Selective detection of homocysteine by laser desorption/ionization mass spectrometry

This article describes the use of 2,3-naphthalenedicarboxaldehyde (NDA) as a selective probe for the determination of homocysteine (HCys) via fluorescence measurement and laser desorption/ionization mass spectrometry (LDI-MS). The derivatives of three aminothiols-HCys, glutathione (GSH), and gamma-glutamylcysteine (gamma-Glu-Cys)-with NDA under alkaline conditions possess different fluorescence emission characteristics, which allow us to identify them from amines, amino acids, and thiols. By selecting appropriate pH and excitation wavelengths, the limits of detection (LODs) at a signal-to-noise ratio of 3 were 5.2, 1.4 and 16 nM for HCys, GSH and gamma-Glu-Cys, respectively. Additionally, strong UV absorption of the NDA-HCys derivative was further observed at 331 nm; it could be directly detected by LDI-MS with a 337-nm nitrogen laser. Selective detection of HCys has been achieved by conducting the LDI-MS of the NDA-HCys derivative, which was found at m/z 406.9. The lowest detectable concentration of the NDA-HCys derivative in this approach was 500 nM. Quantitative determination of HCys in urine samples was accomplished by LDI-MS. Also, a calibration curve was created from plasma samples spiked with standard HCys (20-100 microM). The experimental results suggest that our proposed methods have great potential in clinical diagnosis and metabolomics application.     

High resolution and tandem fourier-transform mass spectrometry with californium-252 plasma desorption

For ions formed by plasma desorption (PD) in a Fourier-transform mass spectrometer, high resolution measurements are demonstrated, such as 65,000 (FWHH) for the protonated molecular ion of gramicidin S (MW 1140.7). Resolution is substantially improved by delaying measurements until a significant ion concentration has built up in the cell, and by collisionally deactivating the orbital kinetic energy of the ions. This also makes the ions available for subsequent dissociation steps, so that tandem mass spectrometry can be demonstrated for PD ions. With this for larger ions, collisionally activated dissociation (CAD) is affected with> 85% efficiency. The CAD spectra of (M + Na)⁺ and of fragment ions from the PD of gramicidin S provide structurally useful information.     

Matrix-assisted laser desorption/ionization of high-mass molecules by Fourier-transform mass spectrometry.

Following the first demonstrations of high-mass analysis using time-of-flight matrix-assisted laser desorption/ionization (MALDI) techniques by Hillenkamp, Tanaka and their co-workers, there have been significant efforts in a number of laboratories to adapt the new methodology to Fourier-transform mass spectrometry (FTMS). The motivation for this research is obvious. Namely, it would be desirable to couple the unparalleled high mass resolution of FTMS with the extended mass range provided by MALDI, particularly for analysis of polymers and biomolecules. Unfortunately, prior to the present work, attempts to mate FTMS and MALDI have met with limited success. The highest mass matrix-assisted laser-desorption-FTMS result previously obtained appears to be the unpublished low resolution spectrum of bovine insulin recently reported by Russell and co-workers. We, Campana and co-workers, and Hettich and Buchanan have had some success with MALDI-FTMS of biomolecules with masses lower than 3000 Da, including melittin, a variety of lower mass peptides, and oligonucleotides with masses lower than 1800 Da. Furthermore, with the single exception of Campana's report of obtaining mass resolution of 5000 for the molecular ion of melittin, such spectra have not displayed high resolution. Here, we report successful development of MALDI-FTMS, demonstrated with spectra obtained from a variety of high-mass polymer and biomolecule samples, using 355 nm radiation from an excimer-pumped dye laser for desorption/ionization and sinapinic acid as matrix. Some of these spectra are of much higher mass resolution than is possible with current time-of flight mass spectrometers.     

Small-molecule analysis by surface-assisted laser desorption/ionization mass spectrometry

In order to meet the challenges facing modern chemistry, biology, and medicine, methods are required capable of performing rapid and reliable analysis of both individual compounds and complex mixtures at the molecular level. Matrix-assisted laser de-sorption/ionization mass spectrometry meets these requirements; however, some limitations complicate its application for the analysis of small molecules. Recently, small-molecule analysis has greatly progressed owing to development of surface-assisted laser desorption/ionization mass spectrometry involving approaches which combine the unique properties of nanostructured surface chemistry and morphology. This review examines such approaches and their specific application in small-molecule mass analysis.     

