Study of synthetic aliphatic copolyamides by time-of-flight matrix assisted laser desorption/ionization mass spectrometry

Synthetic copolyamides based on aliphatic diamines (1,3-propanediamine and 1,4-butanediamine) and dichlorides of aliphatic carboxylic acids (adipic and sebacic acid dichlorides) were investigated using time-of-flight matrix assisted laser desorption/ionization mass spectrometry. Their mass spectra showed peaks for cationized (Na⁺ and K⁺) and protonated (less intense peaks) oligomers with NH₂-NH₂, NH₂-COOH, or COOH-COOH end groups. No cyclic oligomers were detected in the samples. The compositions of oligomers were determined, and the relative reactivities of homologous comonomers in polycondensation were estimated. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1320–1324, July, 2007.     

Automated normal phase nano high performance liquid chromatography/matrix assisted laser desorption/ionization mass spectrometry for analysis of neutral and acidic glycosphingolipids

The coupling of nano high-performance liquid chromatography (nanoHPLC) with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) via an automatic spotting roboter was developed and adapted for the first time for the analysis of complex mixtures of glycosphingolipids (GSLs). The 2,5-dihydroxybenzoic acid and 6-azo-2-thiothymine matrix systems were adjusted to concurrently meet the requirements for reproducible and homogeneous crystal formation with the liquid chromatography (LC) eluent under the variable LC solvent composition over the course gradient and high ionization efficiency of the GSL species, without the need for recrystallization. Precise adjustment of the automatic spotting parameters in terms of matrix flow rate, on-tip collection time of the matrix/LC eluent solution and the matrix spotting mode, i.e., continuous and discontinuous, was accomplished to collect individually nanoHPLC-separated species within distinct spots and consequently recover by MALDI MS screening all major and minor GSL species in the mixtures. The nanoHPLC/MALDI MS coupling protocol was developed and applied to a mixture of neutral GSLs purified from human erythrocytes and a monosialoganglioside mixture expressed by the murine MDAY-D2 cell line. Additionally, on-line nanoHPLC/MALDI doping with lithium cations of individually separated neutral GSLs was introduced to enhance data interpretation of the GSL MS pattern, while preserving the same level of information and ultimately to enhance structural assignment of components of interest. The method is demonstrated to be highly sensitive, reaching the low femtomole level of detection of individual GSL species and is highlighted as a versatile analytical tool for glycolipidomic studies. Figure Automatic LC/MALDI MS profiling of glycosphingolipids Mostafa Zarei and Stephan Kirsch contributed equally to this work.     

基质辅助激光解吸电离飞行时间质谱研究2: 四硫 富瓦烯化合物

采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS),对四硫富瓦烯化合物进行质谱表征。在所用的实验条件下,样品很容易解吸电离生成单电荷分子离子,得到单同位素分辨的质谱图。26种实际样品的质谱分析结果表明;MALDI-TOF-MS可以比其它质谱方法更有效、更方便地用于此类化合物的质谱分析,解决了此类化合物不易进行质谱鉴定的难题。     

Rapid identification and classification of Mycobacterium spp. using whole-cell protein barcodes with matrix assisted laser desorption ionization time of flight mass spectrometry in comparison with multigene phylogenetic analysis

The need of quick diagnostics and increasing number of bacterial species isolated necessitate development of a rapid and effective phenotypic identification method. Mass spectrometry (MS) profiling of whole cell proteins has potential to satisfy the requirements. The genus Mycobacterium contains more than 154 species that are taxonomically very close and require use of multiple genes including 16S rDNA for phylogenetic identification and classification. Six strains of five Mycobacterium species were selected as model bacteria in the present study because of their 16S rDNA similarity (98.4–99.8%) and the high similarity of the concatenated 16S rDNA, rpoB and hsp65 gene sequences (95.9–99.9%), requiring high identification resolution. The classification of the six strains by MALDI TOF MS protein barcodes was consistent with, but at much higher resolution than, that of the multi-locus sequence analysis of using 16S rDNA, rpoB and hsp65. The species were well differentiated using MALDI TOF MS and MALDI BioTyper™ software after quick preparation of whole-cell proteins. Several proteins were selected as diagnostic markers for species confirmation. An integration of MALDI TOF MS, MALDI BioTyper™ software and diagnostic protein fragments provides a robust phenotypic approach for bacterial identification and classification.     

