Study of the acid hydrolysis of (3-methacryloxypropyl)trimethoxysilane by capillary electrophoresis-ion-trap mass spectrometry

The sol-gel method is a widely used technique for the synthesis of various functional coating films. Alkoxysilanes such as (3-methacryloxypropyl)trimethoxysilane (MEMO) are largely used as precursors for inorganic-organic hybrid sol-gel materials. Indeed, these compounds can form complex network, through hydrolysis and condensation reactions. The latter have to be perfectly controlled to obtain the required properties. In such a context, we have studied the potentialities of capillary electrophoresis-ion-trap mass spectrometry (CE-MS) coupling to resolve both separation and characterization of the synthesized compounds as a function of the hydrolysis time. The study of acid hydrolysis of MEMO was carried out as an example. After optimization of the running electrolyte in capillary zone electrophoresis (CZE) with UV detection, we characterized the synthesized compounds in CE-MS by using positive detection mode. The obtained resolution in CZE-UV was not entirely satisfactory because of the very closed charge/mass ratio of formed solute but also because of the interaction between the solutes and the capillary walls. Nevertheless, several oligomers were characterized in CE-MS. The absence of detection with regard to oligomers that possess higher molecular masses than octamer is discussed in this work.     

CE-MS Identification of Amino Acid Sequence Inversion as a New Degradation Pathway of an Aspartyl Model Tripeptide

CE-MS was employed to identify two unknown degradation products of the model tripeptide Phe-α-Asp-Gly heated at 80 °C in aqueous solution at pH 7.4. Both compounds displayed essentially identical mass spectra indicating the presence of peptide diastereomers. The [M + H]⁺-ion at m/z 338 suggested a tripeptide composed of the amino acids Phe, Gly and Asp. The fragmentation pattern indicated that Phe was not located at the N-terminus. Subsequently, the linear peptide α-Asp-Phe-Gly and the branched peptide Asp(Gly)-Phe were synthesized and analyzed by CE-MS. The mass spectrum of synthetic α-Asp-Phe-Gly was identical to that of the unknown compounds confirming the structure of the degradation products. Asp(Gly)-Phe displayed a complex fragmentation pattern. In conclusion, amino acid sequence inversion represents another degradation pathway of Phe-α-Asp-Gly at pH 7.4 besides known reactions including isomerization, enantiomerization, cyclization to diketopiperazine derivatives and backbone hydrolysis. The mechanism of the rearrangement of the amino acid sequence is proposed to proceed via an aza-bridged intermediate.

     

Quantitative TOF-SIMS analysis of oligomeric degradation products at the surface of biodegradable poly(alpha-hydroxy acid)s

This paper reports the development of a new method for quantification of the hydrolytic surface degradation kinetics of biodegradable poly(alpha-hydroxy acid)s using time-of-flight secondary ion mass spectrometry (TOF-SIMS). We report results from static SIMS spectra of a series of poly(alpha-hydroxy acid)s including poly(glycolic acid), poly(L-lactic acid), and random poly(D,L-lactic acid-co-glycolic acid) hydrolyzed in various buffer systems. The distribution of the most intense peak intensities of ions generated in high mass range of the spectrum reflects the intact degradation products (oligomeric hydrolysis products) of each biodegradable polymer. First, a detailed analysis of the oligomeric ions is given based on rearrangement of the intact hydrolysis products. The pattern of ions can distinguish both degradation-generated intact oligomers and their fragment ion peaks with a variety of combinations of each repeat unit. Then, the integration and summation of the area of all ion peaks with the same number of repeat units is proposed as a measurement that provides a more accurate MW average than the typically used method which counts only the most intense peak. The multiple ion summation method described in this paper would be practical in the improvement of quantitative TOF-SIMS studies as a better data reduction method, especially in the surface degradation kinetics of biodegradable polymers.     

