Study of an Unusual Advanced Glycation End-Product (AGE) Derived from Glyoxal Using Mass Spectrometry

Glycation is a post-translational modification (PTM) that affects the physiological properties of peptides and proteins. In particular, during hyperglycaemia, glycation by α-dicarbonyl compounds generate α-dicarbonyl-derived glycation products also called α-dicarbonyl-derived advanced glycation end products. Glycation by the α-dicarbonyl compound known as glyoxal was studied in model peptides by MS/MS using a Fourier transform ion cyclotron resonance mass spectrometer. An unusual type of glyoxal-derived AGE with a mass addition of 21.98436 Da is reported in peptides containing combinations of two arginine-two lysine, and one arginine-three lysine amino acid residues. Electron capture dissociation and collisionally activated dissociation results supported that the unusual glyoxal-derived AGE is formed at the guanidino group of arginine, and a possible structure is proposed to illustrate the 21.9843 Da mass addition.

Comparison of different extraction methods for simultaneous determination of B complex vitamins in nutritional yeast using LC/MS-TOF and stable isotope dilution assay

The application of LC/MS-TOF method combined with stable isotope dilution assay was studied for determination of thiamine, riboflavin, nicotinamide, nicotinic acid, pantothenic acid, pyridoxal, and pyridoxine in food. Nutritional yeast powder was used as a model food matrix. Acid extraction was compared with various enzymatic treatments in ammonium formate buffer to find a suitable method for the conversion of more complex vitamers into the same forms as the used isotope-labeled internal standards. The enzyme preparations α-amylase, takadiastase, β-glucosidase, and acid phosphatase were all able to liberate thiamine and riboflavin. The diastatic enzyme preparations α-amylase and takadiastase also expressed proteolytic side activities resulting in the formation of small peptides which interfered with the mass spectra of thiamine and riboflavin. Liberation of nicotinamide and pantothenic acid from NAD⁺ and CoA, respectively, could not be achieved with any of the studied enzyme preparations. Hydrochloric acid extraction at 121 °C for 30 min was found to be destructive to pantothenic acid, but increased the liberation of pyridoxal.

Total synchronous fluorescence spectroscopic data modeled with first- and second-order algorithms for the determination of doxorubicin in human plasma

In this work, we present the development of a method for the determination of doxorubicin in plasma samples in the presence of an unexpected component (riboflavin) by using total synchronous fluorescence spectroscopic data matrices. To the best of our knowledge, this is the first time that the second-order advantage is obtained with this kind of data. Two strategies including unfolding the data and: (a) processing with multivariate curve resolution coupled to alternating least-squares as first-order data or (b) processing with unfolded partial least-squares and exploiting the second-order advantage by the residual bilinearization procedure were considered. The calibration set was built with human plasma samples spiked with doxorubicin, while the validation set was prepared with human plasma samples spiked with both doxorubicin and riboflavin, a drug whose spectrum highly overlaps with the one corresponding to doxorubicin. Both methodologies reached good indicators of accuracy: recoveries of ca. 100?±?8 % and REP of ca. 5 %; and precision: coefficient of variations between 7 and 9 %.

Capacitive sensing of microcystin variants of Microcystis aeruginosa using a gold immunoelectrode modified with antibodies,gold nanoparticles and polytyramine

We report on the application of an automated and easy-to-use device to directly measure the immunoreactions between adda-specific monoclonal antibodies and microcystins. The antibodies were immobilized on a gold electrode whose surface was modified first with polytyramine and then with gold nanoparticles. The immunoreaction leads to a change in the capacitance of the system. Under optimum conditions, the sensor is capable of performing stable regeneration-assay cycles and has a low detection limit at a concentration of 0.01 pM level of microcystin-leucine-arginine (MC-LR). The surface of the biosensor can be regenerated with pH 2.5 glycine buffer which dissociates the antibody-antigen complex. The biosensor was used to monitor the production of microcystins during batch cultivation of Microcystis aeruginosa (isolated from ponds in Botswana). Liquid chromatography coupled to MS/MS detection was used to identify three variants, viz. MC-LR (995.6 Da), DmMC-LR (981.2 Da) and MC-LA (910.5 Da).

