Kinetic studies of AMP-dependent phosphorolysis of amylopectin catalyzed by phosphorylase b on a 27 MHz quartz-crystal microbalance

Catalytic cleavage reactions of phosphorylase b were monitored directly on an amylopectin-immobilized 27 MHz quartz-crystal microbalance (QCM). When the inactivated phosphorylase b was injected into a phosphate buffer solution of amylopectin-immobilized QCM (method A), the binding of the enzyme to amylopectin was observed as a frequency decrease (mass increase). Then, when AMP (adenosine monophosphate) was added to activate the enzyme, the frequency gradually increased (mass decreased) due to the phosphorolysis of amylopectin in the presence of phosphates as buffers. When the AMP-activated phosphorylase b was employed (method B), the continuous reaction was observed which includes both the mass increase due to the enzyme binding to amylopectin at first and then the following mass decrease due to the phosphorolysis by the AMP-activated enzyme. All kinetic parameters for the enzyme binding to the substrate (binding and dissociation rate constants, k(on) and k(off), and dissociation constant, K(d)), the AMP binding to the enzyme as activator (K(AMP)), the catalytic rate constant (k(cat)) were obtained from curve fittings of time-courses of frequency (mass) changes. The obtained kinetic parameters were compared with those from Michaelis-Menten kinetics.     

Improving mass accuracy of high performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of intact antibodies

The glycosylation profile of intact antibody due to the galactose and fucose heterogeneity in the N-linked sugars was determined with instrument resolution of 5000 and 10,000. After deconvolution of electrospray ionization mass spectra to complete convergence, several extra peaks appeared in addition to the peaks observed in the original mass spectra. The artificial peaks were avoided if deconvolution was stopped after a smaller number of iterations. A standard antibody was used as an external calibrant to minimize mass measurement errors during long-period experiments. Precision of four consecutive LC/MS measurements of the same antibody was 10 ppm (+/-1.5 Da). By using this approach, the masses of 11 intact antibodies were measured. All antibodies containing N-terminal glutamines had a negative mass shift due to the formation of pyroglutamate (-17 Da). Although the pyroglutamate variant of intact antibody was not resolved from the unmodified variant, this modification led to a mass shift proportional to the percentage of N-terminal pyroglutamate. By accurately measuring the mass shift we were able to quantify the abundance of pyroglutamic acid on intact antibodies. Mass accuracy in measuring different antibodies was below 30 ppm (+/-4 Da). The accurate mass measurement can be an effective tool for monitoring chemical degradations in therapeutic antibodies.     

Fourier transform ion cyclotron resonance mass spectrometry with NanoLC/microelectrospray ionization and matrix-assisted laser desorption/ionization: analytical performance in peptide mass fingerprint analysis

Protein identifications by peptide mass fingerprint analyses with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) were performed using microelectrospray ionization coupled to nano liquid chromatography (NanoLC), as well as using matrix-assisted laser desorption/ionization (MALDI). Tryptic digests of bovine serum albumin (BSA), diluted down to femtomole quantities, have been desalted by fast NanoLC under isocratic elution conditions as the high resolving power of FT-ICR MS enables peptides to be separated during the mass analysis stage of the experiment. The high mass accuracy achieved with FT-ICR MS (a few ppm with external calibration) facilitated unambiguous protein identification from protein database searches, even when only a few tryptic peptides of a protein were detected. Statistical confidence in the database search results was further improved by internal calibration due to increased mass accuracy. Matrix-assisted laser desorption/ionization and micro electrospray ionization (ESI) FT-ICR showed good mass accuracies in the low femtomole range, yet a better sensitivity was observed with MALDI. However, in higher femtomole ranges slightly lower mass accuracies were observed with MALDI FT-ICR than with microESI FT-ICR due to scan-to-scan variations of the ion population in the ICR cell. Database search results and protein sequence coverage results from NanoLC FT-ICR MS and MALDI FT-ICR MS, as well as the effect of mass accuracy on protein identification for the peptide mass fingerprint analysis are evaluated.     