Capillary electrophoresis-mass spectrometry for the analysis of intact proteins 2007-2010

CE coupled to MS has proven to be a powerful analytical tool for the characterization of intact proteins, as it combines the high separation efficiency of CE with the selectivity of MS. This review provides an overview of the development and application of CE-MS methods within the field of intact protein analysis as published between January 2007 and June 2010. Ongoing technological developments with respect to CE-MS interfacing, capillary coatings for CE-MS, coupling of CIEF with MS and chip-based CE-MS are treated. Furthermore, CE-MS of intact proteins involving ESI, MALDI and ICP ionization is outlined and overviews of the use of the various CE-MS methods are provided by tables. Representative examples illustrate the applicability of CE-MS for the characterization of proteins, including glycoproteins, biopharmaceuticals, protein-ligand complexes, biomarkers and dietary proteins. It is concluded that CE-MS is a valuable technique with high potential for intact protein analysis, providing useful information on protein identity and purity, including modifications and degradation products.     

Characterizations of modified cassava starch with long chain fatty acid chlorides obtained from esterification under low reaction temperature and its PLA blending

In contrast to typical starch esterification in an aqueous solution, which are carried out at elevated to ambient reaction temperatures, a low reaction temperature was applied in this study to minimize the starch chain hydrolysis. The physical properties of the modified starch, obtained from an esterification of cassava starch with long-chain fatty acid chlorides carried out in aqueous media at 4°C, were characterized using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and contact angle measurements. The modified starches show improvement in thermal stability and hydrophobicity, which can be further optimized by varying the types of acid chlorides and the reaction conditions. The starch products have high potential for use as fillers for biodegradable polymers, especially polylactic acid (PLA), as their tunable hydrophobicity can impose strong effect on controlling of the PLA's hydrolytic degradation rate for specific applications. Results on mechanical properties of the blends between the modified starch and PLA show an improvement in modulus of the polymer.     

Capillary electrophoresis-mass spectrometry of peptides from enzymatic protein hydrolysis: simulation and optimization

Two important limitations still exist for the characterization of protein digests by capillary electrophoresis-mass spectrometry (CE-MS): (i) the buffer choice (i.e., the buffer must provide an adequate CE separation without ruining the MS signal), and (ii) the frequent generation of "unexpected" peptidic fragments during the enzymatic protein hydrolysis. In this work, a new approach is used to solve these difficulties, namely a theoretical model that relates the electrophoretic behavior of peptides to their sequence. The effectiveness of this procedure is demonstrated by the fast attainment of good CE-MS conditions for analyzing the peptides obtained from an enzymatic protein hydrolysate in a single run. This strategy can provide useful information for helping to characterize "unexpected" fragments from protein digests.     

Determination of tobacco-specific N-nitrosamines in rabbit serum by capillary zone electrophoresis and capillary electrophoresis-electrospray ionization-mass spectrometry with solid-phase extraction

In this paper, we propose a new strategy for separation and determination of tobacco-specific N-nitrosamines (TSNAs), a group of strong carcinogens found only in tobacco products, by using CZE and CE-MS associated with SPE. Six TSNAs: N'-nitrosonornicotine, N'-nitrosoanatabine, N'-nitrosoanabasine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, and 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol were simultaneously separated by either of two CZE methods, one of which worked with ammonium formate buffer (pH 2.5) and another with citrate buffer (pH 2.4), as well as a CE-MS method. The CZE conditions including pH and concentration of running buffer, capillary length, applied voltage, and capillary temperature were systematically optimized. For CE-MS method, an optimized sheath liquid consisted of methanol-water was used at a flow rate of 10 muL/min. With SPE procedure, our proposed CE-MS method was successfully applied to determine TSNAs after 15 min metabolism in rabbits. A comparison study between CZE and CE-MS methods for quantitative purposes was carried out, showing that both methods provided similar separation efficiency, selectivity, repeatability, linearity, and recovery. However, CE-MS method was better suited for the analysis of TSNAs in complicated biological samples for its sensitivity and extra information on molecular structure. Having good accordance with our previous work by using LC-MS, the new CE-MS method is expected to be an alternative to the LC-MS method and applied to study the metabolism of TSNAs.     

