Accurate molecular mass determination of mycolic acids by MALDI-TOF mass spectrometry

Mycolic acids, major and specific long-chain fatty (C70-C90) acid components of the mycobacterial cell envelope, were analyzed for the first time using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry operating in a reflectron mode. The various types of purified mycolates from representative mycobacterial species were analyzed using 2,5-DHB as matrix, because less than 10 pmol of mycolates was sufficient to obtain well-resolved mass spectra composed exclusively of pseudomolecular [M + Na]+ ions consistent with the structures deduced from the chemical analytical techniques applied to these molecules. Examination of the MALDI mass spectra demonstrated that the chain lengths of the various mycolates correlated with the growth rate of mycobacterial strains. Although slow growers, such as Mycobacterium tuberculosis and Mycobacterium ulcerans, produced a series of odd carbon numbers (C74-C82) of alpha-mycolic acids, rapid growers synthesized both odd and even carbon numbers. In addition, the main chain of oxygenated mycolic acids from slow growers were four to six carbon atoms longer than the corresponding alpha-mycolic acids, whereas rapid growers elaborated oxygenated homologues possessing the same chain lengths as their alpha-mycolic acids. Furthermore, a comparative analysis of the crude fatty acid mixtures from a wild-type strain of M. tuberculosis and its isogenic mutant effected in the synthesis of oxygenated mycolates by MALDI mass spectrometry revealed structural differences between the alpha-mycolates from the two strains. Thus, this technique appeared to be a rapid and highly sensitive technique for the analysis of mycolic acids, not only by providing accurate molecular masses and new structural information, but also by both reducing sample consumption and saving time.     

Coumarin tags for analysis of peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 tag for increased peptide and protein Identification by LC-MALDI-TOF/TOF MS

The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF/TOF MS. Part 1 of the study was focused on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. In part 2, various derivatization approaches for the improvement of peptide fragmentation efficiency in MALDI-TOF/TOF MS are explored. We demonstrate that permanent cation tags, while significantly improving signal intensity in the MS mode, lead to severe suppression of MS/MS fragmentation, making these tags unsuitable for high-throughput MALDI-TOF/TOF MS analysis. In the present work, it was found that labeling with Alexa Fluor 350, a coumarin tag containing a sulfo group, along with guanidation of epsilon-amino groups of Lys, could enhance unimolecular fragmentation of peptides with the formation of a high-intensity y-ion series, while the peptide intensities in the MS mode were not severely affected. LC-MALDI-TOF/TOF MS analysis of tryptic peptides from the SCX fractions of an E. coli lysate revealed improved peptide scores, a doubling of the total number of peptides, and a 30% increase in the number of proteins identified, as a result of labeling. Furthermore, by combining the data from native and labeled samples, confidence in correct identification was increased, as many proteins were identified by different peptides in the native and labeled data sets. Additionally, derivatization was found not to impair chromatographic behavior of peptides. All these factors suggest that labeling with Alexa Fluor 350 is a promising approach to the high-throughput LC-MALDI-TOF/TOF MS analysis of proteomic samples.     

Stable-isotope-assisted MALDI-TOF mass spectrometry for accurate determination of nucleotide compositions of PCR products.

In parallel with a large-scale sequencing effort, the human genome project will need next-generation tools for accurate and efficient analyses of the enormous pool of DNA sequences. Such analyses are required for (a) validation of DNA sequences, (b) comparison of a parent (known) sequence with a related (unknown) sequence, and (c) characterization of sequence polymorphisms in various genes, especially those associated with genetically inherited human diseases. Here, we report a novel method that combines stable isotope 13C/15N labeling of PCR products of the target sequences with analysis of the mass shifts by mass spectrometry (MS). The mass shift due to the labeling of a single type of nucleotide (i.e., A, T, G, or C) reveals the number of that type of nucleotide in a given DNA fragment. Using this technique, we have accurately determined nucleotide compositions of DNA fragments. The method has also been applied to score a known single-nucleotide polymorphism (SNP). The comparisons of nucleotide compositions determined by our method among homologous sequences are useful in sequence validation, sequence comparison, and characterizations of sequence polymorphisms.     

A non-linear numerical method for accurate determination of limit and bifurcation points

This paper presents a numerical procedure for accurate determination of a limit or a bifurcation point. The method minimizes simultaneously the first and the second variations of an admissible functional or iterates to satisfy the equilibrium and the semi definite condition for the tangent stiffness matrix. It can be readily incorporated into a computer program for non-linear finite element analysis to improve its accuracy in the location of critical points.     

