Mass spectrometry characterization of 3‐OH butyrated β‐cyclodextrin

Oligo(3‐OH butyrate)‐β‐cyclodextrin esters (PHB‐CD) were obtained through ring opening of β‐butyrolactone (β‐BL) in the presence of β‐cyclodextrin (CD) and (‐)‐sparteine (SP) as nucleophilic activator. The resulted reaction mixture was first separated in two fractions and then investigated through a deep mass spectrometry (MS) study performed on a liquid chromatography‐electrospray ionization‐quadrupole time of flight (LC‐ESI‐QTOF) instrument. LC MS and tandem MS structural assignment of the reaction products was completed by NMR. The performed analysis revealed that poly(3‐OH butyrate) homopolymers (PHB) are formed together with the PHB‐CD products. Secondary reactions resulting in the formation of crotonates were also proved to occur. A comparison between MS and NMR results demonstrated that more than one PHB oligomer is attached to the CD in the PHB‐CD product. The tandem MS fragmentation studies validated the proposed structure of CD derivatives. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Low‐temperature controlled free‐radical polymerization of vinyl monomers in the presence of a novel cyclic dixanthate under γ‐ray irradiation

The free‐radical polymerization of methyl acrylate (MA) has been studied in the presence of a novel cyclic dixanthate under γ‐ray irradiation (80 Gy min^?1) at room temperature (～28 °C), ?30 °C, and ?76 °C respectively. The resultant polymers have controlled molecular weights and relatively narrow molecular weight distributions, especially at low temperatures (i.e., ?30 and ?76 °C). The polymerization control may be associated with the temperature: the lower the temperature is, the more control there is. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry analysis of poly(methyl acrylate) (PMA) samples shows that there are at least three distributions: [3‐(MA)_n‐H]⁺ cyclic polymers, [3‐(MA)_n‐THF‐H]⁺, and [3‐(MA)_n‐(THF)₂‐H]⁺ linear PMAs. The relative content of the cyclic polymers markedly increases at a lower temperature, and this may be related to the reduced diffusion rate and the suppressed chain‐transfer reaction at the low temperature. It is evidenced that the good control of the polymerization at the low temperature may be associated with the suppressed chain‐transfer reaction, unlike reversible addition–fragmentation chain transfer polymerization. In addition, styrene bulk polymerizations have been performed, and gel permeation chromatography traces show that there is only one cyclic dixanthate moiety in the polymer chain. This article is the first to report the influence of a low temperature on controlled free‐radical polymerizations. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 2847–2854, 2007     

[N⋅⋅⋅I+⋅⋅⋅N] Halogen‐Bonded Dimeric Capsules from Tetrakis(3‐pyridyl)ethylene Cavitands

Two [N???I⁺???N] halogen‐bonded dimeric capsules using tetrakis(3‐pyridyl)ethylene cavitands with different lower rim alkyl chains are synthesized and analyzed in solution and the gas phase. These first examples of symmetrical dimeric capsules making use of the iodonium ion (I⁺) as the main connecting module are characterized by ¹H NMR spectroscopy, diffusion ordered NMR spectroscopy (DOSY), electrospray ionization mass spectrometry (ESI‐MS), and ion mobility‐mass spectrometry (TW‐IMS) experiments. The synthesis and effective halogen‐bonded dimerization proceeds through analogous dimeric capsules with [N???Ag⁺???N] binding motifs as the intermediates as evidenced by the X‐ray structures of (CH₂Cl₂)₂@[ 3 a ₂?Ag₄?(H₂O)₂?OTs₄] and (CH₂Cl₂)₂@[ 3 a ₂?Ag₄?(H₂O)₄?OTs₄], two structurally different capsules.     