The determination of penicillins by titrations with mercury(II) solution.

A simple technique, involving two titrations with mercury(II) solutions, is described for the determination of penicillins and their degradation products. The first titration, at pH 4–5 on an untreated penicillin solution, gives the amount of degradation products; the second titration, on a hydrolysed solution at the same pH, gives the sum of the degradation products and penicillin degraded during the hydrolysis. Enzymic hydrolysis is superior to alkaline hydrolysis for penicillinase-sensitive penicillins. Enzyme-resistant penicillins should be hydrolysed with alkali at optimum conditions, e.g. for cloxacillin at pH 13.5 for 5 min. A standard deviation of less than 0.5 % was obtained for the penicillins investigated. The method is absolute; calibration with standard penicillin is not necessary.     

Degradable polyesters through chain linking for packaging and biomedical applications

The major route to convert lactic acid to high-molecular-weight polymers is ring-opening polymerization of lactide. We have investigated alternative synthesis routes based on oligomerization and chain linking to produce high-molecular-weight thermoplastic degradable polymers cost-effectively. Chain linking also offers new possibilities to prepare degradable polyesters for biomedical applications by extending the range of polymer properties achievable. In this paper, we briefly review different chain linking techniques used in our laboratory. Typically, lactic acid prepolymers with molecular weights of around 3,000-15,000 g x mol(-1) have been prepared by direct polycondensation. Hydroxyl terminated oligomers have been chain linked by using diisocyanate coupling agents, preferably 1,4-butane diisocyanate, forming poly(ester-urethanes). Poly(ester-amides) have been prepared by using 2,2'-bis(2-oxazoline) as coupling agent for carboxylic acid telechelic oligomers. Chain linking by end functionalization has been used in the preparation of poly(ester-anhydrides). In addition, a variety of crosslinked degradable polymers and copolymers have been synthesized through different crosslinking routes, by using methacrylic, itaconic or maleic double bonds or triethoxysilane moieties. A biodegradation test and ecotoxicological evaluation of the degradation products were carried out in addition to hydrolysis tests. Lactic acid based chain linked polymers were biodegradable and the degradation products were harmless. In hydrolysis tests, enzymatic degradation was pronounced in the chain linked poly(epsilon-caprolactone).     

聚羟基烷酸酯酶降解动力学研究

研究了羟基丁酸 羟基戊酸共聚物 (PHBV)在脂肪酶中的降解行为 ,用滴定法测定降解速度并进行酶促反应动力学研究 .探讨了降解速度与酶浓度和底物浓度的数学关系和Michaelis Menten常数 ,从实验上和理论上证实了PHBV的物理形态和几何尺寸对酶降解过程的影响 ,以及实验数据与非均相动力学模型的拟合     

Simplified 2-D CE-MS mapping: analysis of proteolytic digests

It has been demonstrated that CE-MS is a very useful hyphenated technique for proteomic studies. However, the huge amount of data stored in a single CE-MS run makes it necessary to account with procedures able to extract all the relevant information made available by CE-MS. In this work, we present a new and easy approach capable of generating a simplified 2-D map from CE-MS raw data. This new approach provides the automatic detection and characterization of the most abundant ions from the CE-MS data including their mass-to-charge (m/z) values, ion intensities and analysis times. It is demonstrated that visualization of CE-MS data in this simplified 2-D format allows: (i) an easy and simultaneous visual inspection of large datasets, (ii) an immediate perception of relevant differences in closely related samples, (iii) a rapid monitoring of data quality levels in different samples, and (iv) a fast discrimination between comigrating polypeptides and ESI-MS fragmentation ions. The strategy proposed in this work does not rely on an excellent mass accuracy for peak detection and filtering, since MS values obtained from an IT analyzer are used. Moreover, the methodology developed works directly with the CE-MS raw data, without interference by the user, giving simultaneously a simplified 2-D map and a much easier and more complete data evaluation. Besides, this procedure can easily be implemented in any CE-MS laboratory. The usefulness of this approach is validated by studying the very similar trypsin digests from bovine, rabbit and horse cytochrome c. It is demonstrated that this simplified 2-D approach allows specific markers for each species to be obtained in a fast and simple way.     