以2, 4, 6-三羟基苯乙酮为基体使用基体辅助激光解吸/电离质 谱法测定核酸分 子的研究

报道以2,4,6-三羟基苯乙酮(2,4,6-THAP)为基体用基体辅助激光解吸/电离飞行时间质谱法(MALDI-TOF-MS)测定核酸分子的研究。2,4,6-THAP解吸和电离DNA分子的效率很高,测定DNA分子d(T)~1~0的[M-H]^-分辨率可达1130.0,信噪比为366.0,检出限达5×10^-^1^4mol,可以测定相对分子质量高达14800以上混合碱基组成的DNA分子。为提高基体对样品解吸/电离效率,对纯化DNA样品的方法进行了讨论。     

Capillary electrophoretic separation and fractionation of hydrophobic peptides onto a pre-structured matrix assisted laser desorption/ ionization target for mass spectrometric analysis

A CE separation of hydrophobic peptides followed by fractionation onto a prestructured MALDI target and off-line MS analysis was performed. An improved and partially automated manufacturing procedure of the previously described MALDI target is presented. This target is structurally coated with silicone and especially developed for hydrophobic peptides and proteins. Here, the target plate was designed specifically for the CE fraction collection. Different solvents were evaluated to meet the requirements of peptide solubility and compatibility to both the CE and MALDI methods and to the fractionation procedure. CE-MALDI-MS analysis of nine highly hydrophobic peptides from cyanogen bromide-digested bacteriorhodopsin is demonstrated.     

Strain and phase identification of the U.S. category B agent Coxiella burnetii by matrix assisted laser desorption/ionization time-of-flight mass spectrometry and multivariate pattern recognition

Accurate bacterial identification is important in diagnosing disease and in microbial forensics. Coxiella burnetii, a highly infective microorganism causative of the human disease Q fever, is now considered a U.S. category B potential bioterrorism agent. We report here an approach for the confirmatory identification of C. burnetii at the strain level which involves the combined use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and supervised pattern recognition via Partial Least Squares-Discriminant Analysis (PLS-DA). C. burnetii isolates investigated in this study included the following prototype strains from different geographical and/or historical origins and with different antigenic properties: Nine Mile I, Australian QD, M44, KAV, PAV, Henzerling, and Ohio. After culture and purification following standard protocols, linear MALDI-TOF mass spectra of pure bacterial cultures were acquired in positive ion mode. Mass spectral data were normalized, baseline-corrected, denoised, binarized and modeled by PLS-DA under crossvalidation conditions. Robustness with respect to uncontrolled variations in the sample preparation and MALDI analysis protocol was assessed by repeating the experiment on five different days spanning a period of 6 months. The method was validated by the prediction of unknown C. burnetii samples in an independent test set with 100% sensitivity and specificity for five out of six strain classes.     

Analysis and classification of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and a chemometric approach

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a useful technique for the identification of bacteria on the basis of their characteristic protein mass spectrum fingerprint. Highly standardized instrumental analytical performance and bacterial culture conditions are required to achieve useful information. A chemometric approach based on multivariate analysis techniques was developed for the analysis of MALDI data of different bacteria to allow their identification from their fingerprint. Principal component analysis, linear discriminant analysis (LDA) and soft independent modelling of class analogy (SIMCA) were applied to the analysis of the MALDI MS mass spectra of two pathogenic bacteria, Escherichia coli O157:H7 and Yersinia enterocolitica, and the non-pathogenic E. coli MC1061. Spectra variability was assessed by growing bacteria in different media and analysing them at different culture growth times. After selection of the relevant variables, which allows the evaluation of an m/z value pattern with high discriminant power, the identification of bacteria by LDA and SIMCA was performed independently of the experimental conditions used. In order to better evaluate the analytical performance of the approach used, the ability to correctly classify different bacteria, six wild-type strains of E. coli O157:H7, was also studied and a combination of different chemometric techniques with a severe validation was developed. The analysis of spiked bovine meat samples and the agreement with an independent chemiluminescent enzyme immunoassay demonstrated the applicability of the method developed for the detection of bacteria in real samples. The easy automation of the MALDI method and the ability of multivariate techniques to reduce interlaboratory variability associated with bacterial growth time and conditions suggest the usefulness of the proposed MALDI MS approach for rapid routine food safety checks. Figure Workflow of the developed MALDI-TOF MS and chemometric approach for the analysis and classification of bacteria Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Determination of polyethylene glycol end group functionalities by combination of selective reactions and characterization by matrix assisted laser desorption/ionization time-of-flight mass spectrometry

End groups play a critical role in macromolecular coupling reactions for building complex polymer architectures, yet their identity and purity can be difficult to ascertain using traditional analytical technique. Recent advances in mass spectrometry techniques have made matrix-assisted laser desorption/ionization time-of-fight (MALDI-TOF) mass spectrometry a rapid and powerful tool for providing detailed information about the identity and purity of homopolymer end groups. In this work, MALDI-TOF mass spectrometry was used to study end groups of linear polyethylene glycols. In particular, the identifications of alcohol, amine and thiol end groups are investigated because these nucleophilic moieties are among the most common within biological and synthetic macromolecules. Through comparative characterization of alcohol, amine, and thiol end groups, the exact identification of these end groups could be confirmed by selective and quantitative modification. The precision of this technique enables the unambiguous differentiation of primary amino groups relative to hydroxyl groups, which differ by only 1 mass unit. In addition, the quantitative conversion of various polyethylene glycol end groups using highly efficient coupling reactions such as the thiol-ene and azide-alkyne click reactions can be confirmed using MALDI-TOF mass spectrometry.     

