Yi-Sheng Wang

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.

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 β‐casein, and complex β‐casein/BSA mixture. The TiO2‐coated Fe@CNPs out‐performed the commercial TiO2‐coated magnetic beads for detection of phosphopeptides from tryptic digests of β‐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.

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.

Initial Ionization Reaction in Matrix-Assisted Laser Desorption/Ionization

The initial ionization reaction in matrix-assisted laser desorption/ionization (MALDI) was examined on the basis of the appearance of photoelectrons. The threshold laser fluence for the ejection of photoelectrons from 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), and trihydroxyacetopheone (THAP) on stainless steel (SS) substrates was 0.05, 0.41, and 8.39 mJ/cm(2), respectively. These values are considerably lower than those for MALDI ions, indicating that the electron detachment likely precedes other ionization reactions. The SS substrate played an insignificant role in the production of photoelectrons because suspended DHB produced a photoelectron signal similar to DHB on the SS surface, and decreasing the DHB thickness on the SS reduced the photoelectron intensity. For crystalline DHB and SA, the photoelectron intensity increased with the laser (337 nm) fluence in a relationship of less than second order, suggesting considerable reductions of ionization potentials in comparison with free molecules. According to ab initio calculations, the ionization potential of DHB clusters reduces as the cluster size increases from monomer to octamer. The impact of these abundant electrons on the ion production in MALDI is discussed.

Solid-Phase Thermodynamic Interpretation of Ion Desorption in Matrix-Assisted Laser Desorption/Ionization

This work demonstrates a quantitative interpretation of ion desorption in matrix-assisted laser desorption/ionization (MALDI). The theoretical modeling incorporates transition state theory for the desorption of surface ions, assuming chemical and thermal equilibrium in the solid state prior to desorption. It is distinct from conventional models that assume chemical equilibrium in the gas phase. This solid-state thermodynamic interpretation was used to examine the desorption of pure 2,4,6-trihydroxyacetophenone (THAP) and of angiotensin I mixed with THAP. It successfully described the changes in ion yield with the effective temperature under various laser fluence and initial temperature conditions. The analysis also revealed the key role played by ion concentration in the modeling to provide the best fit of the model to observations. On the other hand, divergence of the ion beam with laser fluence was examined using an imaging detection method, and the signal saturation normally seen at high fluence was appropriately reduced by ion focusing. Simplified but deceptive theoretical interpretations were obtained when the analysis was conducted without adequate calibration of the instrument bias.

Hydrogel Micropatches for Sampling and Profiling Skin Metabolites

Metabolites excreted by skin have a huge potential as disease biomarkers. However, due to the shortage of convenient sampling/analysis methods, the analysis of sweat has not become very popular in the clinical setting (pilocarpine iontophoresis being a prominent exception). In this report, a facile method for sampling and rapid chemical profiling of skin metabolites excreted with sweat is proposed. Metabolites released by skin (primarily the constituents of sweat) are collected into hydrogel (agarose) micropatches. Subsequently, they are extracted in an online analytical setup incorporating nanospray desorption electrospray ionization and an ion trap mass spectrometer. In a series of reference measurements, using bulk sampling and electrospray ionization mass spectrometry, various low-molecular-weight metabolites are detected in the micropatches exposed to skin. The sampling time is as short as 10 min, while the desorption time is 2 min. Technical precision of micropatch analysis varies within the range of 3-42%, depending on the sample and the method of data treatment; the best technical precision (≤10%) has been achieved while using an isotopically labeled internal standard. The limits of detection range from 7 to 278 pmol. Differences in the quantities of extracted metabolites are observed for the samples obtained from healthy individuals (intersubject variabilities: 30-89%; n = 9), which suggests that this method may have the potential to become a semiquantitative assay in clinical analysis and forensics.

Selective enhancement of carbohydrate ion abundances by diamond nanoparticles for mass spectrometric analysis.

Diamond nanoparticles (DNPs) were incorporated into matrix-assisted laser desorption/ionization (MALDI) samples to enhance the sensitivity of the mass spectrometer to carbohydrates. The DNPs optimize the MALDI sample morphology and thermalize the samples for thermally labile compounds because they have a high thermal conductivity, a low extinction coefficient in UV-vis spectral range, and stable chemical properties. The best enhancement effect was achieved when matrix, DNP, and carbohydrate solutions were deposited and vacuum-dried consecutively to form a trilayer sample morphology. It allows the direct identification of underivatized carbohydrates mixed with equal amount of proteins because no increase in the ion abundance of proteins was achieved. For dextran with an average molecular weight of 1500, the trilayer method typically improves the sensitivity by 79- and 7-fold in comparison to the conventional dried-droplet and thin-layer methods, respectively.

Analysis of initial reactions of MALDI based on chemical properties of matrixes and excitation condition.

This investigation concerns the initial chemical reactions that affect the ionization of matrixes in matrix-assisted laser desorption/ionization (MALDI). The study focuses on the relaxations of photon energy that occur on a comparable time scale to that of ionization, in which the available laser energy is shared and the ionization condition is changed. The relaxations include fluorescence, fragmentation, and nonradiative relaxation from the excited state to the ground state. With high absorption cross section and long excited-state lifetime, photoionization of matrix plays an important role if sufficient laser energy is used. Under other conditions, thermal ionization of the molecule in the ground state is predicted to be one of the important reactions. Evidence of change in the branching ratio of initial reactions with the matrix and the excitation wavelength was obtained with α-cyano-4-hydroxycinnamic acid, sinapinic acid, 2,5-dihydroxybenzoic acid, and 2,4,6-trihydroxyacetophenone. These matrixes are studied by obtaining their mixed crystal absorption spectra, fluorescence properties, laser-induced infrared emission, and product ions. The exact ionization pathway depends on the chemical properties of matrixes and the excitation conditions. This concept may explain the diversity of experimental results observed in MALDI experiments, which provides an insight into the ensemble of chemical reactions that govern the generation of ions.

Ultrasound ionization of biomolecules

To date, mass spectrometric analysis of biomolecules has been primarily performed with either matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI). In this work, ultrasound produced by a simple piezoelectric device is shown as an alternative method for soft ionization of biomolecules. Precursor ions of proteins, saccharides and fatty acids showed little fragmentation. Cavitation is considered as a primary mechanism for the ionization of biomolecules.

Incoherent production reactions of positive and negative ions in matrix-assisted laser desorption/ionization

Utilizing synchronized dual-polarity matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, we found good evidence of the incoherent production of positive and negative matrix ions. Using thin, homogeneous 2,5-dehydroxybenzoic acid (DHB) matrix films, positive and negative matrix ions were found to appear at different threshold laser fluences. The presence of molecular matrix ions of single charge polarity suggests that the existence of DHB ion-pairs may not be a prerequisite in MALDI. Photoelectrons induced by the laser excitation may assist the production of negative DHB ions, as shown in experiments conducted with stainless steel and glass substrates. At high laser fluences, the relative yield of positive and negative matrix ions remained constant when homogeneous matrix films were used, but it fluctuated significantly with inhomogeneous crystal morphology. This result is also inconsistent with the hypothesis that matrix ion-pairs are essential primary ions. Evidence from both low and high laser fluences suggests that the productions of positive and negative matrix ions in MALDI may occur via independent pathways.