Matrix-assisted laser desorption/ionisation mass spectrometry of oligosaccharides and glycoconjugates

The technique of matrix-assisted laser desorption/ionization mass spectrometry (MALDI) is described and examples are given of its use for the examination of glycoproteins, glycopeptides, glycolipids and oligosaccharides. Abundant [M + H]⁺ ions are produced by the glycoproteins and glycopeptides, whereas glycolipids and oligosaccharides give mainly [M + Na]⁺ ions. Resolution on time-of-flight (TOF) instruments is poor but improved resolution can be obtained by use of ion cyclotron resonance or magnetic sector instruments. Although the technique gives mainly [M + Na]⁺ ions from neutral, underivatised oligosaccharides, with little fragmentation when implemented on TOF systems, the use of a reflectron enables fragment ions produced by post-source decay to be obtained. Acidic sugars give less satisfactory positive ion spectra with TOF analysers. but generally produce abundant negative ions. Extensive fragmentation is observed with these compounds when the spectra are recorded with magnetic sector instruments. Neutral glycolipids produce strong spectra from several matrices but acidic glycolipids show extensive fragmentation as the result of sialic acid loss.     

Matrix-assisted laser desorption/ionization mass spectrometry of taxanes

Taxanes are biologically active compounds that have been extensively used in pharmacology for their powerful anticancer properties. High specificity and low level sensitivity for analysis of these compounds have been obtained with reversed-phase high-pressure liquid chromatography/mass spectrometry (RP-HPLC/MS), but the number of applications of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for low molecular weight analytes is rapidly growing. A new MALDI-MS approach for the rapid screening of a variety of taxanes and a tandem mass spectrometric (MS/MS) analysis of the most important and diagnostic taxane fragmentation pathways are proposed. A solid-phase extraction method followed by preliminary quantification is also reported.     

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。     

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the analysis of polydienes

An analytical method based on matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry has been developed to provide information on oligomer structure, average molecular weight, and molecular weight distributions of polydienes (e.g., polybutadiene and polyisoprene), an important class of industrial polymers. This MALDI method involves the use of all-trans-retinoic acid as the matrix, copper (II) nitrate as the cationization reagent, and tetrahydrofuran as the solvent. The incorporation of this copper salt generates Cu⁺ adducts with the polymer chains. It also improves the signal strength and extends the upper mass range when used with all-trans-retinoic acid, as compared to silver nitrate. With this formulation, it is demonstrated that polybutadienes of narrow polydispersity with masses up to 300,000 u and polyisoprenes of narrow polydispersity with masses up to 150,000 u can be analyzed. The upper molecular weight limit is set by the requirement of using higher matrix-to-polymer ratios with increasing polymer molecular weight, to the point where the instrument can no longer detect the small quantity of polymer present in the matrix host. It is also shown that this sample preparation generates previously unreported adduction behavior. The practical implications of this adduction behavior on polymer structural analysis, accuracy of molecular weight determination, and the upper molecular weight limit of oligomer resolution are discussed. It is illustrated that, in a linear time-lag focusing MALDI instrument, oligomer resolution can be obtained for polydienes with molecular weights up to 24,000, providing structural confirmation of the end-groups and the repeat unit. The average molecular weights of a number of polydienes of narrow polydispersity determined by MALDI are compared to those obtained by gel permeation chromatography, and discrepancies are noted.     

Matrix-assisted laser desorption/ionization mass spectrometry using a visible laser

Visible matrix-assisted laser desorption/ionization (VIS-MALDI) was performed using 2-amino-3-nitrophenol as matrix. The matrix is of near-neutral pH, and has an optical absorption band in the near-UV and visible region. A frequency-doubled Nd:YAG laser operated at 532 nm wavelength was used for matrix excitation and comparisons were made with a frequency-tripled Nd:YAG laser (355 nm). Visible and ultraviolet (UV)-MALDI produce similar mass spectra for peptides, polymers, and small proteins with comparable sensitivities. Due to the smaller optical absorption coefficient of the matrix at 532 nm wavelength, the optical penetration depth is larger, and the sample consumption per laser shot in VIS-MALDI is higher than that of UV-MALDI. Nevertheless, VIS-MALDI using 2-amino-3-nitrophenol as matrix may offer a complementary technique to the conventional UV-MALDI method in applications where deeper laser penetration is required.     