Branched Multifunctional Polyether Polyketals: Variation of Ketal Group Structure Enables Unprecedented Control over Polymer Degradation in Solution and within Cells

Multifunctional biocompatible and biodegradable nanomaterials incorporating specific degradable linkages that respond to various stimuli and with defined degradation profiles are critical to the advancement of targeted nanomedicine. Herein we report, for the first time, a new class of multifunctional dendritic polyether polyketals containing different ketal linkages in their backbone that exhibit unprecedented control over degradation in solution and within the cells. High-molecular-weight and highly compact poly(ketal hydroxyethers) (PKHEs) were synthesized from newly designed α-epoxy-ω-hydroxyl-functionalized AB(2)-type ketal monomers carrying structurally different ketal groups (both cyclic and acyclic) with good control over polymer properties by anionic ring-opening multibranching polymerization. Polymer functionalization with multiple azide and amine groups was achieved without degradation of the ketal group. The polymer degradation was controlled primarily by the differences in the structure and torsional strain of the substituted ketal groups in the main chain, while for polymers with linear (acyclic) ketal groups, the hydrophobicity of the polymer may play an additional role. This was supported by the log?P values of the monomers and the hydrophobicity of the polymers determined by fluorescence spectroscopy using pyrene as the probe. A range of hydrolysis half-lives of the polymers at mild acidic pH values was achieved, from a few minutes to a few hundred days, directly correlating with the differences in ketal group structures. Confocal microscopy analyses demonstrated similar degradation profiles for PKHEs within live cells, as seen in solution and the delivery of fluorescent marker to the cytosol. The cell viability measured by MTS assay and blood compatibility determined by complement activation, platelet activation, and coagulation assays demonstrate that PKHEs and their degradation products are highly biocompatible. Taken together, these data demonstrate the utility this new class of biodegradable polymer as a highly promising candidate in the development of multifunctional nanomedicine.     

Characterization of proteins from Spirulina platensis microalga using capillary electrophoresis-ion trap-mass spectrometry and capillary electrophoresis-time of flight-mass spectrometry

In this work, a new capillary electrophoresis-mass spectrometry (CE-MS) procedure is developed to analyze proteins in Spirulina platensis microalgae. It is demonstrated that a fine optimization of several separation parameters is essential in order to achieve suitable CE-MS analysis of these proteins in natural extracts from microalgae. Namely, optimization of the composition of the separation buffer, electrospray conditions, and washing routine between runs are required in order to obtain reliable and reproducible CE-MS analyses of the main proteins found in this microalga (namely, allophycocyanin-alpha chain, allophycocyanin-beta, c-phycocyanin-alpha, and c-phycocyanin-beta). The relative molecular mass of these biopolymers is determined using two different MS instruments coupled to CE, i.e., CE-ion trap-MS and CE-time of flight-MS (CE-TOF-MS). A comparison between the results obtained with both instruments is carried out. The high resolution of the TOF-MS enables the distinction of small modifications in proteins and, thus, a more accurate mass determination. Interestingly, molecular mass values obtained by both CE-MS procedures agree very well while these experimental values are only in partial agreement with those theoretically expected (i.e., genetically derived masses). Some protein modifications due to amino acids exchange induced by nucleotide codon mutations are proposed to explain this difference.     

