large glycoprotein present in plasma and extracellular matrix and is important for many processes. Within Fn the 70 kDa N-terminal region (70k-Fn) is involved in cell-mediated Fn assembly, a process that contributes to embryogenesis, development, and platelet thrombus formation. In addition, major human pathogens including Staphlycoccus aureus and Streptococcus pyogenes bind the 70k-Fn region by a novel form of protein-protein interaction called ß-zipper formation, facilitating bacterial spread and colonization. Knowledge of blood plasma and platelet proteins that interact with 70k-Fn by ß-zipper formation is incomplete. In the current study, we aimed to characterize these proteins through affinity purification. For this affinity purification, we used a novel purification technique termed immiscible filtration assisted by surface tension (IFAST). The foundation of this technology is immiscible phase filtration, using a magnet to draw paramagnetic particle (PMP)-bound analyte through an immiscible barrier (oil or organic solvent) that separates an aqueous sample from an aqueous eluting buffer. The immiscible barrier functions to remove unbound proteins via exclusion rather than dilutive washing used in traditional isolation methods. We identified 31 interactors from plasma, of which only seven were previously known to interact with Fn. Furthermore, five proteins were identified to interact with 70k-Fn from platelet lysate, of which one was previously known. These results demonstrate that IFAST offers advantages for proteomic studies of interacting molecules in that the technique requires small sample volumes, can be done with high enough throughput to sample multiple interaction conditions, and is amenable to exploratory mass spectrometric and confirmatory immuno-blotting read-outs.

Identifying post-translational modifications of NEMO by tandem mass spectrometry after high affinity purification.

An integral component of NF-κB signalling is NEMO, NF-κB essential modulator, a regulatory protein of the IκB kinase (IKK) complex. Post-translational modifications of NEMO, including phosphorylation, SUMOylation, and ubiquitination are critical events during stimuli induced NF-κB activation. Here we demonstrate a method to detect post-translational modifications of NEMO using cells stably expressing polyhistidine tagged NEMO which allows for high-affinity purification of NEMO following rapid denaturing lysis and characterization by MS/MS. We identified a previously uncharacterized basal phosphorylation of NEMO at Serine 387 and tested the biological significance of this phosphorylation through a somatic genetic complementation analysis using the NEMO mutants S387A, S388D, and P388I in 1.3E2 NEMO-deficient murine pre-B cells. NF-κB signalling induced by bacterial lipopolysaccharide, Interleukin-1ß or the DNA damaging agent etoposide was not perturbed by these mutations of NEMO. Thus, S387 phosphorylation of NEMO is not a general requirement to mediate efficient NF-κB signalling and therefore may have cell type and/or stimulus-specific activity in vivo.

Smaller CpG-Conjugated Gold Nanoconstructs Achieve Higher Targeting Specificity of Immune Activation.

This study describes a side-by-side comparison of the in vitro immunostimulatory activity of cytosine-phosphate-guanine (CpG)-conjugated gold nanoparticles. Three different gold nanoparticle cores (13 nm spheres, 50 nm spheres, and 40 nm stars) with the same CpG surface density were investigated for toll-like receptor 9 activation. For this parameter set, 13 nm spheres displayed significantly higher specificity for targeting immune receptors and larger nanoparticles (50 nm spheres and 40 nm stars) showed higher cellular uptake and higher immune activation because of off-target effects. Changes in nanoparticle size and presentation of activating ligands affect construct-induced immune responses at different levels, and care must be taken when considering practical and global design rules for CpG delivery.

Erythropoietin signaling regulates heme biosynthesis.

Heme is required for survival of all cells, and in most eukaryotes, is produced through a series of eight enzymatic reactions. Although heme production is critical for many cellular processes, how it is coupled to cellular differentiation is unknown. Here, using zebrafish, murine, and human models, we show that erythropoietin (EPO) signaling, together with the GATA1 transcriptional target, AKAP10, regulates heme biosynthesis during erythropoiesis at the outer mitochondrial membrane. This integrated pathway culminates with the direct phosphorylation of the crucial heme biosynthetic enzyme, ferrochelatase (FECH) by protein kinase A (PKA). Biochemical, pharmacological, and genetic inhibition of this signaling pathway result in a block in hemoglobin production and concomitant intracellular accumulation of protoporphyrin intermediates. Broadly, our results implicate aberrant PKA signaling in the pathogenesis of hematologic diseases. We propose a unifying model in which the erythroid transcriptional program works in concert with post-translational mechanisms to regulate heme metabolism during normal development.

One-hour proteome analysis in yeast.

Recent advances in chromatography and mass spectrometry (MS) have made rapid and deep proteomic profiling possible. To maximize the performance of the recently produced Orbitrap hybrid mass spectrometer, we have developed a protocol that combines improved sample preparation (including optimized cellular lysis by extensive bead beating) and chromatographic conditions (specifically, 30-cm capillary columns packed with 1.7-µm bridged ethylene hybrid material) and the manufacture of a column heater (to accommodate flow rates of 350-375 nl/min) that increases the number of proteins identified across a single liquid chromatography-tandem MS (LC-MS/MS) separation, thereby reducing the need for extensive sample fractionation. This strategy allowed the identification of up to 4,002 proteins (at a 1% false discovery rate (FDR)) in yeast (Saccharomyces cerevisiae strain BY4741) over 70 min of LC-MS/MS analysis. Quintuplicate analysis of technical replicates reveals 83% overlap at the protein level, thus demonstrating the reproducibility of this procedure. This protocol, which includes cell lysis, overnight tryptic digestion, sample analysis and database searching, takes ∼24 h to complete. Aspects of this protocol, including chromatographic separation and instrument parameters, can be adapted for the optimal analysis of other organisms.