Oligonucleotide analysis by nanoparticle-assisted laser desorption/ionization mass spectrometry

We analyzed oligonucleotides by nanoparticle-assisted laser desorption/ionization (nano-PALDI) mass spectrometry (MS). To this end, we prepared several kinds of nanoparticles (Cr-, Fe-, Mn-, Co-based) and optimized the nano-PALDI MS method to analyze the oligonucleotides. Iron oxide nanoparticles with diammonium hydrogen citrate were found to serve as an effective ionization-assisting reagent in MS. The mass spectra showed both [M - H](-) and [M + xMe(2+)- H](-) (Me: transition metal) peaks. The number of metal-adducted ion signals depended on the length of the oligonucleotide. This phenomenon was only observed using bivalent metal core nanoparticles, not with any other valency metal core nanoparticles. Our pilot study demonstrated that iron oxide nanoparticles could easily ionize samples such as chemical drugs and peptides as well as oligonucleotides without the aid of an oligonucleotide-specific chemical matrix (e.g., 3-hydroxypicolinic acid) used in conventional MS methods. These results suggested that iron-based nanoparticles may serve as the assisting material of ionization for genes and other biomolecules.     

Matrix-assisted laser desorption of neutral organic molecules

Results of initial investigations of matrix-assisted laser desorption (LD) of neutral molecules are presented. The investigations were performed using gramicidine D as the test molecule, since both the wavelength-dependent LD beharioor and the ionization and fragmentation behaviour of the pure material had been studied previously. Comparisons between previously reported results for the pure compound and the matrix mixtures used in these studies are made. The results show that, whereas no signal could be observed from the pure sample material at non-resonant desorbing wavelengths, addition of a strongly absorbing matrix to the sample initiated LD of abundant intact neutral sample molecules. The results are discussed in the light of previous studies of the LD mechanism and support an explosive, mechanically driven model for the LD process.     

Determination of neutral oligosaccharides in vegetables by matrix-assisted laser desorption/ionization mass spectrometry

In this work, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS) was used for characterization of oligosaccharides in some vegetable samples (Jerusalem artichoke, red onion, glucose syrup from potatoes). The selection of suitable matrix has critical importance for quality of MALDI–TOF spectra. Therefore six selected matrices (2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, -cyano-4-hydroxycinnamic acid, 2-(4-hydroxyphenylazo)benzoic acid, 3-aminoquinoline and sinapinic acid) were tested. The optimization of experimental conditions was carried out for two model carbohydrates that are important in food chemistry—inulin and starch. The experiments were performed in both positive linear and reflectron mode. The signals of the standard samples in reflectron mode were weak and repeatability of the measurements was lower than in linear mode. 2,4,6-Trihydroxyacetophenone for inulin and 2,5-dihydroxybenzoic acid for starch were found as the best matrices. Therefore, the real samples were analyzed with these two matrices in linear mode. The distribution of oligosaccharides from Jerusalem artichoke showed the degree of polymerization (DP) of the oligosaccharides in the range from 2 to 25. Red onion contained the saccharides with DP from 1 to 14. Glucose syrup from potatoes had DP from 2 to 48. MALDI–TOF–MS was found more sensitive for detection of oligosaccharides than the chromatographic methods used for the some purpose.     

Surfactant-mediated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of small molecules

A variety of surfactants have been tested as matrix-ion suppressors for the analysis of small molecules by matrix-assisted laser desorption/ionization time-of flight mass spectrometry. Their addition to the common matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) greatly reduces the presence of matrix-related ions when added at the appropriate mole ratio of CHCA/surfactant, while still allowing the analyte signal to be observed. A range of cationic quaternary ammonium surfactants, as well as a neutral and anionic surfactant, was tested for the analysis of phenolics, phenolic acids, peptides and caffeine. It was found that the cationic surfactants, particularly cetyltrimethylammonium bromide (CTAB), were suitable for the analysis of acidic analytes. The anionic surfactant, sodium dodecyl sulfate, showed promise for peptide analysis. For trialanine, the detection limit was observed to be in the 100 femtomole range. The final matrix/surfactant mole ratio was a critical parameter for matrix ion suppression and resulting intensity of analyte signal. It was also found that the mass resolution of analytes was improved by 25-75%. Depth profiling of sample spots, by varying the number of laser shots, revealed that the surfactants tend to migrate toward the top of the droplet during crystallization, and that it is likely that the analyte is also enriched in this surface region. Here, higher analyte/surfactant concentration would reduce matrix-matrix interactions (known to be a source of matrix-derived ions).     

Multiphoton ionization detected by fourier-transform mass spectrometry

Ions produced by multiphoton ionization (MPI) of naphthalene, fluoranthene and triphenylene have been detected by Fourier-transform mass spectrometry (FT MS). Paret ions are produced very efficiently at 250 and 222 nm with pulse energies as low as 1 mJ. With FT MS a complete, high-resolution mass spectrum is obtained for each laser pulse.     