New approaches to mapping through-thickness variations in the degradation in poly (lactide-co-glycolide)

Biodegradable poly (lactide-co-glycolide) (PLGA) copolymers have been used for many years for biomedical applications such as soluble sutures, orthopaedic implants and more recently as potential tissue scaffold materials. The rate at which the copolymers degrade can be manipulated from a period of days to months by changing the lactide/glycolic acid ratio. Degradation of PLGA copolymers occurs by hydrolysis of the ester bonds in the polymer backbone. The hydrolysis reaction is autocatalytic and is accelerated by the build up of degradation products in the bulk of the material. As a consequence, material degradation is expected to be non-uniform through the specimen thickness with the material at the centre degrading at a faster rate than at the surface. Despite many studies of PLGA degradation, information on this local variance is sparse as the techniques used to track the process are usually bulk measures. In this study, two new approaches for monitoring degradation have been developed that enable local measurements of degradation to be made throughout the specimen over an extended period of time. Chemical and mechanical variations in the structure of the polymer have been mapped using attenuated total reflectance infrared spectroscopy (ATR-FTIR) and nanoindentation. These have produced comparable results and show that the degradation rate at the centre of the specimens is almost an order of magnitude higher than at the surface.     

Identification of novel angiotensin-converting enzyme-inhibitory peptides from ovine milk proteins by CE-MS and chromatographic techniques

In this report, we present the use of CE-MS as complement to RP separation for the identification of novel angiotensin-converting enzyme-inhibitory (ACEI) peptides from a complex milk protein hydrolysate. As preliminary step, fast protein LC (FPLC) was used to isolate the different casein fractions from raw ovine milk. Enzymatic hydrolysis of these fractions was performed by using proteolytic enzymes of gastrointestinal origin. The most active hydrolysate, corresponding to kappa-casein hydrolyzed with pepsin, chymotrypsin, and trypsin, was fractionated by RP-HPLC and the peptides contained in the active fractions were sequenced by CE coupled to IT-MS (CE-MS). The use of CE-MS allowed the identification of short peptides with ACEI activity included in the scarcely retained fraction obtained by semipreparative RP-HPLC. Among the identified peptides, those with hydrophobic or positively charged residues at the C-terminal tripeptide were chemically synthesized to determine their ACEI activity. This procedure allowed us to identify four novel potent ACEI peptides from kappa-casein with sequences IAK, YQQRPVA, WQVLPNAVPAK, and HPHPHLSF. These active sequences could be obtained by enzymatic hydrolysis either of the individual kappa-casein fraction or the total casein fraction from ovine milk.     

Insight into industrial PLA aging process by complementary use of rheology,HPLC, and MALDI

Nowadays, there is a growing availability of biodegradable industrial materials intended to food contact applications whose service life behavior needs to be further investigated. This article is focused on the degradation of two materials based on polylactic acid. The correlation between the rate of degradation and the amount of trapped degradation products was investigated applying three characterization techniques in parallel, namely rheology, high‐performance liquid chromatography (HPLC), and matrix‐assisted laser desorption/ionization (MALDI). The rate of degradation was studied through the evaluation of their rheological properties and calculation of the number of average molecular weights, and weight‐average molecular weights. Water‐soluble oligomers and lactic acid were quantified by HPLC‐ultraviolet. Changes in cyclic and linear oligomers were monitored by MALDI‐time‐of‐flight mass spectrometry. Specimens of 4‐mm thickness of each biopolymer were subjected to hydrolysis in deionized water up to 6 months at two temperatures, simulating service conditions of food packaging. The diminution in viscosity and consequently in molecular weight distribution (20–60%) showed the degradation of the molecular structure of both polylactic acids. The chain scission was followed through the increasing values of lactic acid and hydrolyzed oligomers (twofold to eightfold), and the predominant signal of the linear oligomers over the cyclic ones with aging. Rheology, HPLC, and MALDI showed to be complementary tools to better understand the changes in the molecular structure. The obtained results showed the necessity of adding suitable stabilizers for each particular food packaging application. Copyright © 2013 John Wiley & Sons, Ltd.     

