Measuring Nanoscale Thermostability of Cell Membranes with Single Gold-Diamond Nanohybrids.

Much of the current understanding of thermal effects in biological systems is based on macroscopic measurements. There is little knowledge about the local thermostability or heat tolerance of subcellular components at the nanoscale. Herein, we show that gold nanorod-fluorescent nanodiamond (GNR-FND) hybrids are useful as a combined nanoheater/nanothermometer in living cells. With the use of a 594 nm laser for both heating and probing, we measure the temperature changes by recording the spectral shifts of the zero-phonon lines of negatively charged nitrogen-vacancy centers in FNDs. The technique allows us to determine the rupture temperatures of individual membrane nanotubes in human embryonic kidney cells, as well as to generate high temperature gradients on the cell membrane for photoporation and optically controlled hyperthermia. Our results demonstrate a new paradigm for hyperthermia research and application.

Quantitative analysis of oligosaccharides derived from sulfated glycosaminoglycans by nanodiamond-based affinity purification and matrix-assisted laser desorption/ionization mass spectrometry.

Degraded fragments of sulfated glycosaminoglycans (GAGs) are key reporters for profiling the burden of mucopolysaccharidosis (MPS) disease at baseline and during therapy. Here, we present a high-throughput assay, which combines microwave-assisted degradation, solid-phase affinity purification, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), for quantitative analysis of sulfated oligosaccharides in biological samples. First, sulfated oligosaccharides such as chondroitin-4-sulfate (CS) were efficiently isolated from highly diluted solutions or spiked artificial cerebrospinal fluid (aCSF) using polyarginine-coated nanodiamonds (PA-coated NDs) as affinity sorbents. Next, they were degraded to disaccharides through microwave-assisted methanolysis or enzymatic digestion for subsequent MALDI-TOF MS analysis. The reaction times for GAG depolymerization were significantly reduced from a few hours to less than 7 min under the microwave irradiation. Deuterium-labeled internal standards were then mixed with the CS-derived disaccharides for quantitative analysis by MALDI-TOF MS using the N-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) matrix. The new assay is facile, specific (with distinct chlorine-isotope trait markers), sensitive (with a detection limit of ~70 pg), and potentially useful for clinical diagnosis of MPS.

Efficient enrichment of phosphopeptides by magnetic TiO2-coated carbon-encapsulated iron nanoparticles.

Titanium dioxide (TiO2) has been widely used for phosphopeptide enrichment. Several approaches have been reported to produce magnetic TiO2 affinity probes. In this report, we present a facile approach to immobilize TiO2 onto poly(acrylic acid)-functionalized magnetic carbon-encapsulated iron nanoparticles as affinity probes for efficient enrichment of phosphopeptides. By using the new magnetic TiO2 affinity probes, denoted as TiO2-coated Fe@CNPs, rapid and effective MALDI-TOF MS profiling of phosphopeptides was demonstrated in different model systems such as tryptic digests of ß-casein, and complex ß-casein/BSA mixture. The TiO2-coated Fe@CNPs out-performed the commercial TiO2-coated magnetic beads for detection of phosphopeptides from tryptic digests of ß-casein/BSA mixture with a molar ratio of 1:100. The new TiO2-coated magnetic probes were also proven to be applicable for real life samples. The magnetic TiO2-coated Fe@CNPs were employed to selectively isolate phosphopeptides from tryptic digests of HeLa cell lysates and out-performed the commercial magnetic TiO2 beads in the number of identified phosphopeptides and phosphorylation sites. In a 200-µg equivalent of HeLa cell lysates, we identified 1415 unique phosphopeptides and 1093 phosphorylation sites, indicating the good performance of the new approach.

Comprehensive molecular imaging of photolabile surface samples with synchronized dual-polarity time-of-flight mass spectrometry.