A simple procedure for the accurate determination of stress intensity factors by finite element method

A simple procedure for the accurate determination of stress intensity factors K_I, K_II by the conventional finite element method is proposed. The first step of the method is to calculate the stress σ₂ of the plate without a crack. The second step is to calculate the stress σ_tip, of the plate with the crack. The value of (σ_tip−σ_g) at the crack tip element is regarded to have the intimate relation with K_I, K_II K_I, and K_II are determined from the value of (σ_tip−σ_g) and a standard solution. It is shown that the results obtained for many problems by the proposed method are in excellent coincidence with the analytical solutions. The error is below 1–3% for the most cases.     

A MALDI-TOF MS study of oligomeric poly(m-phenyleneisophthalamide)

MALDI-TOF MS was used to study the end-group distribution of a series of poly(m-phenyleneisophthalamide) oligomers which were synthesized using various mole percent ratios of diamine to diacid chloride (90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, and 10:90) to clarify results obtained in previous work published in this journal. Oligomers synthesized with excess diamine or excess diacid chloride were found to contain abundances of amine or carboxylate end groups, respectively, as expected. Oligomers synthesized with equal molar ratios of reactants produced cyclic species which were also found in a previous publication as an oligomer in commercially produced, high molecular mass Nomex.     

Quantification of proteins on gold nanoparticles by combining MALDI-TOF MS and proteolysis

Protein-coated nanoparticles have been used in many studies, including those related to drug delivery, disease diagnosis, therapeutics, and bioassays. The number and density of proteins on the particles' surface are important parameters that need to be calculable in most applications. While quantification methods for two-dimensional surface-bound proteins are commonly found, only a few methods for the quantification of proteins on three-dimensional surfaces such as nanoparticles have been reported. In this paper, we report on a new method of quantifying proteins on nanoparticles using matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS). In this method, the nanoparticle-bound proteins are digested by trypsin and the resulting peptide fragments are analyzed by MALDI-TOF MS after the addition of an isotope-labeled internal standard (IS) which has the same sequence as a reference peptide of the surface-bound protein. Comparing the mass intensities between the reference peptide and the IS allows the absolute quantification of proteins on nanoparticles, because they have the same molecular milieu. As a model system, gold nanoparticles were examined using bovine serum albumin (BSA) as a coating protein. We believe that our strategy will be a useful tool that can provide researchers with quantitative information about the proteins on surfaces of three-dimensional materials.     

Coumarin tags for improved analysis of peptides by MALDI-TOF MS and MS/MS. 1. Enhancement in MALDI MS signal intensities

The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF MS and MS/MS. The proposed tags, commercially available fluorescent derivatives of coumarin, can be advantageous for peptide analysis in both MS and MS/MS modes. This paper, part 1, will focus on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. Labeling peptides with tags containing the coumarin core was found to enhance the intensities of peptide peaks (in some cases over 40-fold) in MALDI-TOF MS using CHCA and 2,5-DHAP matrixes. The signal enhancement was found to be peptide- and matrix-dependent, being the most pronounced for hydrophilic peptides. No correlation was found between the UV absorptivity of the tags at the excitation wavelengths typical for UV-MALDI and the magnitude of the signal enhancement. Interestingly, peptides labeled with Alexa Fluor 350, a coumarin derivative containing a sulfo group (i.e., bearing strong negative charge), showed a 5-15-fold increase in intensity of MALDI MS signal in the positive ion mode, relative to the underivatized peptides, when 2,5-DHAP was used as the matrix. The Alexa Fluor 350 tag yielded a significantly higher signal relative to that for the CAF tag, likely due to the increased hydrophobicity of the coumarin structure. With 2,5-DHB, a decrease of MALDI MS signal was observed for all coumarin-labeled peptides, again relative to the unlabeled species. These findings support the hypothesis that derivatization with coumarin, a relatively hydrophobic structure, improves incorporation of hydrophilic peptides into hydrophobic MALDI matrixes, such as CHCA and 2,5-DHAP.     