Effect of matrices and additives on phosphorylated and ketodeoxyoctonic acid lipids A analysis by matrix‐assisted laser desorption ionization‐mass spectrometry

Lipid A is a major compound of the outer membrane of gram‐negative bacteria and is a key factor of bacterial virulence. As lipid A's structure differs among bacterial species and varies between strains of the same species, knowing its modifications is essential to understand its implications in the infectious process. To analyze these lipids, matrix‐assisted laser desorption ionization‐mass spectrometry (MALDI‐MS) is a well‐suited method that is fast and efficient. However, there are limitations with the matrix and additives used, such as the suppression of signal or prompt fragmentations that could give a false overview of lipid A composition in biological samples. For a comprehensive analysis of the entire lipid A species present in a sample, we tested 16 matrices and 11 additives on two commercial lipids A. The first commercial one contains single phosphorylation group, and the second contains two phosphorylation and two ketodeoxyoctonic acid (KDO) groups. The lipid A containing KDO groups was essentially detected by the 3‐hydroxypicolinic acid (3‐HPA) matrix, whereas the monophosphorylated lipid A could be detected by 13 matrices out of the 16. We also demonstrated that the signal of diphosphorylated lipid A can be enhanced with the use of additives in the matrix. Our study indicated that the best conditions to obtain a clear signal of both lipids A without prompt fragmentation was the use of 3‐HPA with 10mM trifluoroacetic acid (TFA).     

Determination of block size in poly(ethylene oxide)‐b‐polystyrene block copolymers by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Characterization of block size in poly(ethylene oxide)‐b‐poly(styrene) (PEO‐b‐PS) block copolymers could be achieved by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) after scission of the macromolecules into their constituent blocks. The performed hydrolytic cleavage was demonstrated to specifically occur on the targeted ester function in the junction group, yielding two homopolymers consisting of the constitutive initial blocks. This approach allows the use of well‐established MALDI protocols for a complete copolymer characterization while circumventing difficulties inherent to amphiphilic macromolecule ionization. Although the labile end‐group in PS homopolymer was modified by the MALDI process, PS block size could be determined from MS data since polymer chains were shown to remain intact during ionization. This methodology has been validated for a PEO‐b‐PS sample series, with two PEO of number average molecular weight (M_n) of 2000 and 5000 g mol^?1 and M_n(PS) ranging from 4000 to 21,000 g mol^?1. Weight average molecular weight (M_w), and thus polydispersity index, could also be reached for each segment and were consistent with values obtained by size exclusion chromatography. This approach is particularly valuable in the case of amphiphilic copolymers for which M_n values as determined by liquid state nuclear magnetic resonance might be affected by micelle formation. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 3380–3390, 2009     

Characterization of different poly(2‐oxazoline) block copolymers by MALDI‐TOF MS/MS and ESI‐Q‐TOF MS/MS

A complete library of poly(2‐oxazoline) block copolymers was synthesized via cationic ring opening polymerization for the characterization by two different soft ionization techniques, namely matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF MS). In addition, a detailed characterization was performed by tandem MS to gain more structural information about the block copolymer composition and its fragmentation behavior. The fragmentation of the poly(2‐oxazoline) block copolymers revealed the desired polymer structure and possible side reactions, which could be explained by different mechanisms, like 1,4‐ethylene or hydrogen elimination and the McLafferty +1 rearrangement. Polymers with aryl side groups showed less fragmentation due to their higher stability compared to polymers with alkyl side groups. These insights represent a further step toward the construction of a library with fragments and their fragmentation pathways for synthetic polymers, following the successful examples in proteomics. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

MALDI‐MS analysis and imaging of small molecule metabolites with 1,5‐diaminonaphthalene (DAN)

1,5‐diaminonaphthalene (DAN) has previously been reported as an effective matrix for matrix‐assisted laser desorption ionization‐mass spectrometry of phospholipids. In the current work, we investigate the use of DAN as a matrix for small metabolite analysis in negative ion mode. DAN was found to provide superior ionization to the compared matrices for MW < ~400 Da; however, 9‐aminoacridine (9‐AA) was found to be superior for a uridine diphosphate standard (MW 566 Da). DAN was also found to provide a more representative profile of a natural phospholipid mixture than 9‐AA. Finally, DAN and 9‐AA were applied for imaging of metabolites directly from corn leaf sections. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

Functionalization of polymeric organolithium compounds with 3,4‐epoxy‐1‐butene: Precursors for diene‐functionalized macromonomers