Quantification of Competing H3PO4 Versus HPO3 + H2O Neutral Losses from Regioselective 18O-Labeled Phosphopeptides

Abundant neutral losses of 98 Da are often observed upon ion trap CID-MS/MS of protonated phosphopeptide ions. Two competing fragmentation pathways are involved in this process, namely, the direct loss of H₃PO₄ from the phosphorylated residue and the combined losses of HPO₃ and H₂O from the phosphorylation site and from an additional site within the peptide, respectively. These competing pathways produce product ions with different structures but the same m/z values, potentially limiting the utility of CID-MS³ for phosphorylation site localization. To quantify the relative contributions of these pathways and to determine the conditions under which each pathway predominates, we have examined the ion trap CID-MS/MS fragmentation of a series of regioselective ¹⁸O-phosphate ester labeled phosphopeptides prepared using novel solution-phase amino acid synthesis and solid-phase peptide synthesis methodologies. By comparing the intensity of the –100 Da (–H₃PO₃ ¹⁸O) versus –98 Da (–[HPO₃ + H₂O]) neutral loss product ions formed upon MS/MS, quantification of the two pathways was achieved. Factors that affect the extent of formation of the competing neutral losses were investigated, with the combined loss pathway predominantly occurring under conditions of limited proton mobility, and with increased combined losses observed for phosphothreonine compared with phosphoserine-containing peptides. The combined loss pathway was found to be less dominant under ion activation conditions associated with HCD-MS/MS. Finally, the contribution of carboxylic acid functional groups and backbone amide bonds to the water loss in the combined loss fragmentation pathway was determined via methyl esterification and by examination of a phosphopeptide lacking side-chain hydroxyl groups.

Synergistic extraction of some divalent metal cations into nitrobenzene by using strontium dicarbollylcobaltate and electroneutral macrocyclic lactam receptor

From extraction experiments and $ \gamma $ -activity measurements, the exchange extraction constants corresponding to the general equilibrium M²⁺(aq) + 1·Sr²⁺(nb) $ \Leftrightarrow $ 1·M²⁺(nb) + Sr²⁺(aq) taking place in the two-phase water–nitrobenzene system (M²⁺ = Mg²⁺, Ca²⁺, Ba²⁺, Pb²⁺, Cu²⁺, Zn²⁺, Cd²⁺, $ {\hbox{UO}}_{2}^{2 + } $ , Mn²⁺, Co²⁺, Ni²⁺; 1 = macrocyclic lactam receptor–see Scheme 1; aq = aqueous phase, nb = nitrobenzene phase) were evaluated. Moreover, the stability constants of the 1·M²⁺ complexes in nitrobenzene saturated with water were calculated; they were found to increase in the following cation order: Mg²⁺ < Co²⁺ < Cu²⁺, Mn²⁺, Ni²⁺ < Cd²⁺ < Ca²⁺ < Ba²⁺, Zn²⁺ < Pb²⁺ <  $ {\hbox{UO}}_{2}^{2 + } $ .

Laser-Induced Hydrogen Radical Removal in UV MALDI-MS Allows for the Differentiation of Flavonoid Monoglycoside Isomers

Negative-ion matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectra and tandem mass spectra of flavonoid mono-O-glycosides showed the irregular signals that were 1 and/or 2 Da smaller than the parent deprotonated molecules ([M – H]^–) and the sugar-unit lost fragment ions ([M – Sugar – H]^–). The 1 and/or 2 Da mass shifts are generated with the removing of a neutral hydrogen radical (H*), and/or with the homolytic cleavage of the glycosidic bond, such as [M – H* – H]^–, [M – Sugar – H* – H]^–, and [M – Sugar – 2H* – H]^–. It was revealed that the hydrogen radical removes from the phenolic hydroxy groups on the flavonoids, not from the sugar moiety, because the flavonoid backbones themselves absorb the laser. The glycosyl positions depend on the extent of the hydrogen radical removals and that of the homolytic cleavage of the glycosidic bonds. Flavonoid mono-glycoside isomers were distinguished according to their TOF MS and tandem mass spectra.