Assessment of the molecular weight distribution of tannin fractions through MALDI-TOF MS analysis of protein-tannin complexes

An innovative mass spectrometry method was developed for determining mass distributions of tannin fractions that cannot be approached through direct MALDI-TOF analysis. It was applied to three procyanidin fractions with average degrees of polymerizations = 3, 9, and 28, respectively, and one gallotannin fraction (Tara tannin). The proposed approach consists of MALDI-TOF analysis of the soluble complexes formed between these tannin fractions and bovine serum albumin (BSA). Complexes were detected as an unresolved "hump" following the BSA signal, and spectra were mathematically processed to determine the parameters relative to the protein-tannin complexes, which are the number-average molecular weight (Mn), the weight-average molecular weight (Mw), and the polydispersity index (PI) for each tannin fraction. Regarding condensed tannins, results are consistent with those of the standard method (thiolysis followed by HPLC separation) for all tested fractions. The method was successfully applied to a hydrolyzable tannin fraction but no standard method is available for comparison.     

A statistical method for assessing peptide identification confidence in accurate mass and time tag proteomics

Current algorithms for quantifying peptide identification confidence in the accurate mass and time (AMT) tag approach assume that the AMT tags themselves have been correctly identified. However, there is uncertainty in the identification of AMT tags, because this is based on matching LC-MS/MS fragmentation spectra to peptide sequences. In this paper, we incorporate confidence measures for the AMT tag identifications into the calculation of probabilities for correct matches to an AMT tag database, resulting in a more accurate overall measure of identification confidence for the AMT tag approach. The method is referenced as Statistical Tools for AMT Tag Confidence (STAC). STAC additionally provides a uniqueness probability (UP) to help distinguish between multiple matches to an AMT tag and a method to calculate an overall false discovery rate (FDR). STAC is freely available for download, as both a command line and a Windows graphical application.     

Novel, fourier filtering method that reuses interference-patterned spectra to extend the calibration set for thickness determination

Determining the thickness of plastic sheets on the basis of near-infrared spectra by building a multivariate calibration model requires a relatively large sample set. In the thickness region, where just a few non-interference-patterned samples are available, it is a waste of information if interference-patterned spectra are excluded. After eliminating the interference pattern from the spectra (filtering), the calibration set can be extended with these filtered spectra. Fourier transformation of an interference-patterned spectrum versus wavenumber leads to a Fourier spectrum as a function of the optical path length containing an easily recognizable interference peak. Unfortunately, this peak coincides with components of the spectral information of absorbance, on which multivariate calibration is based. Hence, replacing the interference peak is a cardinal step of the filtering process. Since the Fourier spectrum versus optical path length function is not known, it has been shown that interpolated data over the remaining Fourier components can be substituted for the missing part of the spectrum. In this paper, a novel method is proposed that uses a linear approximation between the Fourier spectra and the thickness values so that the regression coefficients are calculated on components of all but the interference-patterned Fourier spectra and the corresponding thicknesses, and then the deleted components in the filtered spectrum are replaced. This latter method yields more detailed Fourier spectra. Reducing the disturbing effect of scattering is also discussed. The effectiveness of the filtering was tested on low-density polyethylene sheets. The performance of different calibration models with or without filtering was compared by significance tests on standard error of prediction values. Application of the new Fourier type filtering technique led to significant improvements in the calibration performance.     

球幕点目标投影跟踪系统的精确标定方法

针对球幕点目标投影与跟踪系统中多个子系统相对位置关系探测复杂的问题,提出了一种适合现场测量的精确标定方法。将球幕作为世界坐标系,利用子系统对球幕球心标定,得到子系统在球幕坐标系下的坐标,从而实现目标点在子系统之间的坐标转换。研究了球幕球心标定原理及标定点投射方法,建立了球幕球心在标定系统坐标系下的高斯-马尔科夫(G-M)求解模型,仿真分析了影响标定误差的因素。结果表明,通过减小子系统到球心的距离和改进投射的标定点空间分布能够有效提高标定精度,最后提出基于整体最小二乘(TLS)的球幕标定方法。根据仿真结果,设计模拟球幕和标定器并完成实验,标定结果满足现场快速精确标定的要求。     

A method for the analysis of low-mass molecules by MALDI-TOF mass spectrometry

We report a novel method termed matrix suppressed laser desorption/ionization to improve the analysis of low-mass molecules by MALDI-TOF mass spectrometry. In this method, the surfactant of cetrimonium bromide (CTAB) is added to the conventional matrix of alpha-cyano-4-hydroxycinnamic acid solution to prepare the MALDI samples. During the MALDI process, the presence of CTAB could substantially or even completely suppress the matrix-related ion background. As a result, very clean mass spectra can be routinely obtained in the low-mass range. In addition, the presence of CTAB can significantly improve the mass resolution of low-mass molecules. It is seen that high-quality spectra were routinely obtained at a matrix/CTAB ratio of 1000:1. This method has been successfully used to analyze a variety of low-mass molecules.     