Fabrication of nanostructured silicon by metal-assisted etching and its effects on matrix-free laser desorption/ionization mass spectrometry

A matrix-free, high sensitivity, nanostructured silicon surface assisted laser desorption/ionization mass spectrometry (LDI-MS) method fabricated by metal-assisted etching was investigated. Effects of key process parameters, such as etching time, substrate resistance and etchant composition, on the nanostructured silicon formation and its LDI-MS efficiency were studied. The results show that the nanostructured silicon pore depth and size increase with etching time, while MS ion intensity increases with etching time to 300 s then decreases until 600 s for both low resistance (0.001–0.02 Ω cm) and high resistance (1–100 Ω cm) silicon substrates. The nanostructured silicon surface morphologies were found to directly affect the LDI-MS signal ion intensity. By characterizing the nanostructured silicon surface roughness using atomic force microscopy (AFM) and sample absorption efficiency using fluorescence microscopy, it was further demonstrated that the nanostructured silicon surface roughness was highly correlated to the LDI-MS performance.     

MoS2/Ag nanohybrid: A novel matrix with synergistic effect for small molecule drugs analysis by negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

This paper reports a facile synthesis of molybdenum disulfide nanosheets/silver nanoparticles (MoS₂/Ag) hybrid and its use as an effective matrix in negative ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The nanohybrid exerts a strong synergistic effect, leading to high performance detection of small molecule analytes including amino acids, peptides, fatty acids and drugs. The enhancement of laser desorption/ionization (LDI) efficiency is largely attributed to the high surface roughness and large surface area for analyte adsorption, better dispersibility, increased thermal conductivity and enhanced UV energy absorption as compared to pure MoS₂. Moreover, both Ag nanoparticles and the edge of the MoS₂ layers function as deprotonation sites for proton capture, facilitating the charging process in negative ion mode and promoting formation of negative ions. As a result, the MoS₂/Ag nanohybrid proves to be a highly attractive matrix in MALDI-TOF MS, with desired features such as high desorption/ionization efficiency, low fragmentation interference, high salt tolerance, and no sweet-spots for mass signal. These characteristic properties allowed for simultaneous analysis of eight different drugs and quantification of acetylsalicylic acid in the spiked human serum. This work demonstrates for the first time the fabrication and application of a novel MoS₂/Ag hybrid, and provides a new platform for use in the rapid and high throughput analysis of small molecules by mass spectrometry.     

Gold nanoparticle-enhanced target (AuNPET) as universal solution for laser desorption/ionization mass spectrometry analysis and imaging of low molecular weight compounds

Preparation is described of a durable surface of cationic gold nanoparticles (AuNPs), covering commercial and custom-made MALDI targets, along with characterization of the nanoparticle surface properties and examples of the use in MS analyses and MS imaging (IMS) of low molecular weight (LMW) organic compounds. Tested compounds include nucleosides, saccharides, amino acids, glycosides, and nucleic bases for MS measurements, as well as over one hundred endogenous compounds in imaging experiment. The nanoparticles covering target plate were enriched in sodium in order to promote sodium-adduct formation. The new surface allows fast analysis, high sensitivity of detection and high mass determination accuracy. Example of application of new Au nanoparticle-enhanced target for fast and simple MS imaging of a fingerprint is also presented.     

The characterization of dendronized poly(ethylene glycol)s and poly(ethylene glycol) multi-arm stars using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The synthesis of branched poly(ethylene glycol) (PEG) derivatives, namely star PEG and dendronized PEG, can be challenging and their purity can be difficult to ascertain using traditional techniques, such as NMR and GPC. Herein, the detailed characterization of these branched PEGs using MALDI-ToF MS was investigated in order to confirm their structural purity. In this light, mass spectrometry offers a number of advantages for polymer characterization, including the ability to get detailed structural data, such as end group masses, from microgram-scale samples. In addition, the ability to rapidly acquire data from crude reaction aliquots makes MALDI-ToF MS ideal for monitoring end group transformations.     

Effects of different alkali metal ions on the cationization of poly(ethylene glycol)s in matrix-assisted laser desorption/ionization mass spectrometry: a new selectivity parameter

The cationization of poly(ethylene glycol)s, PEG 4000 and PEG 6000, under matrix-assisted laser desorption/ionization conditions was studied by using different concentration ratios of the sodium ion, as the reference ion, and another alkali metal ion (Li(+), K(+), Rb(+), Cs(+)). A linear correlation was found between the intensity ratio of the sodiated PEGs and PEGs cationized with alkali metal ions versus the initial concentration ratio of sodium and alkali metal ions. The slopes of these straight lines are proposed as a novel selectivity ratio for the ionization process. The intensity distribution of the cationized PEGs was also investigated. It was found that the cationized oligomers follow Poisson statistics. The M(n) and M(w) values were also evaluated. An explanation for the observed effects is given.