Origin of mass shifts in the quadrupole ion trap: dissociation of fragile ions observed with a hybrid ion trap/mass filter instrument

A novel hybrid tandem mass analyzer, coupling a quadrupole ion trap with a quadrupole mass filter, has been constructed to permit mass analysis of ions ejected from the ion trap. The initial application of this instrument is the investigation of the origin of mass shifts in the ion trap due to ion fragility. We hypothesize that fragile ions undergo mass shifts, characterized by peak fronting, due to early ejection from the quadrupole ion trap. As these ions come into resonance with the ejection frequency, they gain kinetic energy, collide with buffer gas molecules and thus can dissociate to produce fragment ions. These fragment ions will not be stable within the ion trap as they are situated past the stability boundary at q(z) = 0. 908. Consequently the fragment ions are ejected prematurely. This results in an apparent mass shift due to peak fronting. The experiments reported here clearly document the production of fragment ions as the origin of mass shifts during the resonant ejection of fragile ions. Copyright 2000 John Wiley & Sons, Ltd.     

Mass spectral studies of perfluorooctane sulfonate derivatives separated by high-resolution gas chromatography

The mass spectral characteristics of perfluorooctane sulfonate (PFOS, C(8)F(17)SO(3)-) isomers present in technical PFOS were obtained using high-resolution gas chromatography (HRGC) combined with mass spectrometry (MS). To make PFOS amenable to HRGC separation, a simple derivatization procedure was developed. The method involved the conversion of PFOS into the iso-propyl ester using iso-propanol as the derivatization reagent under acidic conditions. Mass spectra were generated employing electron ionization (EI) and negative chemical ionization (NCI). Interpretation of fragment ions was possible due to the use of deuterium-labeled iso-propanol as derivatization reagent, which induced mass shifts in the electron ionization (EI) and negative chemical ionization (NCI) mass spectra. HRMS allowed the accurate mass measurement of important EI fragments and confirmed the derivatization reaction as well as the proposed fragmentation pathway involving rearrangement. Moreover, the high resolution provided by HRGC enabled the separation of eleven PFOS isomers present in the technical product. This is an improvement over the previously reported high-performance liquid chromatography (HPLC) separation. A complete identification of all isomers was not possible due to lack of pure reference materials. Finally, the developed derivatization procedure was successfully applied to perfluoroalkyl carboxylates (PFCA) and corresponding fragmentation involving rearrangement of the derivatized PFCA was observed. The described qualitative derivatization offers a promising alternative technique for the separation and identification of isomers of perfluoroalkyl sulfonates and carboxylates by HRGC/MS.     

Mass calibration of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer including the rise time of the delayed extraction pulse

To utilize fully modern MALDI-TOF and TOF/TOF mass spectrometers with mass resolution exceeding 10,000 and 2 ppm precision of flight time measurements for high mass accuracy, the model of ion motion used in the mass calibration equation must be expanded. The standard three-term equation providing up to 5-10 ppm (rms) mass accuracy with internal standards was modified with an additional term accounting for the finite rise time of the high-voltage extraction pulse. This new four-term calibration equation minimizes the effect of systematic error resulting from the fact that ion velocities are mass dependent due to the rise time of the extraction pulse. Applying this new calibration equation to a mass spectrum obtained in an axial MALDI-TOF MS containing 70 peaks (sodiated PEG), each with a signal-to-noise ratio greater than 100, a mass accuracy of 1.6 ppm (rms) was obtained over the mass range 1.0-4.0 kDa compared with 3.6 ppm (rms) with the standard three-term equation. The physical basis of the effects of the finite extraction pulse rise time on mass calibration is examined for axial MALDI-TOF mass spectrometers, as well as for orthogonal acceleration TOF mass spectrometers.     

Investigation of adsorbed polymer layers by flocculation of oppositely charged sols III. Effect of molecular mass of polymers

The stabilizing effect of water-soluble neutral polymers with different chemical structure and relative molecular mass on AgI sol has been studied. It was shown that the stabilizing effect of the polymers used is independent of the relative molecular mass of the polymer when equal amounts of the adsorbed polymers are compared with each other as stabilizers. The high stabilizing effect of polymers with high relative molecular mass is presumably due to the larger adsorbed amount.Among polyvinyl alcohol, polyvinyl pyrrolidone and methylcellulose, polyvinyl pyrrolidone exhibits the smallest stabilizing layer thickness at equal adsorbed amounts. According to our earlier results this is due to the fact that the polyvinyl pyrrolidone shows the strongest affinity for AgI surfaces, forming short loops or tails.     