HPLC and MALDI-TOF MS analysis of highly branched polystyrene: resolution enhancement by branching

Temperature gradient interaction chromatography (TGIC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were applied for the characterization of highly branched polystyrenes (PS) prepared by linking living polystyryl anions using 4-chlorodimethylsilylstyrene. Reversed-phase (RP)-TGIC showed an unexpectedly high resolution according to the number of branches despite significant overlap of the molecular weight as confirmed by MALDI-TOF MS. The enhancement of the resolution is ascribed to the contribution of the nonpolar groups in the branched PS: the dimethylsilyl groups in the branching unit as well as the sec-butyl initiator groups. As the number of branches increases, the number of nonpolar groups increases, which in turn increases the RP-TGIC retention synergistically with increasing molecular weight. In contrast, a poorer resolution was found in normal-phase-TGIC, in which the nonpolar groups reduce the retention. The resolution in RP-TGIC appears superior to that of liquid chromatography at the chromatographic critical condition (LCCC) of PS. It is seemingly due to the synergistic contribution of the incremental PS molecular weight to the functionality in the branched PS in RP-TGIC while only the functionality contributes to the separation in LCCC. This type of resolution enhancement could be utilized efficiently for the analysis of highly branched polymers such as dendrimers or hyperbranched polymers.     

LC/MS/MS method for quantitative determination of long-chain fatty acyl-CoAs

Long-chain acyl-CoA esters (LCACoAs) are activated lipid species that represent key substrates in lipid metabolism. The relationship between lipid metabolism disorders and type 2 diabetes has attracted much attention to this class of metabolites. This paper presents a highly sensitive and robust on-line LC/MS(2) procedure for quantitative determination of LCACoAs from rat liver. A fast SPE method has been developed without the need for time-consuming evaporation steps for sample preparation. LCACoAs were separated with high resolution using a C18 reversed-phase column at high pH (10.5) with an ammonium hydroxide and acetonitrile gradient. Five LCACoAs (C16:0, C16:1, C18:0 C18:1, C18:2) were quantified by selective multireaction monitoring using a triple quadrupole mass spectrometer in positive electrospray ionization mode. It is possible to perform a neutral loss scan of 507 for lipid profiling of complex LCACoA mixtures in tissue extracts. The method presented was validated according to ICH guidelines for quantitative determination of five LCACoAs for physiological concentrations in 100-200 mg of tissue with accuracies ranging from 94.8 to 110.8%, interrun precisions between 2.6 and 12.2%, and intrarun precisions between 1.2 and 4.4%. Due to the high sensitivity of the developed method, the amount of tissue biopsied for reliable quantification can be reduced. This may be advantageous in the quantification of LCACoAs in humans.     

A LC/MS method for the determination of cyanobacteria toxins in water

The cyanobacteria toxins anatoxin-a, microcystin-LR, microcystin-RR, microcystin-YR, and nodularin were separated in less than 30 min on several 1 mm x 15 cm reversed-phase liquid chromatography (LC) columns, and their electrospray mass spectra were measured using injections of 50 ng or less with a benchtop time-of-flight (TOF) mass spectrometer. New data from this work include the following: (a) the impact of acetic acid concentrations in the methanol-water mobile phase on measured ion abundances; (b) the performance of the electrospray-TOF mass spectrometer as an LC detector; (c) the accuracy and precision of exact m/z measurements after LC separation with a routinely used mass spectrometer resolving power of 5000; and (d) recoveries of the five toxins from reagent water, river waters, and sewage treatment plant effluent samples extracted with C-18 silica particles enmeshed in thin Teflon membrane filter disks. This technique has the potential of providing a relatively simple and reasonable-cost sample preparation and LC/MS method that provides the sensitivity, selectivity, reliability, and information content needed for source and drinking water occurrence and human exposure studies.     

