Gas Phase Ion Chemistry to Determine Isoaspartate in a Peptide Backbone.

Proof of concept evidence is presented for a new method for the determination of isoaspartate, an important post-translational modification. Chemical derivatization is performed using common reagents for the modification of carboxylic acids and shown to yield suitable diagnostic information with regard to isomerization at the aspartate residue. The diagnostic gas phase chemistry is probed by collision-induced dissociation mass spectrometry, on the timescale of the MS experiment and semi-quantitative calibration of the percentage of isoaspartate in a peptide sample is demonstrated. Graphical Abstract ᅟ.

Chemical profiling of cerebrospinal fluid by multiple reaction monitoring mass spectrometry.

We report an accelerated biomarker discovery workflow and results of sample screening by mass spectrometry based on multiple reaction monitoring (MRM). This methodology shows promising initial results for the currently unsolved challenge of Parkinson's disease (PD) laboratory diagnosis by biomarker screening. Small molecules present in cerebrospinal fluid (CSF) at low parts per million levels are monitored using specific transitions connecting ion pairs. A set of such transitions constitutes a multidimensional chemical profile used to distinguish and characterize different CSF samples using multivariate statistical methods.

Molecular labels for analysis of amines and diols by spray based ionization-mass spectrometry.

A family of imidazolium and pyridinium salts was synthesized for the purpose of labeling amines and 1,2-diols for highly sensitivite analysis by mass spectrometry. The chosen mass labels are shown to serve as effective reporters when bound to particles functionalized with amines or 1,2-diols and the binding is reversible. The straightforward synthetic route allows analogous internal standards to be generated quickly. Solvents amenable to electrospray ionization facilitate the rapid liberation of imidazolium and pyridinium mass labels from particles by hydrolysis in aqueous acid, while the acetal bond remains stable in anhydrous or buffered aqueous solution. Detection of the labels is demonstrated at 1 nM and quantitation of mass labels released from particles is also demonstrated.

Lipid dynamics in zebrafish embryonic development observed by DESI-MS imaging and nanoelectrospray-MS.

The zebrafish Danio rerio is a model vertebrate organism for understanding biological mechanisms. Recent studies have explored using zebrafish as a model for lipid-related diseases, for in vivo fish bioassays, and for embryonic toxicity experiments. Mass spectrometry (MS) and MS imaging are established tools for lipid profiling and spatial mapping of biomolecules and offer rapid, sensitive, and simple analytical protocols for zebrafish analysis. When ambient ionization techniques are used, ions are generated in native environmental conditions, requiring neither sample preparation nor separation of molecules prior to MS. We used two direct MS techniques to describe the dynamics of the lipid profile during zebrafish embryonic development from 0 to 96 hours post-fertilization and to explore these analytical approaches as molecular diagnostic assays. Desorption electrospray ionization (DESI) MS imaging followed by nanoelectrospray (nESI) MS and tandem MS (MS/MS) were used in positive and negative ion modes, allowing the detection of a large variety of phosphatidylglycerols, phosphatidylcholines, phosphatidylinositols, free fatty acids, triacylglycerols, ubiquinone, squalene, and other lipids, and revealed information on the spatial distributions of lipids within the embryo and on lipid molecular structure. Differences were observed in the relative ion abundances of free fatty acids, triacylglycerols, and ubiquinone - essentially localized to the yolk - across developmental stages, whereas no relevant differences were found in the distribution of complex membrane glycerophospholipids, indicating conserved lipid constitution. Embryos exposed to trichloroethylene for 72 hours exhibited an altered lipid profile, indicating the potential utility of this technique for testing the effects of environmental contaminants.

Biogenic aldehyde determination by reactive paper spray ionization mass spectrometry.

Ionization of aliphatic and aromatic aldehydes is improved by performing simultaneous chemical derivatization using 4-aminophenol to produce charged iminium ions during paper spray ionization. Accelerated reactions occur in the microdroplets generated during the paper spray ionization event for the tested aldehydes (formaldehyde, n-pentanaldehyde, n-nonanaldehyde, n-decanaldehyde, n-dodecanaldehyde, benzaldehyde, m-anisaldehyde, and p-hydroxybenzaldehyde). Tandem mass spectrometric analysis of the iminium ions using collision-induced dissociation demonstrated that straight chain aldehydes give a characteristic fragment at m/z 122 (shown to correspond to protonated 4-(methyleneamino)phenol), while the aromatic aldehyde iminium ions fragment to give a characteristic product ion at m/z 120. These features allow straightforward identification of linear and aromatic aldehydes. Quantitative analysis of n-nonaldehyde using a benchtop mass spectrometer demonstrated a linear response over 3 orders of magnitude from 2.5 ng to 5 µg of aldehyde loaded on the filter paper emitter. The limit of detection was determined to be 2.2 ng for this aldehyde. The method had a precision of 22%, relative standard deviation. The experiment was also implemented using a portable ion trap mass spectrometer.