Halohydrination of epoxy resins using sodium halides as cationizing agents in MALDI-MS and DIOS-MS

Halohydrination of epoxy resins using sodium halides as cationizing agents in matrix-assisted laser desorption/ionization (MALDI) and desorption ionization on porous silicon mass spectrometry (DIOS-MS) were investigated. Different mass spectra were observed when NaClO(4) and NaI were used as the cationizing agents at the highest concentration of 10.0 mM, which is much higher than that normally used in MALDI-MS. MALDI mass spectra of epoxy resins using NaI revealed iodohydrination to occur as epoxy functions of the polymers. The halohydrination also occurred using NaBr, but not NaCl, due to the differences in their nucleophilicities. On the basis of the results of experiments using deuterated CD(3)OD as the solvent, the hydrogen atom source was probably ambient water or residual solvent, rather than being derived from matrices. Halohydrination also occurred with DIOS-MS in which no organic matrix was used; in addition, reduction of epoxy functions was observed with DIOS. NaI is a useful cationizing agent for changing the chemical form of epoxy resins due to iodohydrination and, thus, for identifying the presence of epoxy functions.     

Size exclusion chromatography-gradients, an alternative approach to polymer gradient chromatography: 2. Separation of poly(meth)acrylates using a size exclusion chromatography-solvent/non-solvent gradient

A gradient ranging from methanol to tetrahydrofuran (THF) was applied to a series of poly(methyl methacrylate) (PMMA) standards, using the recently developed concept of SEC-gradients. Contrasting to conventional gradients the samples eluted before the solvent, i.e. within the elution range typical for separations by SEC, however, the high molar mass PMMAs were retarded as compared to experiments on the same column using pure THF as the eluent. The molar mass dependence on retention volume showed a complex behaviour with a nearly molar mass independent elution for high molar masses. This molar mass dependence was explained in terms of solubility and size exclusion effects. The solubility based SEC-gradient was proven to be useful to separate PMMA and poly(n-butyl crylate) (PnBuA) from a poly(t-butyl crylate) (PtBuA) sample. These samples could be separated neither by SEC in THF, due to their very similar hydrodynamic volumes, nor by an SEC-gradient at adsorbing conditions, due to a too low selectivity. The example shows that SEC-gradients can be applied not only in adsorption/desorption mode, but also in precipitation/dissolution mode without risking blocking capillaries or breakthrough peaks. Thus, the new approach is a valuable alternative to conventional gradient chromatography.     

Validation of the mass response of a quartz crystal microbalance coated with Prussian Blue film for ac electrogravimetry

Prussian Blue (PB) films have been considerably studied for many research applications such as electrochromic material development, new material for batteries, etc. Many analytical techniques were employed for examining PB electrochemical behaviour in solution and the quartz crystal microbalance (QCM) used in the alternative regime (ac electrogravimetry) appeared as an attractive in situ mass sensor due to its low cost and its high mass sensitivity. Unfortunately, the validity of the common Sauerbrey equation was questionable with these films or in other terms if the QCM was used as a pure mass sensor. In this work PB film is examined through acoustic measurements and the response can be interpreted as a pure mass change if the thickness is around 0.15 μm. Over this limit, film viscoelastic contributions can affect drastically the mass change estimation: if the thickness is two times larger, the mass error reaches 40%.     

Thermogravimetric analysis of the aluminum-polypropylene mixtures after plastic deformation under high pressure

The thermogravimetric analysis of the aluminum-polypropylene mixture of varied composition was performed to determine the effect of plastic deformation under the high pressure. It was found that a loss of mass by the mixed samples up to 400°C is due to decomposition of a polymer and the mass gain above 500°C, due to aluminum oxidation.     