This work presents the unique features of a novel configuration of a synchronized dual-polarity time-of-flight mass spectrometer for comprehensive surface imaging. Mass spectrometry imaging of surface samples covering positive and negative ion modes is difficult due to rapid signal depletion. This limitation is overcome here by dual-polarity time-of-flight mass spectrometry (DP-TOFMS) via two separate TOF mass analyzers that are installed above a sample surface. The new instrument eliminates the polarity bias characteristic of most mass spectrometers, which is important for the analysis of samples with diverse physical and chemical properties. The experimental results show for the first time that the spatial distribution of positive and negative ions of various photolabile samples can be distinguished, including pigments and conventional matrix-assisted laser desorption/ionization samples. The different positive and negative ion distributions suggest that accurate quantitative information can only be obtained when the entire sample region is examined by DP-TOFMS, which was unfeasible in the past. Such a comprehensive diagnostic method is essential for the molecular imaging of trace compositions in delicate biological tissues, as demonstrated here with a Phyllanthus urinaria leaf that only produced ion signals in the first examination and not in the subsequent measurements.

Nanodiamond-based two-step sampling of multiply and singly phosphorylated peptides for MALDI-TOF mass spectrometry analysis.

Simultaneous detection of multiply and singly phosphorylated peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is challenging because of suppression effects during ionization. In oder to overcome this problem, this study presents a new approach to improve the detection of phosphopeptides by stepwise enrichment using polyarginine-coated (PA-coated) and titanium dioxide-coated (TiO(2)-coated) nanodiamonds for fractionation of multiply and singly phosphorylated peptides prior to on-probe MALDI MS analysis. The feasibility of this approach was demonstrated using synthetic peptides containing different numbers of phosphate groups, tryptic digests of α-casein, ß-casein, and complex protein mixtures. The high specificity of the approach is shown in its effective enrichment and fractionation of phosphopeptides from the digest of ß-casein and bovine serum albumin at a molar ratio as low as 1 : 1000, which out-performs the commercial Fe(3+)-IMAC and TiO(2) isolation kits. It offers a simple and effective alternative for the fractionation and identification of multiply and singly phosphorylated peptides by MALDI MS and allows for deduction of more information from limited starting materials.

Mapping protein cysteine sulfonic acid modifications with specific enrichment and mass spectrometry: an integrated approach to explore the cysteine oxidation.

Oxidation of thiol proteins, which results in conversion of cysteine residues to cysteine sulfenic, sulfinic or sulfonic acids, is an important posttranslational control of protein function in cells. To facilitate the analysis of this process with MALDI-MS, we have developed a method for selective enrichment and identification of peptides containing cysteine sulfonic acid (sulfopeptides) in tryptic digests of proteins based on ionic affinity capture using polyarginine-coated nanodiamonds as high-affinity probes. The method was applied to selectively concentrate sulfopeptides from either a highly dilute solution or a complex peptide mixture in which the abundance of the sulfonated analyte is as low as 0.02%. The polyarginine-coated probes exhibit a higher affinity for peptides containing multiple sulfonic acids than peptides containing single sulfonic acid. The limit of the detection is in the femtomole range, with the MALDI-TOF mass spectrometer operating in the negative ion mode. The results show that the new approach has good specificity even in the presence of phosphopeptides. An application of this method for selective enrichment and structural identification of sulfopeptides is demonstrated with the tryptic digests of performic-acid-oxidized BSA.

Hydration of alkaline earth metal dications: effects of metal ion size determined using infrared action spectroscopy.