Differentiation of prostate cancer from normal tissue in radical prostatectomy specimens by desorption electrospray ionization and touch spray ionization mass spectrometry.

Radical prostatectomy is a common treatment option for prostate cancer before it has spread beyond the prostate. Examination for surgical margins is performed post-operatively with positive margins reported to occur in 6.5-32% of cases. Rapid identification of cancerous tissue during surgery could improve surgical resection. Desorption electrospray ionization (DESI) is an ambient ionization method which produces mass spectra dominated by lipid signals directly from prostate tissue. With the use of multivariate statistics, these mass spectra can be used to differentiate cancerous and normal tissue. The method was applied to 100 samples from 12 human patients to create a training set of MS data. The quality of the discrimination achieved was evaluated using principal component analysis - linear discriminant analysis (PCA-LDA) and confirmed by histopathology. Cross validation (PCA-LDA) showed >95% accuracy. An even faster and more convenient method, touch spray (TS) mass spectrometry, not previously tested to differentiate diseased tissue, was also evaluated by building a similar MS data base characteristic of tumor and normal tissue. An independent set of 70 non-targeted biopsies from six patients was then used to record lipid profile data resulting in 110 data points for an evaluation dataset for TS-MS. This method gave prediction success rates measured against histopathology of 93%. These results suggest that DESI and TS could be useful in differentiating tumor and normal prostate tissue at surgical margins and that these methods should be evaluated intra-operatively.

Quadrupole ion traps and trap arrays: geometry, material, scale, performance.

Quadrupole ion traps are reviewed, emphasizing recent developments, especially the investigation of new geometries, guided by multiple particle simulations such as the ITSIM program. These geometries include linear ion traps (LITs) and the simplified rectilinear ion trap (RIT). Various methods of fabrication are described, including the use of rapid prototyping apparatus (RPA), in which 3D objects are generated through point-by-point laser polymerization. Fabrication in silicon using multilayer semi-conductor fabrication techniques has been used to construct arrays of micro-traps. The performance of instruments containing individual traps as well as arrays of traps of various sizes and geometries is reviewed. Two types of array are differentiated. In the first type, trap arrays constitute fully multiplexed mass spectrometers in which multiple samples are examined using multiple sources, analyzers and detectors, to achieve high throughput analysis. In the second, an array of individual traps acts collectively as a composite trap to increase trapping capacity and performance for a single sample. Much progress has been made in building miniaturized mass spectrometers; a specific example is a 10 kg hand-held tandem mass spectrometer based on the RIT mass analyzer. The performance of this instrument in air and water analysis, using membrane sampling, is described.

Forensic analysis of inks by imaging desorption electrospray ionization (DESI) mass spectrometry.

Desorption electrospray ionization mass spectrometry (DESI-MS) is employed in the forensic analysis of documents. Blue ballpoint pen inks applied to ordinary writing paper are examined under ambient conditions without any prior sample preparation. When coupled to an automated moving stage, two-dimensional molecular images are generated. Proof-of-principle experiments include characterization of a simulated forged number and examination of older written records. This application of DESI has advantages over extractive techniques in terms of speed and sample preservation. The effects of the desorbing solvent composition, in this case a mixture of methanol and water, and of flow rate, are evaluated. Results suggest that the solubility of the analyte (dyes Basic Blue 7, Basic Violet 3 and Solvent Blue 26) plays an important role in desorption from the paper surface.

Chiral purity assay for Flindokalner using tandem mass spectrometry: method development, validation, and benchmarking.