Evidence for Sequence Scrambling in Collision-Induced Dissociation of y-Type Fragment Ions

Sequence scrambling from y-type fragment ions has not been previously reported. In a study designed to probe structural variations among b-type fragment ions, it was noted that y fragment ions might also yield sequence-scrambled ions. In this study, we examined the possibility and extent of sequence-scrambled fragment ion generation from collision-induced dissociation (CID) of y-type ions from four peptides (all containing basic residues near the C-terminus) including: AAAAHAA-NH₂ (where “A” denotes carbon thirteen (¹³C₁) isotope on the alanine carbonyl group), des-acetylated-α-melanocyte (SYSMEHFRWGKPV-NH₂), angiotensin II antipeptide (EGVYVHPV), and glu-fibrinopeptide b (EGVNDNEEGFFSAR). We investigated fragmentation patterns of 32 y-type fragment ions, including y fragment ions with different charge states (+1 to +3) and sizes (3 to 12 amino acids). Sequence-scrambled fragment ions were observed from ~50 % (16 out of 32) of the studied y-type ions. However, observed sequence-scrambled ions had low relative intensities from ~0.1 % to a maximum of ~12 %. We present and discuss potential mechanisms for generation of sequence-scrambled fragment ions. To the best of our knowledge, results on y fragment dissociation presented here provide the first experimental evidence for generation of sequence-scrambled fragments from CID of y ions through intermediate cyclic “b-type” ions.

Solvent extraction of europium trifluoromethanesulfonate into nitrobenzene by using three electroneutral receptors

From extraction experiments and $ \gamma $ -activity measurements, the extraction constants corresponding to the general equilibrium Eu³⁺(aq) + 3 A^?(aq) + L(nb) $ \Leftrightarrow $ EuL³⁺(nb) + 3A^?(nb) taking place in the two-phase water–nitrobenzene system ( $ {\text{A}}^{ - } = {\text{CF}}_{ 3} {\text{SO}}_{3}^{ - } $ ; L = electroneutral receptors denoted by 1, 2, and 3 – see Scheme 1; aq = aqueous phase, nb = nitrobenzene phase) were evaluated. Further, the stability constants of the EuL³⁺ complexes in nitrobenzene saturated with water were calculated; they were found to increase in the series of 3 < 2 < 1.

Protected Amine Labels: A Versatile Molecular Scaffold for Multiplexed Nominal Mass and Sub-Da Isotopologue Quantitative Proteomic Reagents

We assemble a versatile molecular scaffold from simple building blocks to create binary and multiplexed stable isotope reagents for quantitative mass spectrometry. Termed Protected Amine Labels (PAL), these reagents offer multiple analytical figures of merit including, (1) robust targeting of peptide N-termini and lysyl side chains, (2) optimal mass spectrometry ionization efficiency through regeneration of primary amines on labeled peptides, (3) an amino acid-based mass tag that incorporates heavy isotopes of carbon, nitrogen, and oxygen to ensure matched physicochemical and MS/MS fragmentation behavior among labeled peptides, and (4) a molecularly efficient architecture, in which the majority of hetero-atom centers can be used to synthesize a variety of nominal mass and sub-Da isotopologue stable isotope reagents. We demonstrate the performance of these reagents in well-established strategies whereby up to four channels of peptide isotopomers, each separated by 4 Da, are quantified in MS-level scans with accuracies comparable to current commercial reagents. In addition, we utilize the PAL scaffold to create isotopologue reagents in which labeled peptide analogs differ in mass based on the binding energy in carbon and nitrogen nuclei, thereby allowing quantification based on MS or MS/MS spectra. We demonstrate accurate quantification for reagents that support 6-plex labeling and propose extension of this scheme to 9-channels based on a similar PAL scaffold. Finally, we provide exemplar data that extend the application of isotopologe-based quantification reagents to medium resolution, quadrupole time-of-flight mass spectrometers.

Advantages of Monodisperse and Chemically Robust “SpheriCal” Polyester Dendrimers as a “Universal” MS Calibrant

The utilization of dendrimer calibrants as an alternative to peptides and proteins for high mass calibration is explored. These synthetic macromolecules exhibited a number of attractive advantages, including exceptional shelf-lives, broad compatibility with a wide range of matrices and solvents, and evenly spaced calibration masses across the mass range examined, 700–30,000 u. The exceptional purity of these dendrimers and the technical simplicity of this calibration platform validate their broad relevance for high molecular weight mass spectrometry.