Capillary electrophoresis off-line matrix-assisted laser desorption/ionisation mass spectrometry of intact and digested proteins using cationic-coated capillaries

Capillary electrophoresis (CE) was coupled off-line with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the analysis of proteins and peptides. CE fractions were collected directly on a matrix-coated MALDI target, using a sheath-flow interface. Protein adsorption during CE separations was prevented by coating the capillaries with the physically adsorbed, cationic polymer PolyE-323. The CE/MALDI-MS system was used for the analysis of model proteins and peptides at physiological pH as well as analysis of proteins in tear fluid. Moreover, tryptic on-target digestion of the collected protein fractions, with subsequent MALDI-MS and MS/MS peptide analysis, was demonstrated.     

Matrix-assisted laser desorption ionization mass spectrometry for identification of shrimp

Matrix-assisted laser desorption ionization (MALDI) time of flight mass spectrometry was used to identify shrimp at the species level using commercial mass spectral fingerprint matching software (Bruker Biotyper). In the first step, a mass spectrum reference database was constructed from the analysis of six commercially important shrimp species: Litopenaeus setiferus, Farfantepenaeus aztecus, Sicyonia brevirostris, Pleoticus robustus, Pandalopsis dispar and Pandalus platyceros. This step required a desalting procedure for optimum performance. In the second step, the reference database was tested using 74 unknown shrimp samples from these six species. Correct identification was achieved for 72 of 74 samples (97%): 72 samples were identified at the species level and 2 samples were identified at the genus level using the manufacturer's log score specifications. The MALDI fingerprinting method for the identification of shrimp species was found to be reproducible and accurate with rapid analysis.     

Matrix-assisted laser desorption/ionization mass spectrometry of collected bioaerosol particles

A method was developed for collection and analysis of bioaerosols by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry using a modified Andersen N6 bioaerosol collector. The overall goal of the study was to develop methods for obtaining mass spectra with minimal reagents and treatment steps for potential use in remote collection and analysis systems. Test bioaerosol particles were generated from a nebulized E. coli bacterial suspension and collected on MALDI targets placed in an Andersen N6 single-stage aerosol impactor. The bioaerosols were mixed with matrix either by deposition on a bare target with the matrix solution added later, or by deposition on a target pre-coated with matrix. The matrix compounds alpha-cyano-4-hydroxycinnamic acid (CHCA) and sinapic acid (SA) were tested and the SA matrix was found to give the best results in number of peaks, resolution, and signal-to-noise ratio. Deposition of bioaerosol particles onto the matrix pre-coated target did not produce signal in the m/z region above 1000, but the signal could be recovered with the addition of a 1:1 (v/v) acetonitrile/water solvent. Addition of solvent by pipette to the pre-coated targets after particle deposition recovered signal comparable to the dried-droplet sample preparations, whereas solvent sprayed into the impactor recovered fewer peaks. Deposition on pre-coated targets with post-collection solvent addition was superior to deposition on bare target followed by post-collection addition of matrix solution.     

Matrix-assisted laser desorption/ionization imaging mass spectrometry for direct measurement of clozapine in rat brain tissue

Matrix-assisted laser desorption/ionization hyphenated with quadrupole time-of-flight (QTOF) mass spectrometry (MS) has been used to directly determine the distribution of pharmaceuticals in rat brain tissue slices which might unravel their disposition for new drug development. Clozapine, an antipsychotic drug, and norclozapine were used as model compounds to investigate fundamental parameters such as matrix and solvent effects and irradiance dependence on MALDI intensity but also to address the issues with direct tissue imaging MS technique such as (1) uniform coating by the matrix, (2) linearity of MALDI signals, and (3) redistribution of surface analytes. The tissue sections were coated with various matrices on MALDI plates by airspray deposition prior to MS detection. MALDI signals of analytes were detected by monitoring the dissociation of the individual protonated molecules to their predominant MS/MS product ions. The matrices were chosen for tissue applications based on their ability to form a homogeneous coating of dense crystals and to yield greater sensitivity. Images revealing the spatial localization in tissue sections using MALDI-QTOF following a direct infusion of (3)H-clozapine into rat brain were found to be in good correlation with those using a radioautographic approach. The density of clozapine and its major metabolites from whole brain homogenates was further confirmed using fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) procedures.     

Matrix-assisted laser desorption mass spectrometry of oligodeoxythymidylic acids.