The present work demonstrates the application and validation of a mass spectrometry method for quantitative chiral purity determination. The particular compound analyzed is Flindokalner, a Bristol-Myers Squibb drug candidate for post-stroke neuroprotection. Chiral quantification of Flindokalner was achieved using tandem mass spectrometry (MS/MS) and the kinetic method, a gas phase method used for thermochemical and chiral determinations. The MS/MS method was validated and benchmarked against two separate chromatographic techniques, chiral high performance liquid chromatography with ultra-violet detection (LC/UV) and achiral high performance liquid chromatography with circular dichroism detection (LC/CD). The chiral purity determination of Flindokalner using MS/MS proved to be rapid (3 min run time for each sample) and to have accuracy and precision comparable to the chiral LC/UV and achiral LC/CD methods. This method represents an alternative to commonly used chromatographic techniques as a means of chiral purity determination and is particularly useful in rapid screening experiments.

Kinetic method for the simultaneous chiral analysis of different amino acids in mixtures.

The kinetic method has been extended to enantiomeric excess (ee) determinations on amino acids present in mixtures. Singly charged trimeric clusters [Cu(II)(ref*)(2)(A(m)) - H](+) are readily generated by electrospraying solutions containing Cu(II), a chiral reference ligand (ref*), and the amino acids (analytes A(m), m = 1-3). A trimeric cluster ion for each amino acid is individually mass-selected and then collisionally activated to cause dissociation by competitive loss of either the reference ligand or the analyte. For each analyte in the mixture, as shown from separate experiments, the logarithm of the ratio of the fragment abundances for the complex containing one enantiomer of this analyte expressed relative to that for the fragments of the corresponding complex containing the other enantiomer is linearly related to the enantiomeric composition of the amino acid. Formation and dissociation of each trimeric complex ion are shown to occur independently of the presence of other analytes. Chiral selectivity appears to be an intrinsic property and the chiral selectivity R(chiral(m)) measured from the mixture of analytes is equal to R(chiral) measured for the pure analyte. The sensitive nature of the methodology and the linear relationship between the logarithm of the fragment ion abundance ratio and the optical purity, characteristic of the kinetic method, allow the determination of chiral impurities of less than 2% ee in individual compounds present in mixtures by simply recording the ratios of fragment ion abundances in a tandem mass spectrum.

Ligand and metal-ion effects in metal-ion clusters used for chiral analysis of alpha-hydroxy acids by the kinetic method.

Chiral recognition of alpha-hydroxy acids has been achieved, and mixtures of enantiomers have been quantified in the gas phase, by using the kinetics of competitive unimolecular dissociation of singly-charged transition metal ion-bound trimeric complexes, [M(II)(A)(ref*)(2)-H](+) (M(II)=divalent transition metal ion; A=alpha-hydroxy acid; ref*=chiral reference ligand), to form the dimeric complexes [M(II)(A)(ref*)-H](+) and [M(II)(ref*)(2)-H](+). Chiral selectivity, the ratio of these two fragment ion abundances for the complex containing the analyte in one enantiomeric form expressed relative to that for the fragments of the corresponding complex containing the other enantiomer, ranges from 0.65 to 7.32. Chiral differentiation is highly dependent on the choice of chiral reference compound and central metal ion. The different coordination geometry of complexes resulting from the different d-orbital electronic configurations of these transition metal ions plays a role in chiral discrimination. Of all the transition metal ions examined chiral recognition is lowest for Cu(II), because of large distortion of the coordination complexes, and hence weak metal-ligand interactions and small stereochemical effects. It seems that two independent pi-cation interactions occur when N-acetyl-substituted aromatic amino acids used as the reference ligands and this accounts for improved chiral discrimination. If both metal-ligand and ligand-ligand interactions are optimized, large chiral selectivity is achieved. The sensitive nature of the methodology and the linear relationship between the logarithm of the fragment ion abundance ratio and the optical purity, which are intrinsic to the kinetic method, enable mixtures to be analyzed for small enantiomeric excess ( ee) by simply recording the ratios of fragment ion abundances in a tandem mass spectrum.

Quantitative chiral analysis of sugars by electrospray ionization tandem mass spectrometry using modified amino acids as chiral reference compounds.