Infrared laser action spectroscopy is used to characterize divalent Mg, Ca, and Ba ions solvated by discrete numbers of water molecules in the gas phase. The spectra of the hexahydrated ions are very similar and indicate that all six water molecules directly solvate the metal ion. The spectra of the heptahydrated ions indicate the presence of populations of structures that have a water molecule in the outer shell for all ions and an average coordination number (CN) for Ba that is higher than that for Ca or Mg. Comparisons between CN values obtained from M06 density functional and local MP2 theory indicate that the B3LYP density functional favors smaller CN values. The spectra of clusters containing at least 12 water molecules indicate that the relative abundance of single-acceptor water molecules for a given cluster size decreases with increasing metal ion size, indicating that tighter water binding to smaller metal ions disrupts the hydrogen bond network and results in fewer net hydrogen bonds. The spectra of the largest clusters (n = 32) are very similar, suggesting that intrinsic water properties are more important than ion-water interactions by that size, but subtle effects of Mg on surface water molecules are observed even for such large cluster sizes.

Selective extraction and enrichment of multiphosphorylated peptides using polyarginine-coated diamond nanoparticles.

Despite recent advances in phosphopeptide research, detection and characterization of multiply phosphorylated peptides have been a challenge. This work presents a new strategy that not only can effectively extract phosphorylated peptides from complex samples but also can selectively enrich multiphosphorylated peptides for direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Polyarginine-coated diamond nanoparticles are the solid-phase extraction supports used for this purpose. The supports show an exceptionally high affinity for multiphosphorylated peptides due to multiple arginine-phosphate interactions. The efficacy of this method was demonstrated by analyzing a small volume (50 microL) of tryptic digests of proteins such as beta-casein, alpha-casein, and nonfat milk at a concentration as low as 1 x 10 (-9) M. The concentration is markedly lower than that can be achieved by using other currently available technologies. We quantified the enhanced selectivity and detection sensitivity of the method using mixtures composed of mono- and tetraphosphorylated peptide standards. This new affinity-based protocol is expected to find useful applications in characterizing multiple phosphorylation sites on proteins of interest in complex and dilute analytes.

Facile MALDI-MS analysis of neutral glycans in NaOH-doped matrixes: microwave-assisted deglycosylation and one-step purification with diamond nanoparticles.

A streamlined protocol has been developed to accelerate, simplify, and enhance matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) of neutral underivatized glycans released from glycoproteins. It involved microwave-assisted enzymatic digestion and release of glycans, followed by rapid removal of proteins and peptides with carboxylated/oxidized diamond nanoparticles, and finally treating the analytes with NaOH before mixing them with acidic matrix (such as 2,5-dihydroxybenzoic acid) to suppress the formation of both peptide and potassiated oligosaccharide ions in MS analysis. The advantages of this protocol were demonstrated with MALDI-TOF-MS of N-linked glycans released from ovalbumin and ribonuclease B.

Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors.

Brain diseases such as autism and Alzheimer's disease (each inflicting >1% of the world population) involve a large network of genes displaying subtle changes in their expression. Abnormalities in intraneuronal transport have been linked to genetic risk factors found in patients, suggesting the relevance of measuring this key biological process. However, current techniques are not sensitive enough to detect minor abnormalities. Here we report a sensitive method to measure the changes in intraneuronal transport induced by brain-disease-related genetic risk factors using fluorescent nanodiamonds (FNDs). We show that the high brightness, photostability and absence of cytotoxicity allow FNDs to be tracked inside the branches of dissociated neurons with a spatial resolution of 12 nm and a temporal resolution of 50 ms. As proof of principle, we applied the FND tracking assay on two transgenic mouse lines that mimic the slight changes in protein concentration (∼30%) found in the brains of patients. In both cases, we show that the FND assay is sufficiently sensitive to detect these changes.

N-(1-naphthyl) ethylenediamine dinitrate: a new matrix for negative ion MALDI-TOF MS analysis of small molecules.