Ferulic acid, sinapinic acid and 2,5-dihydroxybenzoic acid (DHBA) have been tested as matrix materials for matrix-assisted laser desorption of the pure oligonucleotide pd(T)12 and a mixture of oligonucleotides pd(T)12 through pd(T)18 using pulsed 337 nm radiation combined with reflecting time-of-flight mass spectrometry. The three matrix materials are compared with respect to obtainable mass resolution, degree of fragmentation, and adduct formation for these oligonucleotides. DHBA was found to produce the least fragmentation and adduct formation, as well as the highest mass resolution.     

Matrix-assisted laser desorption/ionization mass spectrometry analysis of glycans with co-derivatization of asparaginyl-oligosaccharides

As one of the most prevalent and complex post-translational modifications in biological systems, proteins glycosylation has drawn considerable attention in recent decades. Dissociation of the carbohydrates from glycoproteins may be the prerequisite step of glycomics experiments, which commonly performed by specific proteolysis. In this study, an alternative strategy was reported with nonspecific proteolysis in coupling with co-derivatization of TMPP-Ac and methylamidation for glycan moieties analysis by MALDI-MS. With the co-derivatization, a permanent positive charge was introduced to the Asn-glycans and the carboxylic groups were neutralized by methylamidation simultaneously. As a result, approximately 20 and 50-fold enhancement in the detection sensitivity was achieved for asialo-Asn and disialo-Asn respectively in comparison to their native counterparts. Ultimately, this developed strategy was successfully validated using three model glycoproteins, including ribonuclease B, ovalbumin and transferrin.     

Matrix-assisted laser desorption/ionisation mass spectrometry imaging of lipids in rat brain tissue with integrated unsupervised and supervised multivariant statistical analysis

To date matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) analysis has been largely concerned with mapping the distribution of known analytes in tissues. An important step in the progression of its applications is the determination of unknown variants for metabolite and protein profiling in both clinical studies and studies of disease. Principal component analysis (PCA) is a statistical approach which can be used as a means of determining latent variables in multivariate data sets. In the work reported here, PCA, in both unsupervised and supervised modes, has been used to differentiate brain regions based on their lipid composition determined by MALDI-MSI. PCA has been shown to be useful in the determination of hidden variables between spectra taken from six regions of brain tissue. It is possible to identify ions of interest from the loadings plot which are likely to be more prominent in the different regions of the brain and thus differentiating between white and grey matter. It is also possible to distinguish between the grey Cerebellar Cortex and the Hippocampal formation, due to the grey Cerebellar Cortex having a positive PC2 and the Hippocampal formation having a negative PC2 score; this is only possible in supervised PCA with this data set because with unsupervised PCA the two regions overlap.     

基质辅助激光解吸离子化质谱及其在生物大分子分析中的应用

近年来质谱离子化技术方面有两项重要成果：基质辅助激光解吸离子化(matrix-assisted laser desorption ionization，MALDI)和电喷雾离子化(electrospray ionization，ESI)。MALDI和ESI的应用使质谱在生物大分子研究方面取得重大突破。本文仅就MALDI的原理、特点、样品准备方法、基质的选择、仪器条件及其在生物大分子应用方面的最新进展进行简要的综述。     

Matrix-assisted laser desorption/ionization tandem reflectron time-of-flight mass spectrometry of fullerenes

Atandem reflectron time-of-flight mass spectrometer developed in our laboratory provides a unique opportunity to investigate the collision-induced dissociation of fullerene ions formed by matrix-assisted laser desorption/ionization (MALDI). Specifically, this opportunity arises from the ability to utilize high energy collisional activation (normally available only on tandem sector instruments by using continuous ionization techniques) for ions formed by pulsed laser desorption, whereas most MALDI time-of-flight instruments record product ion mass spectra of ions formed by metastable or postsource decay. In this study we investigate the products of mass-selected and collisionally activated C ₆₀ ⁺ and C ₇₀ ⁺ ions by using different target gases over a range of target gas pressures. In general, heavier target gases produce more extensive fragmentation and improve the mass resolution of lower mass ionic products because a greater portion of these ions are formed by single collisions. Additionally, the tandem time-of-flight instrument utilizes a nonlinear (curved-field) reflectron in the second mass analyzer that enables high energy collision-induced dissociation spectra to be recorded without scanning or stepping the reflectron voltage.     

