Bacillus licheniformis peptidoglycan characterization by CZE–MS: Assessment with the benchmark RP‐HPLC‐MS method

Peptidoglycan or murein is an essential polymer found in bacterial cell wall. It is a dynamic structure that is continuously remodeled or modified during bacterial cell growth or in presence of cell wall stresses. These modifications are still poorly understood mainly due to the peptidoglycan, which is rather non‐soluble, and the difficulties to separate the hydrophilic glycopeptides (muropeptides) by reversed phase liquid chromatography, generated by the enzymatic digestion using mutanolysin, an N‐acetyl‐muramidase, cleaving the β_1→4 bound between N‐acetylglucosamine and N‐acetylmuramic acid. Here, we report the use of CZE–MS for an easy and fast screening of muropeptides generated by the action of muramidase on the Bacillus licheniformis cell wall. Electron transfer and CID–MS were also used to unambiguously identify and localize the presence or the absence of amidation and acetylation moieties on muropeptide variants. The reference method to analyse muropeptides by reversed phase chromatography was also tested and the advantages and disadvantages of both methods were evaluated.     

A Generalization Of Reifenberg’s Theorem In {\mathbb{R}}^3

In 1960 Reifenberg proved the topological disc property. He showed that a subset of which is well approximated by m-dimensional affine spaces at each point and at each (small) scale is locally a bi-H?lder image of the unit ball in . In this paper we prove that a subset of which is well approximated in the Hausdorff distance sense by one of the three standard area-minimizing cones at each point and at each (small) scale is locally a bi-H?lder deformation of a minimal cone. We also prove an analogous result for more general cones in . Received: July 2006, Revised: August 2007, Accepted: January 2008     

G-quadruplex DNA assemblies: loop length, cation identity, and multimer formation

G-rich DNA sequences are able to fold into structures called G-quadruplexes. To obtain general trends in the influence of loop length on the structure and stability of G-quadruplex structures, we studied oligodeoxynucleotides with random bases in the loops. Sequences studied are dGGGW(i)GGGW(j)GGGW(k)GGG, with W = thymine or adenine with equal probability, and i, j, and k comprised between 1 and 4. All were studied by circular dichroism, native gel electrophoresis, UV-monitored thermal denaturation, and electrospray mass spectrometry, in the presence of 150 mM potassium, sodium, or ammonium cations. Parallel conformations are favored by sequences with short loops, but we also found that sequences with short loops form very stable multimeric quadruplexes, even at low strand concentration. Mass spectrometry reveals the formation of dimers and trimers. When the loop length increases, preferred quadruplex conformations tend to be more intramolecular and antiparallel. The nature of the cation also has an influence on the adopted structures, with K(+) inducing more parallel multimers than NH4(+) and Na(+). Structural possibilities are discussed for the new quadruplex higher-order assemblies.     

Stabilization and structure of telomeric and c-myc region intramolecular G-quadruplexes: the role of central cations and small planar ligands

A promising approach for anticancer strategies is the stabilization of telomeric DNA into a G-quadruplex structure. To explore the intrinsic stabilization of folded G-quadruplexes, we combined electrospray ionization mass spectrometry, ion mobility spectrometry, and molecular modeling studies to study different DNA sequences known to form quadruplexes. Two telomeric DNA sequences of different lengths and two DNA sequences derived from the NHE III1 region of the c-myc oncogene (Pu22 and Pu27) were studied. NH4+ and the ligands PIPER, TMPyP4, and the three quinacridines MMQ1, MMQ3, and BOQ1 were complexed with the DNA sequences to determine their effect on the stability of the G-quadruplexes. Our results demonstrate that G-quadruplex intramolecular folds are stabilized by NH4+ cations and the ligands listed. Furthermore, the ligands can be classified according to their ability to stabilize the quadruplexes and end stacking is shown to be the dominant mode for ligand attachment. In all cases our solvent-free experimental observations and theoretical modeling reveal structures that are highly relevant to the solution-phase structures.     

Mass spectrometric characterization of 3'-imino[60]fulleryl-3'-deoxythymidine by collision-induced dissociation

The primary structure of 3'-imino[60]fulleryl-3'-deoxythymidine ions is studied using mass spectrometry both in the positive and negative modes. Interaction between the subunits is discussed using collision-induced dissociation (CID) spectra. Collisional activation with argon of the sodiated cations leads to the cleavage of the glycosidic bond and the transfer of a radical hydrogen from the deoxyribose to the thymine. The sodiated thymine is the only fragment observed for low collision energies in the positive mode. In the negative mode, two different ionization mechanisms take place, reduction and deprotonation in the presence of triethylamine. The 2.7 eV electron affinity of C60 and its huge cross section compared to the small cross section and predicted 0.44 eV electron affinity of the thymidine subunit most likely localize the radical electron on the fullerene. On the other hand, deprotonation of the 3'-azido-3'-deoxythymidine (AZT) is known to occur in N-3, the most acidic site of the nucleobase. Consequently, deprotonation causes the negative charge to be initially localized on the thymine. Both types of parent anions give the radical anion C60*- as fragment. The other fragments detected are the dehydrogenated 3'-imino[60]fulleryl-3'-deoxyribose anion, C60NH2-, C60N- and C60H-. Since in negative ion mass spectrometry all fragments include the [60]fullerene unit, this suggests that the fragmentation is driven by the electron affinity of the [60]fullerene, likely responsible for a charge transfer between the deprotonated thymine and the C60.     