An organic salt, N-(1-naphthyl) ethylenediamine dinitrate (NEDN), with rationally designed properties of a strong UV absorbing chromophore, hydrogen binding and nitrate anion donors, has been employed as a matrix to analyze small molecules (m/z < 1000) such as oligosaccharides, peptides, metabolites and explosives using negative ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Compared with conventional matrixes such as α-cyano-4-hydroxycinnamic acid (CCA) and 2,5-dihydroxybenzoic acid (DHB), NEDN provides a significant improvement in detection sensitivity and yields very few matrix-associated fragment and cluster ions interfering with MS analysis. For low-molecular-weight saccharides, the lowest detection limit achieved ranges from 500 amol to 5 pmol, depending on the molecular weight and the structure of the analytes. Additionally, the mass spectra in the lower mass range (m/z < 200) consist of only nitrate and nitric acid cluster ions, making the matrix particularly useful for structural identification of oligosaccharides by post-source decay (PSD) MALDI-MS. Such a characteristic is illustrated by using maltoheptaose as a model system. This work demonstrates that NEDN is a novel negative ion-mode matrix for MALDI-MS analysis of small molecules with nitrate anion attachment.

Synchronized dual-polarity electrospray ionization mass spectrometry.

This work describes the synchronized dual-polarity (DP) electrospray ionization (ESI) method and demonstrates the first DP ESI mass spectra obtained using two mass spectrometers. Stable double Taylor cones were produced by applying two counter electric voltages with opposite polarities to one electrosprayer. The development of double Taylor cones required higher extraction voltages than conventional ESI, but DP ESI worked effectively at liquid flow rate range three times wider than conventional ESI. Using pure methanol, the emission currents of the two cones were neutralized and no current was drawn from the sprayer. Synchronized DP mass spectra were obtained using electrospray calibrants dissolved in methanol solution of low water content. For bovine insulin with conventional electrospray solution, the gas-assisted electrospray delivered satisfactory sensitivity and stability for routine mass analyses.

Binding energies of water to doubly hydrated cationized glutamine and structural analogues in the gas phase.

The modes of metal-ion and water binding in doubly hydrated complexes of lithiated and sodiated glutamine (Gln) are probed using blackbody infrared radiative dissociation experiments and density functional theory calculations. Threshold dissociation energies, E0, for loss of a water molecule from these complexes are obtained from master-equation modeling of these data. The values of E0 are 36 +/- 1 and 38 +/- 2 kJ/mol for the lithiated and sodiated glutamine complexes, respectively, and are consistent with calculated water binding energies for the nonzwitterionic form of these complexes. Calculated water binding energies for the zwitterionic forms of these complexes are significantly higher. In contrast, calculations indicate that the zwitterionic form of Gln in these complexes is more stable than the nonzwitterionic form by 8 and 15 kJ/mol when lithiated and sodiated, respectively. Doubly hydrated lithiated and sodiated complexes of asparagine methyl ester (AsnOMe), asparagine ethyl ester (AsnOEt), and glutamine methyl ester (GlnOMe) were also studied for comparison to Gln. Although these clusters lack the acidic group of Gln and therefore have different water coordination behavior, these results further support the conclusion that Gln is nonzwitterionic in these clusters. Surprisingly, the complexes containing sodium are more stable than those containing lithium, a result that is attributed to subtle differences in how these two metal ions bind to the amino acid esters in these complexes.

Structures and hydration enthalpies of cationized glutamine and structural analogues in the gas phase.

The structures of lithiated and sodiated glutamine, both with and without a water molecule, are investigated using experiment and theory. Loss of water from these complexes and from lithiated and sodiated complexes of asparagine methyl ester, asparagine ethyl ester, and glutamine methyl ester is probed with blackbody infrared radiative dissociation experiments performed over a wide temperature range. Threshold dissociation energies, E(o), for loss of a water molecule from these complexes are obtained from master equation modeling of these data. The values of E(o) are 63 +/- 1 and 53 +/- 1 kJ/mol for the lithiated and sodiated glutamine complexes, respectively. These values are similar to those for the nonzwitterionic model complexes and are in excellent agreement with calculated values. In contrast, water binding to the zwitterionic form is calculated to be significantly higher. These results indicate that glutamine in these lithiated and sodiated complexes with a water molecule are nonzwitterionic. Complexes with the asparagine side chain have slightly higher E(o) values than those with the glutamine side chain, a result consistent with more effective solvation of the metal ion due to the slightly longer side chain of glutamine. Calculations indicate that lithiated and sodiated glutamine are nonzwitterionic, with the metal ion interacting with the amine nitrogen and carbonyl oxygen from the amino acid backbone and the amide oxygen of the side chain. Addition of a water molecule does not affect the lowest-energy structure of lithiated glutamine, whereas, for sodiated glutamine, the lowest-energy zwitterionic and nonzwitterionic structures are essentially isoenergetic.