Matrix-suppressed laser desorption/ionisation mass spectrometry and its suitability for metabolome analyses

Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry was investigated for the simultaneous detection of several metabolites, as applicable to global metabolite analysis (metabolomics). The commonly employed organic matrices alpha-cyano-4-hydroxycinnamic acid and 3,5-dihydroxybenzoic acid, in both the crystalline and ionic liquid forms, were investigated. The employment of a low matrix-to-analyte molar ratio suppressed matrix peaks and was effective in detecting all the metabolites with a unique mass in a 30-metabolite synthetic cocktail, albeit to varying degrees. These matrix-suppressed laser desorption/ionisation (MSLDI) analyses were performed in the positive ion mode, and metabolites were detected as the protonated [M+H]+, sodiated [M+Na]+ or potassiated [M+K]+ species. The spectral signals were dominated by basic metabolites. It was possible to detect components of a synthetic cocktail when it was spiked quantitatively into a microbial extract, demonstrating the feasibility of using the technique for detecting metabolite signals in a complex biological matrix. However, analyte suppression effects were noted when the relative proportion of one analyte was allowed to increasingly dominate the others in a mixture. The implications of the findings with respect to applications in metabolomic investigations are discussed.     

Electrospray-assisted laser desorption/ionization mass spectrometry for direct ambient analysis of solids

A new method of electrospray-assisted laser desorption/ionization (ELDI) mass spectrometry, which combines laser desorption with post-ionization by electrospray, was applied to rapid analysis of solid materials under ambient conditions. Analytes were desorbed from solid metallic and insulating substrata using a pulsed nitrogen laser. Post-ionization produced high-quality mass spectra characteristic of electrospray, including protein multiple charging. For the first time, mass spectra of intact proteins were obtained using laser desorption without adding a matrix. Bovine cytochrome c and an illicit drug containing methaqualone were chosen in this study to demonstrate the applicability of ELDI to the analysis of proteins and synthetic organic compounds.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of dextran and dextrin derivatives

Application of matrix-assisted laser desorption/ionization (MALDI) to the analysis of dextran and dextrin derivatives, specifically glucose saccharides, by time-of-flight (TOF) mass spectrometry has been reported. MALDI-TOF analysis was carried out on alpha-, beta- and gamma-cyclodextrin, two O-methylated-beta-cyclodextrins of differing degrees of substitution (DS) and dextrans (a linear glucose saccharide), as pure and doped solutions and as mixtures of two or more of these. Doping was carried out with trace amounts of inorganic salts. The purpose of the analysis of the cyclodextrins was to determine whether they would form inclusion complexes with the various cations added, or whether less specific cation addition/exchange was occurring either prior to desorption or in the gas phase.     

Matrix-assisted laser desorption/ionization mass spectrometry compatible beta-elimination of O-linked oligosaccharides

A new beta-elimination procedure has been introduced to cleave O-linked oligosaccharides from low- to sub-microgram amounts of glycoproteins prior to analysis by mass spectrometry. Borane-ammonia complex in aqueous ammonia is used as a cleaving solution alternative to the sodium borohydride/sodium hydroxide medium conventionally used in beta-elimination. The procedure results in minimum sample purification, leading to minimal sample loss and consequently an overall enhancement in sensitivity. It was applied successfully in the analysis of bovine fetuin and submaxillary mucin, as well as to a complex bile-salt-stimulated lipase glycoprotein isolated from human milk.     

Atmospheric pressure matrix-assisted laser desorption/ionisation ion trap mass spectrometry of synthetic polymers: a comparison with vacuum matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI/QIT) mass spectrometry has been investigated for the analysis of polyethylene glycol (PEG 1500) and a hyperbranched polymer (polyglycidol) in the presence of alkali-metal salts. Mass spectra of PEG 1500 obtained at atmospheric pressure showed dimetallated matrix/analyte adducts, in addition to the expected alkali-metal/PEG ions, for all matrix/alkali-metal salt combinations. The relative intensities of the desorbed ions were dependent on the matrix, the alkali-metal salt added to aid cationisation and the ion trap interface conditions [capillary temperature, in-source collisionally-induced dissociation (CID)]. These data indicate that the adducts are rapidly stabilised by collisional cooling enabling them to be transferred into the ion trap. Experiments using identical sample preparation conditions were carried out on a vacuum MALDI time-of-flight (ToF) mass spectrometer. In all cases, vacuum MALDI-ToF spectra showed only alkali-metal/PEG ions and no matrix/analyte adducts. The tandem mass spectrometry (MS/MS) capability of the ion trap has been demonstrated for a lithiated polyglycol yielding a rich fragment-ion spectrum. Analysis of the hyperbranched polymer polyglycidol by AP-MALDI/QIT reveals the characteristic ion series for these polymers as also observed under vacuum MALDI-ToF conditions.