Rapid quantitative enantiomeric analysis of mannose, glucose, galactose, and ribose is achieved using electrospray ionization and cluster ion dissociation with data analysis by the kinetic method. Several modified amino acids (N-Ac-L-Phe, N-benzoyl-L-Phe, N-t-Boc-L-Phe, N-Ac-L-Pro, N-t-Boc-L-Pro, N-Fmoc-L-Pro, N-Ac-L-Tyr, O-Me-L-Tyr) and four transition divalent metal cations (Co2+, Cu2+, Ni2+, and Zn2+) were tested to select the best system for chiral recognition and quantitation of each sugar. Quantitative determinations of the enantiomeric compositions of sugar solutions were achieved using either multiple- or two-point calibration curves; differences between the actual and experimental values were <2% enantiomeric excess (ee).

External interface for trap-and-release membrane introduction mass spectrometry applied to the detection of inorganic chloramines and chlorobenzenes in water.

Construction and evaluation of an external configuration trap-and-release membrane introduction system for mass spectrometry is described. This novel interface allows independent control of the temperature of the membrane and eliminates the dependence of membrane heating efficiency on its position in the ion source. The external trap-and-release MIMS configuration is successfully applied to detection of inorganic chloramines and chlorobenzenes. The method is shown to give temporal resolution of volatile vs. semi-volatile compounds, which increases its sensitivity for semi-volatiles in the presence of volatiles and provides an additional selectivity parameter. Further selectivity is provided by tandem mass spectrometry.

Direct analysis of semivolatile organic compounds in air by atmospheric pressure chemical ionization mass spectrometry.

Atmospheric pressure chemical ionization is employed for direct air analysis, without ion source modification, by using the sheath gas as the sample transport agent. A simple modification of the sheath gas inlet line allows introduction of gaseous samples into a commercial atmospheric pressure chemical ionization source. Optimization and testing of this novel air sampling method are described and detection of semivolatile compounds is shown. The analytical performance of the technique is established with methyl salicylate, including a limit of quantification of 100 pptr, a limit of detection of 50 pptr, a linear response from 100 pptr to 20 ppb, and rise and fall times of 12 and 20 s, respectively. Using reagent ion monitoring, it is shown that the protonated methanol dimer is the principal CI reagent ion leading to protonated dimethyl methylphosphonate, while the monomer is mainly responsible for protonating methyl salicylate. Since the formation of the CI reagent (methanol clusters) can be controlled by simple variation of experimental parameters, the selectivity of the method can be easily adjusted to suit the targeted analyte. Performance is found to be independent of the choice of air or nitrogen as the sheath gas (and thus as the sample matrix) and this, together with the sensitivity and speed of the technique, make it promising for field studies.

Rapid enantiomeric quantification of an antiviral nucleoside agent (D,L-FMAU, 2'-fluoro-5-methyl-beta,D,L-arabinofurano-syluracil) by mass spectrometry.

A novel mass spectrometric method is applied to rapid, accurate (<1%) quantification of chiral Clevudine (L-FMAU, 2'-fluoro-5-methyl-beta,L-arabinofuranosyluracil), a potent antiviral nucleoside agent against hepatitis B virus. Transition metal bound complex ions containing the chiral drug are generated by electrospray ionization mass spectrometry and subjected to collision-induced dissociation. The ratio of the two competitive dissociation rates is related to the enantiomeric composition of the drug mixture, allowing the determination of enantiomeric contamination in the drug.

Organic chloramine analysis and free chlorine quantification by electrospray and atmospheric pressure chemical ionization tandem mass spectrometry.

Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), together with tandem mass spectrometry (MSn), are used to study the mechanism of chlorination of amines and to develop a method for qualitative and quantitative determination of organic chloramines. Cyclohexylamine and 1,4-butanediamine (putrescine) are used as model compounds to investigate the mechanisms of the reactions between primary aliphatic amines and hypochlorous acid (aqueous Cl2). The chlorination products are identified and characterized by collision-induced dissociation (CID) and H/D exchange. Chlorination occurs by electrophilic addition of Cl+ and may be followed by HCl elimination, hydrolysis, or, in the case of diamines, amine elimination by intramolecular nucleophilic substitution. The relative rates of chlorination at amine and chloramine nitrogens are a function of pH and depend on the basicity of the amine. A novel method for active chlorine quantification using ESI or APCI mass spectrometry is suggested on the basis of the extent of chlorination of a sacrifical amine standard. This measurement has a limit of detection for N-chlorocyclohexylamine in the range of 0.1-10 microM, a linear dynamic range of 10(2)-10(3), and an accuracy of +/-10%, as determined for wastewater samples.