The estimation of pig bone age for forensic application using thermogravimetric analysis

An accurate means of determining bone age is a goal for forensic scientists. In this study, thermogravimetric analysis (TGA) has been used to examine pig bone specimens of different post-mortem age. Analysis of bone in both air and nitrogen atmospheres reveals a decrease in total mass loss as the bones age. Two mass loss steps due to the decomposition of the organic bone components were observed and show decreasing trends with age for decomposition in an air atmosphere. In a nitrogen atmosphere the decomposition was observed to be more complex and age dependence of the mass loss for each step was not identified. The TGA data, however, demonstrates the potential of the technique as a means of estimating post-mortem age of forensic bone specimens.     

The study of the changes in the thermal properties of <Emphasis Type="Italic">Labeo rohita</Emphasis> bones due to arsenic exposure

The paper presents the changes in the thermal properties of control, arsenic exposed and DMSA treated Labeo rohita bones by using thermo analytical techniques. The result shows that the mass loss due to the thermal decomposition occurs in three distinct steps due to loss of water, organic and inorganic materials. The arsenic exposed bones present a different thermal behaviour compared to the control bones. The residue masses are increased due to arsenic exposure, while the DMSA treatment reduces the residue mass level. These thermal characteristics can be used as a qualitative method to check the metal accumulation in samples.     

Adsorption et desorption de l''eau vapeur sur un polymere polyacrylique superabsorbant IV. Effect de la masse sur les cinétiques d''adsorption et de désorption

The effect of steam adsorption-desorption on hydrophilic polymer processes is studied under controlled pressure and temperature. Experiments were performed using a relatively high mass of polymer (2 to 20 g), to examine the influence of different parameters on the adsorption-desorption mechanisms, such as the polymeric adsorbant mass, the steam pressure, the temperature and the number of adsorption-desorption cycles.

The limiting step of these processes varies from a pseudo-first-order step type for the low adsorbant mass (< 1 g), to a diffusive step for the mass range 2 to 20 g. In this last range, the process does not depend upon the adsorbant mass; only the adsorption and desorption rates change due to the modification of the space scale of the solid. The adsorption kinetics was not modified by the number of cycles, even for the first cycle; this is the opposite result as compared to when the experiment was performed with a low mass of polymer.     

Electrospray and matrix-assisted laser desorption/ionization mass spectral characterization of linear single nylon-6 oligomers

Synthetic nylon-6 single molecular mass oligomers were studied by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. These oligomers, considered as model compounds for the study of nylon-6 polymers, gave good mass spectrometric results using both MALDI and ESI. In spite of the gentle nature of both techniques, the MALDI and ESI spectra showed evidence of end-group cleavage from the oligomer chains. MALDI-MS was found to give similar fragmentation patterns for all of the oligomer samples. An increase in doubly charged ion signals with increasing oligomer mass was observed in the ESI mass spectra, as was end-group fragmentation. Signals from oligomer clusters were observed in ESI-MS for the dimer, tetramer and hexamer, most likely due to non-covalent bonding among the low-mass oligomer molecules.     

Selektive chemische Trennverfahren für die Aktivierungsanalyse

Relative sensitivity factors (RSF) of 32 elements in the concentration range 1–10⁴ ppm (mass of element/ mass of electrode) have been determined for spark source mass spectrometric analysis. Graphite and silver powder was used as conductive material. The RSF of about two thirds of the elements under investigation were found to be dependent on their concentration in the electrodes. This is thought to be due to volatilization at high temperatures whereby the concentration of these elements at the electrode surfaces is diminished. In general RSF's at silver powder electrodes are higher and necessary sparking times are shorter than those at graphite electrodes. Therefore the use of silver powder for determinations of concentrations <10³ ppm is advantageous although experimental results show greater scatter due to inhomogeneity of the electrode.     

Proteolysis in microfluidic droplets: an approach to interface protein separation and peptide mass spectrometry

A versatile microreactor protocol based on microfluidic droplets has been developed for on-line protein digestion. Proteins separated by liquid chromatography are fractionated in water-in-oil droplets and digested in sequence. The microfluidic reactor acts also as an electrospray ionization emitter for mass spectrometry analysis of the peptides produced in the individual droplets. Each droplet is an enzymatic micro-reaction unit with efficient proteolysis due to rapid mixing, enhanced mass transfer and automated handling. This droplet approach eliminates sample loss, cross-contamination, non-specific absorption and memory effect. A protein mixture was successfully identified using the droplet-based micro-reactor as interface between reverse phase liquid chromatography and mass spectrometry.     