The functionalization of polymeric organolithiums (PLi) with 3,4‐epoxy‐1‐butene (EPB) in a hydrocarbon solution yielded the corresponding hydroxybutene‐functionalized polymers in high yields (>95%). Three modes of addition of PLi to EPB were observed (1,4, 3,4, and 4,3). The products and chain‐end structures were characterized by ¹H NMR, ¹³C NMR, attached‐proton‐test ¹³C NMR, calculated ¹³C NMR chemical shifts, and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). The regioselectivity of the addition depended on the PLi chain‐end structure, the reaction conditions, and the addition of lithium salts or Lewis bases. In the absence of additives, the functionalization of poly(styryl)lithium (PSli) produced equal amounts of 1,4‐, 3,4‐, and 4,3‐addition, as determined by quantitative ¹³C NMR analysis. The use of a low temperature (6 °C), inverse addition, the addition of triethylamine (TEA; [TEA]/[PSLi] = 20) as a Lewis base, or dienyllithium chain ends produced polymers with only the 1,4‐addition product. Mild dehydration of the hydroxybutene‐functionalized polymer with p‐toluenesulfonic acid produced the corresponding diene‐functionalized macromonomer, as shown by MALDI‐TOF MS. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 947–957, 2003     

The reactivity of human serum albumin toward trans‐4‐hydroxy‐2‐nonenal

Mass spectrometry was used to probe the preferred locations of trans‐4‐hydroxy‐2‐nonenal (HNE) addition to the cysteine, histidine, and lysine residues of human serum albumin (HSA). Considering only those modified peptides supported by high mass accuracy Orbitrap precursor ion measurements (high confidence hits), with HNE:HSA ratios of 1:1 and 10:1, 3 and 15 addition sites, respectively, were identified. Using less stringent criteria, a total of 34 modifications were identified at the higher concentration. To gain quantitative data, iTRAQ labeling studies were completed. Previous work had identified Cys³⁴, the only free cysteine, as the most reactive residue in HSA, and we have found that Lys¹⁹⁹, His^242/7, and His²⁸⁸ are the next most reactive residues. Although the kinetic data indicate that the lysines and histidines can react at relatively similar rates, the results show that lysine addition is much less favorable thermodynamically; under our reaction conditions, lysine addition generally does not go to completion. This suggests that under physiological conditions, HNE addition to lysine is only relevant in situations where unusually high HNE concentrations or access to irreversible secondary reactions are found. Copyright © 2012 John Wiley & Sons, Ltd.     

5‐Methoxytryptophan‐dependent inhibition of oral squamous cell carcinoma metastasis

The metastatic status of oral cancer is highly associated with the overall survival rate of patients. Previous studies have revealed that the endogenous tryptophan metabolite 5‐methoxytryptophan (5‐MTP) can downregulate cyclooxygenase‐2 expression; suppress tumor proliferation, migration, and invasion; and reduce the tumor size. To improve the understanding of the molecular mechanisms involved in the regulation of 5‐MTP in the tumorigenesis of oral cancer, we conducted a comparative wound healing and transwell invasion assays. Our results revealed that 5‐MTP reduce oral cancer cell migration and invasion ability. In addition, the results of an in vivo assay demonstrated that the growth of primary tumors was significantly inhibited by 5‐MTP in OC3 oral cancer cells and in invasive OC3‐I5 oral cancer cells. Moreover, enlarged spleens were observed in OC3‐I5‐implanted severe combined immunodeficiency mice although 5‐MTP can inhibit spleen enlargement. Through comparative proteomics, we identified 32 differentially regulated protein spots by using 2D‐DIGE/MALDI‐TOF MS analyses. Some of the differentially regulated proteins such as amadillo‐repeat‐containing X‐linked protein 1, phosphoglycerate kinase 1, tropomyosin alpha‐1, and tropomyosin alpha‐4 may be associated with the 5‐MTP‐dependent inhibition of oral cancer growth and metastasis. We conclude that 5‐MTP plays a crucial role in inhibiting in vitro and in vivo cancer invasion and metastasis.     