Development of an absolute quantification method targeting growth hormone biomarkers using liquid chromatography coupled to isotope dilution mass spectrometry

A method to perform absolute quantification of two biomarkers (IGF-1 and IGFBP-3) of growth hormone abuse has been developed. Isotope dilution is used with synthetically labelled peptides as internal standards. Peptide selection and multiple reaction monitoring design are discussed. A simple sample preparation based on the reduction and alkylation of cysteine residues followed by tryptic digestion provides a sufficient digestion of proteins. Serum samples fortified with increasing amounts of target proteins are analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a triple quadrupole mass spectrometer. Specificity is ensured by the selection of sequences with no homology in BLAST, as well as retention time deviation check, and ion ratio monitoring. Linearity is studied in terms of calibration curves. These curves for IGFBP-3 and IGF-1 are generated with mean slopes of 0.055 and 0.065, intercepts of 0.107 and -0.011, and with coefficients of correlation of 0.95 and 0.98, respectively. These curves result from the addition of proteins to the serum. Risks of variations related to potential matrix effects are therefore reduced, as well as probable variations related to the digestion steps. The working concentration ranges are 4-10 ng/microl for IGFBP-3 and 2-8 ng/microl for IGF-1. Preliminary data regarding repeatability show that relative standard deviations (RSDs) range between 13 and 32% for IGFBP-3 and between 7 and 29% for IGF-1.     

Development and validation of a multi-residue method for pesticide determination in honey using on-column liquid-liquid extraction and liquid chromatography-tandem mass spectrometry

We report on the development and validation under ISO 17025 criteria of a multi-residue confirmatory method to identify and quantify 17 widely chemically different pesticides (insecticides: Carbofuran, Methiocarb, Pirimicarb, Dimethoate, Fipronil, Imidacloprid; herbicides: Amidosulfuron, Rimsulfuron, Atrazine, Simazine, Chloroturon, Linuron, Isoxaflutole, Metosulam; fungicides: Diethofencarb) and 2 metabolites (Methiocarb sulfoxide and 2-Hydroxytertbutylazine) in honey. This method is based on an on-column liquid-liquid extraction (OCLLE) using diatomaceous earth as inert solid support and liquid chromatography (LC) coupled to mass spectrometry (MS) operating in tandem mode (MS/MS). Method specificity is ensured by checking retention time and theoretical ratio between two transitions from a single precursor ion. Linearity is demonstrated all along the range of concentration that was investigated, from 0.1 to 20 ng g(-1) raw honey, with correlation coefficients ranging from 0.921 to 0.999, depending on chemicals. Recovery rates obtained on home-made quality control samples are between 71 and 90%, well above the range defined by the EC/657/2002 document, but in the range we had fixed to ensure proper quantification, as levels found in real samples could not be corrected for recovery rates. Reproducibility is found to be between 8 and 27%. Calculated CCalpha and CCbeta (0.0002-0.943 ng g(-1) for CCalpha, and 0.0002-1.232 ng g(-1) for CCbeta) show the good sensitivity attained by this multi-residue analytical method. The robustness of the method has been tested in analyzing more than 100 raw honey samples collected from different areas in Belgium, as well as some wax and bee samples, with a slightly adapted procedure.     

High-throughput biomonitoring of dioxins and polychlorinated biphenyls at the sub-picogram level in human serum

We report on the use of a state-of-the-art method for the measurement of selected polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and polychlorinated biphenyls in human serum specimens. The sample preparation procedure is based on manual small size solid-phase extraction (SPE) followed by automated clean-up and fractionation using multi-sorbent liquid chromatography columns. SPE cartridges and all clean-up columns are disposable. Samples are processed in batches of 20 units, including one blank control (BC) sample and one quality control (QC) sample. The analytical measurement is performed using gas chromatography coupled to isotope dilution high-resolution mass spectrometry. The sample throughput corresponds to one series of 20 samples per day, from sample reception to data quality cross-check and reporting, once the procedure has been started and series of samples keep being produced. Four analysts are required to ensure proper performances of the procedure. The entire procedure has been validated under International Organization for Standardization (ISO) 17025 criteria and further tested over more than 1500 unknown samples during various epidemiological studies. The method is further discussed in terms of reproducibility, efficiency and long-term stability regarding the 35 target analytes. Data related to quality control and limit of quantification (LOQ) calculations are also presented and discussed.     

Difference of Electron Capture and Transfer Dissociation Mass Spectrometry on Ni<Superscript>2+</Superscript>-, Cu<Superscript>2+</Superscript>-, and Zn<Superscript>2+</Superscript>-Polyhistidine Complexes in the Absence of Remote Protons

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) in metal-peptide complexes are dependent on the metal cation in the complex. The divalent transition metals Ni²⁺, Cu²⁺, and Zn²⁺ were used as charge carriers to produce metal-polyhistidine complexes in the absence of remote protons, since these metal cations strongly bind to neutral histidine residues in peptides. In the case of the ECD and ETD of Cu²⁺-polyhistidine complexes, the metal cation in the complex was reduced and the recombination energy was redistributed throughout the peptide to lead a zwitterionic peptide form having a protonated histidine residue and a deprotonated amide nitrogen. The zwitterion then underwent peptide bond cleavage, producing a and b fragment ions. In contrast, ECD and ETD induced different fragmentation processes in Zn²⁺-polyhistidine complexes. Although the N–C_α bond in the Zn²⁺-polyhistidine complex was cleaved by ETD, ECD of Zn²⁺-polyhistidine induced peptide bond cleavage accompanied with hydrogen atom release. The different fragmentation modes by ECD and ETD originated from the different electronic states of the charge-reduced complexes resulting from these processes. The details of the fragmentation processes were investigated by density functional theory.

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