Protonated clathrate cages enclosing neutral water molecules: (H+)(H2O)21 and (H+)(H2O)28.

This paper describes a systematic study on the clathrate structure of (H+)(H2O)21 using tandem mass spectrometry, vibrational predissociation spectroscopy, Monte Carlo simulations, and density functional theory calculations. We produced (H+)(H2O)n from a continuous corona-discharged supersonic expansion and observed three anomalies simultaneously at the cluster temperature near 150 K, including (1) the peak at n=21 is more intense than its neighboring ions in the mass spectrum, (2) the size-dependent dissociation fractions show a distinct drop for the 21-mer, and (3) the infrared spectrum of (H+)(H2O)21 exhibits only a single feature at 3699 cm(-1), corresponding to the free-OH stretching of three-coordinated water molecules. Interestingly, the anomalies appear or disappear together with cluster temperature, indicating close correlation of these three observations. The observations, together with Monte Carlo simulations and density functional theory calculations, corroborate the notion for the formation of a distorted pentagonal dodecahedral (5(12)) cage with a H2O molecule in the cage and a H3O+ ion on the surface for this "magic number" water cluster ion. The dodecahedral cage melts at higher temperatures, as evidenced by the emergence of a free-OH stretching feature at 3717 cm(-1) for the two-coordinated water in (H+)(H2O)21 produced in a warmer molecular beam. Extension of this study to larger clusters strongly suggests that the experimentally observed isomer of (H+)(H2O)28 is most likely to consist of a distorted protonated pentakaidecahedral (5(12)6(3)) cage enclosing two neutral water molecules.

Vibrational predissociation spectra and hydrogen-bond topologies of H+(H2O)9-11.

Vibrational predissociation spectra of protonated water clusters H+(H2O)n, n = 9-11, are presented. Examination of the spectra in the free-OH stretching region revealed predominance of a single absorption band at approximately 3690 cm(-1) for three-coordinate H2O acting as a double-proton-acceptor/single-proton-donor in the n = 11 cluster. In contrast, the intensity of the absorption band of two-coordinate H2O acting as a single-proton-acceptor/single-proton-donor at approximately 3715 cm(-1) decreases with cluster size, and that of one-coordinate H2O acting as a single-proton-acceptor at approximately 3740 and approximately 3650 cm(-1) diminishes nearly entirely at n > 10 in the spectrum. To deduce the information about cluster temperature, we measured the spontaneous dissociation rates of the cluster ions inside an octopole ion trap and fitted the measured rate constants to an empirical Arrhenius equation. Temperatures in the range of 150 K were estimated for all H+ (H2O)9-11, suggesting that the thermal effect may populate the structures other than the true ground state. The results, combined with previously acquired spectra for H+ (H2O)5-8 (J.-C. Jiang, Y.-S. Wang, H.-C. Chang, S. H. Lin, Y. T. Lee, G. Niedner-Schatteburg and H.-C. Chang, J. Am. Chem. Soc., 2000, 122, 1398) and Monte Carlo simulations with the OSS2 model potential (L. Ojamlie, I. Shavitt and S. J. Singer, J. Chem. Phys., 1998, 109, 5547), show a systematic change in hydrogen-bond topology from tree-like, single-ring, multiple-ring to cage-like isomers (and their mixtures) as the cluster size increases from n = 5 to n = 11.