The use of thermospray-liquid chromatography/mass spectrometry for the verification of chemical warfare agents

Summary Thermospray-liquid chromatography mass spectrometry (TSP-LC-MS) is a relatively new analytical technique which proved to be useful for the verification of chemical warfare agents and their polar degradation products in aqueous solutions. The principles of the technique are described and comparisons are made with other forms of mass spectrometric analysis. A survey is presented of the results obtained so far at the Prins Maurits Laboratory TNO. The analysis of organophosphorus nerve agents and their hydrolysis products (organophosphorus acids) in various types of water is described. Special attention is paid to the nerve agent VX. Direct analysis of vesicants in water by TSP-LC-MS is limited. However, analysis of their hydrolysis products, as well as related compounds such as adducts of mustard gas with nucleosides and peptides, is possible. Finally, the use of TSP-LC-MS for the analysis of other compounds of chemical warfare interest (toxins) is indicated.     

The degradation and biocompatibility of waterborne biodegradable polyurethanes for tissue engineering

To better investigate the degradation and biocompatibility of waterborne biodegradable polyurethanes for tissue engineering, a series of new waterborne biodegradable polyurethanes (PEGPUs) with low degree of crosslinking was synthesized using IPDI, BDO and L-lysine as hard segments, PCL and PEG as soft segment. The bulk structures and properties of the prepared polyurethanes were characterized by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), tensile mechanical tests and water contact angle (WCA) measurements. The degree of microphase separation was slightly improved because of the lowered crosslinking degree of these PEGPUs in comparison with the high cross-linking degree samples, leading to good mechanical properties, as indicated by DSC and stress-strain data. Moreover, biodegradability of the polyurethanes was evaluated in phosphate buffer solutions (PBS) under different pH values and enzymatic solution at pH 7.4 through weight loss monitoring. The results suggested that the degradation of these PEGPUs was closely related to their bulk and surface properties. And the degradation products didn’t show apparent inhibition effect against fibroblasts in vitro. These studies demonstrated that the waterborne biodegradable polyurethanes could find potential use in soft tissue engineering and tissue regeneration.     

Enhanced hydrolysis resistance of biodegradable polymers and bio-composites

This study investigated the hydrolysis of biodegradable polymers and bio-composites at 50 °C and 90% relative humidity (RH). With increasing hydrolysis time, the mechanical properties of the biodegradable polymers and bio-composites significantly decreased due to the easy hydrolytic degradation of the ester linkage of the biodegradable polymers. With increasing hydrolysis time, the tensile strength of the polybutylene succinate (PBS) treated with anti-hydrolysis agent or with trimethylolpropane triacrylate (TMPTA) significantly increased compared to the non-treated PBS. The same results were observed for the PBS-based bio-composites. This result was confirmed by the Fourier transform infrared-attenuated total reflectance (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS) spectra, which exhibited a less eroded surface, small cracks and fewer holes due to the reduced surface hydrolysis and erosion under high humidity condition.     

A novel strategy for encapsulation and release of proteins: hydrogels and microgels with acid-labile acetal cross-linkers

A new acid-labile acetal cross-linker was synthesized and used to prepare protein-loaded hydrogels and microgels. This cross-linker undergoes an acid-catalyzed degradation with a half-life of 5.5 min at pH 5.0 and 24 h at pH 7.4. Protein-loaded hydrogels were synthesized with this cross-linker, and their release profiles were measured as a function of pH. Hydrogels made with the acetal cross-linker release their contents in a pH-dependent manner. The acetal cross-linker was also used to synthesize microgels with sizes between 1 and 10 mum, a range suitable for phagocytosis. The unique acid sensitivity of the acetal cross-linker should make it a useful synthetic intermediate in the design of acid-sensitive drug or gene delivery systems.