Building an empirical mass spectra library for screening of organic pollutants by ultra-high-pressure liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry

Hybrid quadrupole time-of-flight mass spectrometry (QTOF MS) has gained wide acceptance in many fields of chemistry, for example, proteomics, metabolomics and small molecule analysis. This has been due to the numerous technological advances made to this mass analyser in recent years. In the environmental field, the instrument has proven to be one of the most powerful approaches for the screening of organic pollutants in different matrices due to its high sensitivity in full acquisition mode and mass accuracy measurements. In the work presented here, the optimum experimental conditions for the creation of an empirical TOF MS spectra library have been evaluated. For this model we have used a QTOF Premier mass spectrometer and investigated its functionalities to obtain the best MS data, mainly in terms of mass accuracy, dynamic range and sensitivity. Different parameters that can affect mass accuracy, such as lock mass, ion abundance, spectral resolution, instrument calibration or matrix effect, have also been carefully evaluated using test compounds (mainly pesticides and antibiotics). The role of ultra-high-pressure liquid chromatography (UHPLC), especially when dealing with complex matrices, has also been tested. In addition to the mass accuracy measurements, this analyser allows the simultaneous acquisition of low and high collision energy spectra. This acquisition mode greatly enhances the reliable identification of detected compounds due to the useful (de)protonated molecule and fragment ion accurate mass information obtained when working in this mode. An in-house empirical spectral library was built for approximately 230 organic pollutants making use of QTOF MS in MS(E) mode. All the information reported in this paper is made available to the readers to facilitate screening and identification of relevant organic pollutants by QTOF MS.     

Analysis of the saliva from patients with oral cancer by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), this study analyzed the saliva obtained from patients with oral cancer and compared these mass spectra with those obtained from healthy controls. Saliva without pre-treatment was mixed directly with a sinapinic acid matrix. Alpha-amylase (57 kDa) dominated the high mass range in the MALDI mass spectra of the saliva from healthy subjects, but the peak was suppressed for patients with oral cancer and was replaced by a peak at m/z 66 k in the spectra of patients' samples (15 out of 20). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with in-gel tryptic digestion combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) was employed to characterize this 66-kDa protein, which was thus shown to be albumin. However, based on SDS-PAGE results, concentrations of both alpha-amylase and albumin in patients' saliva were significantly higher than those in healthy subjects. This discrepancy was shown to be due to MALDI suppression effects due to the albumin. MALDI-MS thus has potential as a possible rapid diagnostic screening tool for oral cancer.     

Quantification of amorphous content in mixed systems: amorphous trehalose with lactose

There has been a lot of interest in quantification of the amorphous content of materials, especially when the amorphous content is a small percentage of the total mass. Whilst there has been success in studies on single materials, there has been little work showing how quantification of the amorphous content of one material can be undertaken in the presence of another. In this work isothermal microcalorimetry was used to measure the content of amorphous trehalose following mixing with crystalline lactose. Gravimetric water sorption studies revealed that trehalose did not form a complete dihydrate when exposed to 75% RH, presumably due to the rapid crystallisation of the outer regions of the particles. At 53% RH, the gravimetric studies showed dihydrate formation. The calorimetry data revealed that the crystallisation response was directly related to the mass of amorphous material in the mixture and was not affected by the mass of non-crystallising sample. It was shown that as long as there was a minimum mass of amorphous material (in this case 4 mg), it was possible to measure a crystallisation response with sufficient accuracy to allow quantification. Lower masses of amorphous content allowed detection, but less accurate quantification, as the response was superimposed on the initial calorimetric heat flow response. It was also found that the response at 53% RH in the TAM was less accurate due to the low peak height and long duration (compared to that seen at 75% RH). It can be concluded that the TAM method is well suited to both detection and quantification of amorphous content when there is one amorphous sample mixed with another (and thus presumably more than one) non-crystallising material.