Preparation of PMMA/acid-modified chitosan core-shell nanoparticles and their potential as gene carriers

The core-shell nanoparticles consisting of poly(methyl methacrylate) (PMMA) cores surrounded by various acid-modified chitosan shells were synthesized using a surfactant-free emulsion copolymerization, induced by a tert-butylhydroperoxide (TBHP) solution. Methyl methacrylate (MMA) was grafted onto four acid-modified chitosans (hydrochloric, lactic, aspartic, and glutamic acids) with MMA conversions up to 64%. The prepared nanoparticles had diameter ranging from 100 to 300 nm characterized by atomic force microscopy and displayed highly positive surface charges up to +77 mV. Transmission electron microscopic images clearly revealed well-defined core-shell morphology of the nanoparticles where PMMA cores were coated with acid-modified chitosan shells. The effect of acid-modified chitosans on particle size, intensity of surface charge, morphology, and thermal stability were determined systematically. The plasmid DNA/nanoparticles complexes were investigated with ζ-potential measurement. The results suggested that these nanoparticles can effectively complex with plasmid DNAs via electrostatic interaction and could be used as gene carriers.

On-Plate Self-Desalting and Matrix-Free LDI MS Analysis of Peptides With a Surface Patterned Sample Support

A hydrophobic-hydrophilic-hydrophobic pattern has been produced on the surface of a silicon substrate for selective enrichment, self-desalting, and matrix-free analysis of peptides in a single step. Upon sample application, the sample solution is first confined in a small area by a hydrophobic F-SAM outer area, after which salt contaminants and peptides are selectively enriched in the hydrophilic and hydrophobic areas, respectively. Simultaneously, matrix background noise is significantly reduced or eliminated because of immobilization of matrix molecules. As a result, the detection sensitivity is enhanced 20-fold compared with that obtained using the usual MALDI plate, and interference-free detection is achieved in the low m/z range. In addition, peptide ions can be identified unambiguously in the presence of NH₄HCO₃ (100 mM), urea (1 M), and NaCl (1 M). When the device was applied to the analysis of BSA digests, the peptide recovery and protein identification confidence were greatly improved.

Mass spectrometry characterisation of fatty acids from metabolically engineered soybean seeds

Improving the quality and performance of soybean oil as biodiesel depends on the chemical composition of its fatty acids and requires an increase in monounsaturated acids and a reduction in polyunsaturated acids. Despite its current use as a source of biofuel, soybean oil contains an average of 25 % oleic acid and 13 % palmitic acid, which negatively impacts its oxidative stability and freezing point, causing a high rate of nitrogen oxide emission. Gas chromatography and ion mobility mass spectrometry were conducted on soybean fatty acids from metabolically engineered seed extracts to determine the nature of the structural oleic and palmitic acids. The soybean genes FAD2-1 and FatB were placed under the control of the 35SCaMV constitutive promoter, introduced to soybean embryonic axes by particle bombardment and down-regulated using RNA interference technology. Results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 94.58 %) and a reduction in palmitic acid (to <3 %) in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and non-transgenic oil extracts.

Metabolic pathway elucidation towards time- and dose-dependent electrophoretic screening of stable oxidative phenolic compounds

This study demonstrates an untested link between model phenolic compounds and the formation/electrophoretic separation of stable urinary metabolites. Sterically encumbered carbonyl groups were examined, and mass determination was used to confirm the presence and stability of two oxidative metabolites of pentachlorophenol: tetrachloro-1,2-benzoquinone and tetrachloro-1,4-dihydroquinone. Subsequently, baseline resolved separation of pentachlorophenol and the two oxidative metabolites was demonstrated under the following conditions: 75 mM sodium tetraborate buffer (pH?=?8.5) with 5 % methanol and 50 mM SDS, +10.0 kV running voltage, injection time?=?5.0 s, effective capillary length?=?55 cm, and run temperature?=?20 °C. Results not only provide key metabolic inferences for pentachlorophenol, they also exhibit improvements in the ability to separate and detect changes in urinary metabolites in response to phenolic-related exposure.