Screening disrupted molecular functions and pathways associated with clear cell renal cell carcinoma using Gibbs sampling

ObjectiveTo explore the disturbed molecular functions and pathways in clear cell renal cell carcinoma (ccRCC) using Gibbs sampling.MethodsGene expression data of ccRCC samples and adjacent non-tumor renal tissues were recruited from public available database. Then, molecular functions of expression changed genes in ccRCC were classed to Gene Ontology (GO) project, and these molecular functions were converted into Markov chains. Markov chain Monte Carlo (MCMC) algorithm was implemented to perform posterior inference and identify probability distributions of molecular functions in Gibbs sampling. Differentially expressed molecular functions were selected under posterior value more than 0.95, and genes with the appeared times in differentially expressed molecular functions ≥5 were defined as pivotal genes. Functional analysis was employed to explore the pathways of pivotal genes and their strongly co-regulated genes.ResultsIn this work, we obtained 396 molecular functions, and 13 of them were differentially expressed. Oxidoreductase activity showed the highest posterior value. Gene composition analysis identified 79 pivotal genes, and survival analysis indicated that these pivotal genes could be used as a strong independent predictor of poor prognosis in patients with ccRCC. Pathway analysis identified one pivotal pathway − oxidative phosphorylation.ConclusionsWe identified the differentially expressed molecular functions and pivotal pathway in ccRCC using Gibbs sampling. The results could be considered as potential signatures for early detection and therapy of ccRCC.     

Fragmentation features of intermolecular cross‐linked peptides using N‐hydroxy‐ succinimide esters by MALDI‐ and ESI‐MS/MS for use in structural proteomics

The use of mass spectrometry coupled with chemical cross‐linking of proteins has become one of the most useful tools for proteins structure and interactions studies. One of the challenges in these studies is the identification of the cross‐linked peptides. The interpretation of the MS/MS data generated in cross‐linking experiments using N‐hydroxy succinimide esters is not trivial once a new amide bond is formed allowing new fragmentation pathways, unlike linear peptides. Intermolecular cross‐linked peptides occur when two different peptides are connected by the cross‐linker and they yield information on the spatial proximity of different domains (within a protein) or proteins (within a complex). In this article, we report a detailed fragmentation study of intermolecular cross‐linked peptides, generated from a set of synthetic peptides, using both ESI and MALDI to generate the precursor ions. The fragmentation features observed here can be helpful in the interpretation and identification of cross‐linked peptides present in cross‐linking experiments and be further implemented in search engine's algorithms. Copyright © 2011 John Wiley & Sons, Ltd.     

A new,validated HPLC‐MS/MS method for the simultaneous determination of the anti‐cancer agent capecitabine and its metabolites: 5′‐deoxy‐5‐fluorocytidine, 5′‐deoxy‐5‐fluorouridine, 5‐fluorouracil and 5‐fluorodihydrouracil,in human plasma

A rapid and selective liquid chromatography/tandem mass spectrometric method was developed for the simultaneous determination of capecitabine and its metabolites 5′‐deoxy‐5‐fluorocytidine (5′‐DFCR), 5′‐deoxy‐5‐fluorouracil (5′‐DFUR), 5‐fluorouracil (5‐FU) and dihydro‐5‐fluorouracil (FUH₂) in human plasma. A 200 μL human plasma aliquot was spiked with a mixture of internal standards fludarabine and 5‐chlorouracil. A single‐step protein precipitation method was employed using 10% (v/v) trichloroacetic acid in water to separate analytes from bio‐matrices. Volumes of 20 μL of the supernatant were directly injected onto the HPLC system. Separation was achieved on a 30 × 2.1 mm Hypercarb (porous graphitic carbon) column using a gradient by mixing 10 mm ammonium acetate and acetonitrile–2‐propanol–tetrahydrofuran (1 : 3 : 2.25, v/v/v). The detection was performed using a Finnigan TSQ Quantum Ultra equipped with the electrospray ion source operated in positive and negative mode. The assay quantifies a range from 10 to 1000 ng/mL for capecitabine, from 10 to 5000 ng/mL for 5′‐DFCR and 5′‐DFUR, and from 50 to 5000 ng/mL for 5‐FU and FUH₂ using a plasma sample of 200 μL. Correlation coefficients (r²) of the calibration curves in human plasma were better than 0.99 for all compounds. At all concentration levels, deviations of measured concentrations from nominal concentration were between ?4.41 and 3.65% with CV values less than 12.0% for capecitabine, between ?7.00 and 6.59% with CV values less than 13.0 for 5′‐DFUR, between ?3.25 and 4.11% with CV values less than 9.34% for 5′‐DFCR, between ?5.54 and 5.91% with CV values less than 9.69% for 5‐FU and between ?4.26 and 6.86% with CV values less than 14.9% for FUH₂. The described method was successfully applied for the evaluation of the pharmacokinetic profile of capecitabine and its metabolites in plasma of treated cancer patients. Copyright © 2009 John Wiley & Sons, Ltd.     

