Modification of Rice Straw for Heavy Metal Ion Adsorbents by Microwave Heating

Summary: Cellulose in rice straw was chemically modified by phosphorylation using conventional heating or microwave heating. Rice straw pretreated by NaOH solution gave the highest phosphorus content when it was phosphorylated using microwave heating at 450 watt (7.07%P, ion exchange capacity 2.60 meq/g). The 3 hour-reaction in oil bath yielded the modified rice straw with lower of phosphorus content (6.32%P) and higher ion exchange capacity (2.99 meq/g) than that of microwave heating. The feasibility of the modified rice straw as cation sorbents for removing heavy metal was investigated. Cd²⁺, Cr³⁺ and Pb²⁺ were used as sorbates. In sorption test of 40 ppm with 2.0 g/L of the modified rice straw, both modified rice straws could adsorb metal ions faster than the commercial ion exchange resin (dowax) at the time less than 60 min. The modified rice straw prepared by microwave heating (A-MCW2) could remove 90% of Cd²⁺ and Cr³⁺ in 60 minutes and remove 99% of Pb²⁺ after 30 min.     

Synapsin-1 and tau reciprocal O-GlcNAcylation and phosphorylation sites in mouse brain synaptosomes

O-linked N-acetylglucosamine (O-GlcNAc) represents a key regulatory post-translational modification (PTM) that is reversible and often reciprocal with phosphorylation of serine and threonine at the same or nearby residues. Although recent technical advances in O-GlcNAc site-mapping methods combined with mass spectrometry (MS) techniques have facilitated study of the fundamental roles of O-GlcNAcylation in cellular processes, an efficient technique for examining the dynamic, reciprocal relationships between O-GlcNAcylation and phosphorylation is needed to provide greater insights into the regulatory functions of O-GlcNAcylation. Here, we describe a strategy for selectively identifying both O-GlcNAc- and phospho-modified sites. This strategy involves metal affinity separation of O-GlcNAcylated and phosphorylated peptides, β-elimination of O-GlcNAcyl or phosphoryl functional groups from the separated peptides followed by dithiothreitol (DTT) conjugation (BEMAD), affinity purification of DTT-conjugated peptides using thiol affinity chromatography, and identification of formerly O-GlcNAcylated or phosphorylated peptides by MS. The combined metal affinity separation and BEMAD approach allows selective enrichment of O-GlcNAcylated peptides over phosphorylated counterparts. Using this approach with mouse brain synaptosomes, we identified the serine residue at 605 of the synapsin-1 peptide, ⁶⁰³QASQAGPGPR⁶¹², and the serine residue at 692 of the tau peptide, ⁶⁸⁸SPVVSGDTSPR⁶⁹⁸, which were found to be potential reciprocal O-GlcNAcylation and phosphorylation sites. These results demonstrate that our strategy enables mapping of the reciprocal site occupancy of O-GlcNAcylation and phosphorylation of proteins, which permits the assessment of cross-talk between these two PTMs and their regulatory roles.     

Selective Monophosphorylation of Aliphatic Diamines

This article describes an improved method to synthesize phosphoramidic acid aminoalkyl esters from diamines by the adaptation of industrial patents. Four mono-phosphorylated products having amino sites were obtained in good a yield. Such compounds have potential coordination properties with transition metals and also potential biological activity.     

Phosphopeptide fragmentation and analysis by mass spectrometry

Reversible phosphorylation is a key event in many biological processes and is therefore a much studied phenomenon. The mass spectrometric (MS) analysis of phosphorylation is challenged by the substoichiometric levels of phosphorylation and the lability of the phosphate group in collision‐induced dissociation (CID). Here, we review the fragmentation behaviour of phosphorylated peptides in MS and discuss several MS approaches that have been developed to improve and facilitate the analysis of phosphorylated peptides. CID of phosphopeptides typically results in spectra dominated by a neutral loss of the phosphate group. Several proposed mechanisms for this neutral loss and several factors affecting the extent at which this occurs are discussed. Approaches are described to interpret such neutral loss‐dominated spectra to identify the phosphopeptide and localize the phosphorylation site. Methods using additional activation, such as MS³ and multistage activation (MSA), have been designed to generate more sequence‐informative fragments from the ion produced by the neutral loss. The characteristics and benefits of these methods are reviewed together with approaches using phosphopeptide derivatization or specific MS scan modes. Additionally, electron‐driven dissociation methods by electron capture dissociation (ECD) or electron transfer dissociation (ETD) and their application in phosphopeptide analysis are evaluated. Finally, these techniques are put into perspective for their use in large‐scale phosphoproteomics studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Recent developments of nanoparticle-based enrichment methods for mass spectrometric analysis in proteomics