Disposable amperometric biosensor for the determination of tyramine using plasma amino oxidase

We have developed screen–printed carbon electrodes for the determination of tyramine (Tyr) via plasma amine oxidase. The enzyme was immobilized on the carbon working electrode by cross–linking it with bovine serum albumin using glutaraldehyde. The employment of the mediator hydroxymethylferrocene lowers the working potential to +260 mV (vs. a screen–printed Ag/AgCl reference electrode). The effects of pH, potential and mediator concentration were optimized and resulted in reproducibility and repeatability values of 8.6 % and 8.7 %, respectively. Response is linear in the range from 2 to 164 μM, and the limit of detection is 2.0?±?0.18 μM. The effects of potentially interfering biogenic amines such as putrescine, cadaverine, histamine, spermine, spermidine and tryptamine were also evaluated. The biosensor was successfully applied to the determination of Tyr in cheese.

Analysis of Biomolecules by Atmospheric Pressure Visible-Wavelength MALDI-Ion Trap-MS in Transmission Geometry

We report the development of a new AP visible-wavelength MALDI-ion trap-MS instrument with significantly improved performance over our previously reported system (Int. J. Mass Spectrom. 315, 66–73 (2012)). A Nd:YAG pulsed laser emitting light at 532 nm was used to desorb and ionize oligosaccharides and peptides in transmission geometry through a glass slide. Limits of detection (LODs) achieved in MS mode correspond to picomole quantities of oligosaccharides and femtomole quantities of peptides. Tandem MS (MS/MS) experiments enabled identification of enzymatically digested proteins and oligosaccharides by comparison of MS/MS spectra with data found in protein and glycan databases. Moreover, the softness of ionization, LODs, and fragmentation spectra of biomolecules by AP visible-wavelength MALDI-MS were compared to those obtained by AP UV MALDI-MS using a Nd:YAG laser emitting light at 355 nm. AP visible-wavelength MALDI appears to be a softer ionization technique then AP UV MALDI for the analysis of sulfated peptides, while visible-wavelength MALDI-MS, MS/MS, and MS/MS/MS spectra of other biomolecules analyzed were mostly similar to those obtained by AP UV MALDI-MS. Therefore, the methodology presented will be useful for MS and MSⁿ analyses of biomolecules at atmospheric pressure. Additionally, the AP visible-wavelength MALDI developed can be readily used for soft ionization of analytes on various mass spectrometers.

Desorption Mass Spectrometry for Nonvolatile Compounds Using an Ultrasonic Cutter

In this work, desorption of nonvolatile analytes induced by friction was studied. The nonvolatile compounds deposited on the perfluoroalkoxy substrate were gently touched by an ultrasonic cutter oscillating with a frequency of 40 kHz. The desorbed molecules were ionized by a dielectric barrier discharge (DBD) ion source. Efficient desorption of samples such as drugs, pharmaceuticals, amino acids, and explosives was observed. The limits of detection for these compounds were about 1 ng. Many compounds were detected in their protonated forms without undergoing significant fragmentation. When the DBD was off, no ions for the neutral samples could be detected, meaning that only desorption along with little ionization took place by the present technique.

On-line species-unspecific isotope dilution analysis in the picomolar range reveals the time- and species-depending mercury uptake in human astrocytes

In order to reveal the time-depending mercury species uptake by human astrocytes, a novel approach for total mercury analysis is presented, which uses an accelerated sample introduction system combined on-line with an inductively coupled plasma mass spectrometer equipped with a collision/reaction cell. Human astrocyte samples were incubated with inorganic mercury (HgCl₂), methylmercury chloride (MeHgCl), and thimerosal. After 1-h incubation with Hg²⁺, cellular concentrations of 3 μM were obtained, whereas for organic species, concentrations of 14–18 μM could be found. After 24 h, a cellular accumulation factor of 0.3 was observed for the cells incubated with Hg²⁺, whereas the organic species both showed values of about 5. Due to the obtained steady-state signals, reliable results with relative standard deviations of well below 5 % and limits of detection in the concentration range of 1 ng L^?1 were obtained using external calibration and species-unspecific isotope dilution analysis approaches. The results were further validated using atomic fluorescence spectrometry.