Analysis of cigarette smoke by laser desorption mass spectrometry

Laser desorption ionization Fourier transform ion cyclotron mass spectrometry (LDI–FTICRMS) has been successfully applied for the characterization of tobacco smokes. The versatily of analysis allows inorganic and organic tobacco smoke constituents to be identified. As a function of ion detection mode and laser irradiance experimental conditions, inorganic nitrate and sulfate as well as nicotine and associated nicotine, tobacco-specific nitrosamine, polycyclic aromatic hydrocarbon, polyoxygenated compounds are highlighted. The high mass resolution and the high mass accuracy attained by LDI–FTICRMS measurements allow the unambiguous assignment of detected species for m/z < 250 to be achieved.     

Surface analysis of sol-gel coatings on glass by secondary neutral mass spectrometry

Secondary neutral mass spectrometry (SNMS) has been used to profile coatings of the systems SiO₂ and 65SiO₂.20TiO₂.15ZrO₂ (STZ). The coatings have been deposited on float glass and heat insulating glass by dip coating from alkoxide solutions. The microporous gel coatings have been densified by heat treatment. The SBM method (separate bombardment mode) has been applied to characterize the systems and the HFM method (high frequency mode) to check for matrix effects in the SBM depth profiles. Both methods show sodium diffusion from the float glass substrate into the STZ coating and no significant sodium diffusion into the SiO₂ coating. Measurements of the coatings on the heat insulating glass indicate that the SnO₂ interlayer acts as a diffusion barrier. The diffusion of sodium from the float glass substrate into the STZ coating during consolidation has been analyzed by SBM-SNMS. The sputtering rate decreases with increasing consolidation. Due to large differences between sputtering rates of the substrate and of the microporous coatings, the calibration of sodium intensities from time to depth at the interface has not been possible. However, a correlation between the final temperature of heat treatment and the depth of the Na₂O depletion in the substrate surface under the coating can be obtained.     

Improved peptide detection with matrix-assisted laser desorption/ionization mass spectrometry by trimethylation of amino groups

Trimethyl alpha-amino derivatives of peptides (penta to deca) with a permanent positive charge on their alpha-amino groups were prepared by in vacuo reaction with iodomethane and subjected to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Compared to the unmodified peptide, the signal intensity of the trimethyl alpha-amino derivative in MALDI-MS is increased by at least an order of magnitude. Similarly, an octapeptide with a trimethylated epsilon-amino group derived from the solitary lysine residue of the B-chain of insulin also shows the same relative increase in signal intensity. Another advantage of the in vacuo methylation procedure is that trimethylation of a peptide amino group can be carried out readily with a combination of isotopes (13)CH(3)I and (12)CH(3)I or CD(3)I and CH(3)I, yielding a doublet signal either 3 or 9 units apart, respectively. The presence or absence of such a doublet signal can be used as a criterion to discriminate between peptide and non-peptide signals in the mass spectrum.     

Quantitative analysis of small molecules by desorption electrospray ionization mass spectrometry from polytetrafluoroethylene surfaces

Desorption electrospray ionization mass spectrometry (DESI-MS) is an emerging technique for ambient analysis. However, its application to routine quantitation has not been explored extensively and this is undertaken here. We present studies that utilize a particularly suitable surface, porous polytetrafluoroethylene (porous PTFE), which shows less cross contamination between samples and improved sensitivity and signal stability compared to other surfaces. Quantitative experiments for 1 microL spotted solutions of the beta-blocker propranolol, using isotopically labeled propranolol as internal standard, showed a good linear correlation (r2 > 0.996) over the range 0.01-100 microM. The inter-day precision, based on the relative standard deviation, for replicates analyzed on three different days was 13% for 0.01 microM and better than 7% for the remainder of the calibration points. The inter-day accuracy, expressed as relative error, was better than +/-7% for all calibration points along the curve. These day-to-day measurements suggest that DESI-MS can be successfully employed for routine quantitative analysis. The use of the analog atenolol as an internal standard and further considerations that should improve quantitation by DESI-MS are also presented.     

Analysis of small molecules by ultra thin-layer chromatography-atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

The feasibility of ultra thin-layer chromatography atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry (UTLC-AP-MALDI-MS) has been studied in the analysis of small molecules. Because of a thinner adsorbent layer, the monolithic UTLC plates provide 10-100 times better sensitivity in MALDI analysis than conventional high performance thin-layer chromatography (HPTLC) plates. The limits of detection down to a low picomole range are demonstrated by UTLC-AP-MALDI-MS. Other advantages of UTLC over HPTLC include faster separations and lower solvent consumption. The performances of AP-MALDI-MS and vacuum MALDI-MS have been compared in the analysis of small drug molecules directly from the UTLC plates. The desorption from the irregular surface of UTLC plates with an external AP-MALDI ion source combined with an ion trap instrument provides clearly less variation in measurements of m/z values when compared with a vacuum MALDI-time-of-flight (TOF) instrument. The performance of the UTLC-AP-MALDI-MS method has been applied successfully to the purity analysis of synthesis products produced by solid-phase parallel synthesis method.     