Monitoring time‐dependent degradation of phospholipids in sectioned tissues by MALDI imaging mass spectrometry

Imaging mass spectrometry (IMS) is useful for visualizing the localization of phospholipids on biological tissue surfaces creating great opportunities for IMS in lipidomic investigations. With advancements in IMS of lipids, there is a demand for large‐scale tissue studies necessitating stable, efficient and well‐defined sample handling procedures. Our work within this article shows the effects of different storage conditions on the phospholipid composition of sectioned tissues from mouse organs. We have taken serial sections from mouse brain, kidney and liver thaw mounted unto ITO‐coated glass slides and stored them under various conditions later analyzing them at fixed time points. A global decrease in phospholipid signal intensity is shown to occur and to be a function of time and temperature. Contrary to the global decrease, oxidized phospholipid and lysophospholipid species are found to increase within 2 h and 24 h, respectively, when mounted sections are kept at ambient room conditions. Imaging experiments reveal that degradation products increase globally across the tissue. Degradation is shown to be inhibited by cold temperatures, with sample integrity maintained up to a week after storage in ?80 °C freezer under N₂ atmosphere. Overall, the results demonstrate a timeline of the effects of lipid degradation specific to sectioned tissues and provide several lipid species which can serve as markers of degradation. Importantly, the timeline demonstrates oxidative sample degradation begins appearing within the normal timescale of IMS sample preparation of lipids (i.e. 1–2 h) and that long‐term degradation is global. Taken together, these results strengthen the notion that standardized procedures are required for phospholipid IMS of large sample sets, or in studies where many serial sections are prepared together but analyzed over time such as in 3‐D IMS reconstruction experiments. Copyright © 2014 John Wiley & Sons, Ltd.     

Biotin α‐End‐Functionalized Gradient Glycopolymers Synthesized by RAFT Copolymerization

Biotinylated gradient glycopolymers have been synthesized via RAFT copolymerization of an acrylamide derivative of galactose with N‐acryloylmorpholine in the presence of a biotin CTA. The polymerization was controlled with a linear increase in molecular weights up to 80% conversion. Copolymer chains have a gradient microstructure with an increasing proportion of galactose units towards the ω chain end. The presence of the biotin ligand at the α end of the chains was confirmed by ¹H NMR and MALDI‐ToF MS. This strategy based on the use of a biotin‐CTA instead of a post‐polymerization labelling of the chains resulted in a high percentage of α‐functionalized chains (92–95%). Such α‐end‐functionalized glycopolymer chains may interact with streptavidin‐modified surfaces.

     

MALDI‐based intact spore mass spectrometry of downy and powdery mildews

Fast and easy identification of fungal phytopathogens is of great importance in agriculture. In this context, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has emerged as a powerful tool for analyzing microorganisms. This study deals with a methodology for MALDI‐TOF MS‐based identification of downy and powdery mildews representing obligate biotrophic parasites of crop plants. Experimental approaches for the MS analyses were optimized using Bremia lactucae, cause of lettuce downy mildew, and Oidium neolycopersici, cause of tomato powdery mildew. This involved determining a suitable concentration of spores in the sample, selection of a proper MALDI matrix, looking for the optimal solvent composition, and evaluation of different sample preparation methods. Furthermore, using different MALDI target materials and surfaces (stainless steel vs polymer‐based) and applying various conditions for sample exposure to the acidic MALDI matrix system were investigated. The dried droplet method involving solvent evaporation at room temperature was found to be the most suitable for the deposition of spores and MALDI matrix on the target and the subsequent crystallization. The concentration of spore suspension was optimal between 2 and 5 × 10⁹ spores per ml. The best peptide/protein profiles (in terms of signal‐to‐noise ratio and number of peaks) were obtained by combining ferulic and sinapinic acids as a mixed MALDI matrix. A pretreatment of the spore cell wall with hydrolases was successfully introduced prior to MS measurements to obtain more pronounced signals. Finally, a novel procedure was developed for direct mass spectra acquisition from infected plant leaves. Copyright © 2012 John Wiley & Sons, Ltd.