Optimization of sample preparation for peptide sequencing by MALDI-TOF photofragment mass spectrometry

This paper describes the optimization of sample preparation for MALDI 193-nm photofragment ion time-of-flight mass spectrometry to sequence small to medium-sized peptides from peptide mixtures. We show that matrix additives, such as fructose and phenylbutyric acid have a dramatic effect on the abundance of fragment ions observed in the post-source decay spectra. A dried-droplet MALDI matrix consisting of 1:1 alpha-cyano-4-hydroxycinnamic acid/fructose proves to be an excellent matrix for photodissociation because [M + H]+ ions are formed with low internal energies, and the photofragment ion spectrum contains high abundances of sequence-informative ions. The addition of fructose appears to improve overall sample homogeneity and durability, as compared to conventional alpha-cyano-4-hydroxycinnamic acid dried-droplet preparations. MALDI-TOF photodissociation is then used to selectively sequence the peptides bradykinin (RPPGFSPFR), des-Arg9 bradykinin (RPPGFSPF), and substance P-amide (RPKPQQFFGLM-NH2) from a mixture of five peptides.     

Improvement of measurement precision of SPME-GC/MS determination of tributyltin using isotope dilution calibration

A unique approach was developed to improve the precision of quantification of tributyltin (TBT) in sedimentsby solid phase microextraction (SPME) using isotope dilution GC/MS. The precision of the analytical technique was initially evaluated using standard calibration solutions. In selective ion monitoring (SIM) mode, the relative standard deviation (RSD) obtained for TBT based on peak area response was 18% (n = 11). When an internal standard, tripropyltin (TPrT), was used, the RSD decreased to 12%. A significant improvement in the precision using SPME was noted when a 117Sn-enriched TBT spike was employed; the RSD decreased 4-fold to 3%. Detection limits of 0.2 and 20 ng(Sn) L(-1) were achieved with SPME sampling and liquid-liquid extraction, respectively. Six analyses were performed for determination of TBT in PACS-2 sediment Certified Reference Material using both standard additions and isotope dilution procedures. For the latter, a 117Sn-enriched TBT spike was used. A concentration of 0.88 +/- 0.03 microg g(-1) (RSD 3.4%) obtained using standard additions was in good agreement with the certified value of 0.98 +/- 0.13 microg g(-1) as tin. Concentrations found using isotope dilution were 0.895 +/- 0.015 microg g(-1) (RSD 1.73%) as tin and 0.874 +/- 0.014 microg g(-1) (RSD 1.66%) as tin using a liquid-liquid extraction and SPME sampling, respectively. A 2-fold improvement in the precision of TBT concentration measurement using isotope dilution was clearly achieved, demonstrating its superiority in providing more accurate and precise results as compared to the method of standard additions. The isotope dilution technique eliminated the problem of poor reproducibility, which typically plagues SPME.     

Method for screening and MALDI-TOF MS sequencing of encoded combinatorial libraries

We describe a new method for encoded synthesis, efficient on-resin screening, and rapid unambiguous sequencing of combinatorial peptide libraries. An improved binary tag system for encoding peptide libraries during synthesis was designed to facilitate unequivocal assignment of isobaric residues by MALDI-TOF MS analysis. The improved method for encoded library synthesis was combined with a new versatile on-resin screening strategy that permitted multiple stages and types of screening to be employed successively on one library under mild conditions. The new method facilitated a combinatorial study of transglutaminase (TGase) enzyme substrate peptides, revealing new details of the effect of amino acid composition on TGase substrates. The approach was first demonstrated for an encoded library (130,321 compounds) of lysine pentapeptide substrates of TGase, synthesized using the "split-mix" method. The library was reacted on-resin with TGase enzyme and a soluble desthiobiotin labeled glutamine substrate. Initial screening was performed by adsorbing streptavidin-coated magnetic microparticles onto library beads, followed by magnetic separation. The differential binding affinities of desthiobiotin and biotin for streptavidin were exploited to release the magnetic microparticles and regenerate the desthiobiotin-labeled resin beads for further screening by flow-cytometry-based automated bead sorting, resulting in 345 beads that were sequenced by MALDI-TOF MS analysis. A second library consisted of encoded glutamine hexapeptide substrates, which was reacted on-resin with TGase enzyme and a soluble desthiobiotin-labeled cadaverine. Two-stage screening identified 267 glutamine peptides as TGase-reactive, of which 21 were further analyzed by solution-phase enzyme kinetics. Kinetic results indicated that the peptide PQQQYV from the library has a 68-fold greater substrate specificity than the best known glutamine substrate QQIV. The new encoding and screening strategies described here are expected to be broadly applicable to synthesis and screening of combinatorial peptide libraries in the future.