基体辅助激光解吸质谱法测定蛋白质分子量

本文叙述用自行研制成功的激光微探针飞行时间质谱仪及采用基体辅助激光解吸的新方法, 对溶菌酶、细胞色素C、肌红蛋白、胰蛋白酶、蛋白酶、白蛋白等多种蛋白质的分子量进行测定, 并对蛋白质混合物进行分析, 得一以满意的结果。此方法测定蛋白质分子量具有速度快(十分钟一个样品), 准确度高(±1%-0.1%), 灵敏度高(10^-^1^2～10^-^1^5mol)等优点, 是传统生物方法难以比拟的。     

Atmospheric pressure matrix-assisted laser desorption/ionization with analysis by ion mobility time-of-flight mass spectrometry

The use of an atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) source was employed with an atmospheric pressure ion mobility spectrometer (APIMS) and an orthogonal acceleration reflector time-of-flight mass spectrometer (TOFMS) to analyze dipeptide and biogenic amine mixtures from a liquid glycerol 2,5-dihydroxybenzoic acid (DHB) matrix. Improved sensitivities were obtained by the addition of a localized electrical (corona) discharge in conjunction with the AP-MALDI source. Enhanced sample ionization efficiency created by this combination provided an overall elevation in signal intensity of approximately 1.3 orders in magnitude. Combinations of three dipeptides (Gly-Lys, Ala-Lys, and Val-Lys) and nine biogenic amines (dopamine, serotonin, B-phenylethylamine, tyramine, octopamine, histamine, tryptamine, spermidine, and spermine) were resolved in less than 18 ms. In many cases, reduced mobility constants (K(o)) were determined for these analytes for the first time. Ion mobility drift times, flight times, arbitrary signal intensities, and collision-induced dissociation (CID) fragmentation product signatures are reported for each of the samples.     

Ion detection limitations to mass resolution in matrix-assisted laser desorption time-of-flight mass spectrometry.

The ion detection process in a discrete-dynode electron multiplier can result in significant mass resolution losses in time-of-flight mass spectrometry (TOF-MS) for higher mass-to-charge (m/z) ion species. This resolution loss is attributed to propagation time delays and signal broadening in the ion detector. This is presumed to be due to the generation of a distribution of secondary ion species produced initially upon impact of a primary ion with the first dynode surface of the ion detector. Comparisons are made between the signals produced by a standard discrete dynode ion detector (which amplifies the negatively charged species produced by impact of a primary ion) and a detector modified to respond to only the positively charged secondary ion species produced by a primary ion impact. Ion signals for higher m/z ions with the standard detector geometry are seen to be due to a narrow signal component, most likely due to the generation of secondary electrons and/or very low mass secondary ions (H-), and a broad signal component, apparently due to secondary ions which take significant amounts of time to traverse the low potential fields between the first and second detector dynode. This results in ion signal tailing for higher m/z ion species. Numerical subtraction of the ion signal obtained with the standard and modified detector geometries (singly protonated molecular ion species of equine myoglobin) results in an improvement in mass resolution, such that a new adduct ion species (from trifluoroacetic acid) can be resolved.     

Benefits of 2.94 micron infrared matrix-assisted laser desorption/ionization for analysis of labile molecules by Fourier transform mass spectrometry

A 2.94 microm Er:YAG laser was used together with a commercial Fourier transform mass spectrometer to study labile biomolecules. The combination has shown superior performance over conventional 337 nm ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI) Fourier transform mass spectrometry (FTMS), especially for the analysis of peptides with post-translational modifications. With succinic acid as a matrix, the sensitivity of the single-shot analysis was increased by an order of magnitude to the low femtomole level, with significantly less fragmentation observed. Intact molecular ions of a range of O-glycosylated and sulfated peptides were detected. Urea was found to induce even less fragmentation, although at the expense of the total ion yield. Molecular ions of a noncovalent complex (vancomycin + diacetyl-L-Lys-D-Ala-D-Ala) have been observed for the first time in MALDI-FTMS. 2.94 microm infrared (IR) MALDI also produced abundant molecular ions of a range of nonbiological samples, including C60 and C70 fullerenes as well as dimetal coordination complexes.