In proteome research, rapid and effective separation strategies are essential for successful protein identification due to the broad dynamic range of proteins in biological samples. Some important proteins are often expressed in ultra low abundance, thus making the pre-concentration procedure before mass spectrometric analysis prerequisite. The main purpose of enrichment is to isolate target molecules from complex mixtures to reduce sample complexity and facilitate the subsequent analyzing steps. The introd...     

Synthesis of caged myo-inositol 1,3,4,5-tetrakisphosphate

The total synthesis of an enantiomerically pure Ins(1,3,4,5)P₄ derivative equipped with a photosensitive nitroveratryl group at the 3-O-phosphate is reported.     

Quantitative monitoring of His and Asp phosphorylation in a bacterial signaling system by using Phos‐tag Magenta/Cyan fluorescent dyes

In the bacterial signaling mechanisms known as two‐component systems (TCSs), signals are generally conveyed by means of a His–Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation status. Here, we show that the Phos‐tag dye technology is suitable for the fluorescent detection of His‐ and Asp‐phosphorylated proteins separated by SDS‐PAGE. The dynamics of the His–Asp phosphorelay of recombinant EnvZ‐OmpR, a TCS derived from Escherichia coli, were examined by SDS‐PAGE followed by simple rapid staining with Phos‐tag Magenta fluorescent dye. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of EnvZ and OmpR in the presence of adenosine triphosphate (ATP) or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from EnvZ to OmpR, which occurs within 1 min in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos‐tag Cyan gel staining. We believe that the Phos‐tag dye technology provides a simple and convenient fluorometric approach for screening of HK inhibitors that have potential as new antimicrobial agents.     

Continuous‐Flow Reactor‐Based Enzymatic Synthesis of Phosphorylated Compounds on a Large Scale

Acid phosphatase, an enzyme that is able to catalyze the transfer of a phosphate group from cheap pyrophosphate to alcoholic substrates, was covalently immobilized on polymethacrylate beads with an epoxy linker (Immobeads‐150 or Sepabeads EC‐EP). After immobilization 70 % of the activity was retained and the immobilized enzyme was stable for many months. With the immobilized enzyme we were able to produce and prepare D ‐glucose‐6‐phosphate, N‐acetyl‐D ‐glucosamine‐6‐phosphate, allyl phosphate, dihydroxyacetone phosphate, glycerol‐1‐phosphate, and inosine‐5′‐monophosphate from the corresponding primary alcohol on gram scale using either a fed‐batch reactor or a continuous‐flow packed‐bed reactor.     

Nanoelectrospray-based detection and sequencing of substoichiometric amounts of phosphopeptides in complex mixtures

A novel immobilized metal affinity chromatography (IMAC)-based protocol was successfully used for sample preparation prior to nanoelectrospray-based sequencing of phosphopeptides. In a first step, phosphorylated peptides are detected in an unseparated peptide mixture using precursor ion scanning in the negative ion mode on a triple-quadrupole mass spectrometer. Then the phosphorylated peptides are enriched by passing the sample over an IMAC column and sequenced in the positive ion mode. The IMAC-based enrichment allows the sequencing of phosphorylated peptides even if other, much more abundant, peptides are present at the same m/z value in the original mixture. Using this two-step approach, we are able to combine the simplicity, sensitivity and specificity of precursor ion scanning-based detection of phosphopeptides using the nanoelectrospray ion source with the ability to sequence phosphorylated peptides even if they are present in substoichiometric amounts.