Search

The rapid evolution of the flow cytometry field, currently allowing the measurement of 30-50 parameters per cell, has led to a marked increase in deep multivariate information. Manual gating is insufficient to extract all this information. Therefore, multivariate analysis (MVA) methods have been developed to extract information and efficiently analyze the high-density multicolour flow cytometry (MFC) data. To aid interpretation, MFC data are often logarithmically transformed before MVA. We studied the consequences of different transformations of flow cytometry data in datasets containing negative intensities caused by background subtractions and spreading error, as logarithmic transformation of negative data is impossible. Transformations such as logicle or hyperbolic arcsine transformations allow linearity around zero, whereas higher (positive and negative) intensities are logarithmically transformed. To define the linear range, a parameter (or cofactor) must be chosen. We show how the chosen transformation parameter has great impact on the MVA results. In some cases, peak splitting is observed, producing two distributions around zero in an actual homogeneous population. This may be misinterpreted as the presence of multiple cell populations. Moreover, when performing arbitrary transformation before MVA analysis, biologically relevant and statistically significant information might be missed. We present a new algorithm, Optimal Transformation for flow cytometry data (OTflow), which uses various statistical methods to optimally choose the parameter of the transformation and prevent artifacts such as peak splitting. Arbitrary or unconsidered transformation can lead to wrong conclusions for the MVA cluster methods, dimensionality reduction methods, and classification methods. We recommend transformation of flow cytometry data by using OTflow-defined parameters estimated per channel, in order to prevent peak splitting and other artifacts in the data.

Multicolor Flow Cytometry (MFC)-based gating allows the selection of cellular (pheno)types based on their unique marker expression. Current manual gating practice is highly subjective and may remove relevant information to preclude discovery of cell populations with specific co-expression of multiple markers. Only multivariate approaches can extract such aspects of cell variability from multi-dimensional MFC data. We describe the novel method ECLIPSE (Elimination of Cells Lying in Patterns Similar to Endogeneity) to identify and characterize aberrant cells present in individuals out of homeostasis. ECLIPSE combines dimensionality reduction by Simultaneous Component Analysis with Kernel Density Estimates. A Difference between Densities (DbD) is used to eliminate cells in responder samples that overlap in marker expression with cells of controls. Thereby, subsequent data analyses focus on the immune response-specific cells, leading to more informative and focused models. To prove the power of ECLIPSE, we applied the method to study two distinct datasets: the in vivo neutrophil response induced by systemic endotoxin challenge and in studying the heterogeneous immune-response of asthmatics. ECLIPSE described the well-characterized common response in the LPS challenge insightfully, while identifying slight differences between responders. Also, ECLIPSE enabled characterization of the immune response associated to asthma, where the co-expressions between all markers were used to stratify patients according to disease-specific cell profiles.

The molecular characterization of castor oil ethoxylates (CASEOs) was studied by reverse-phase liquid chromatography (RPLC) mass spectrometry (MS) and multistage mass spectrometry (MS(n)). The developed RPLC method allowed the separation of the various CASEO components, and especially, the baseline separation of multiple nominal isobars (same nominal mass) and isomers (same exact mass). MS and MS(n) were used for the determination and structure elucidation of various structures and for the discrimination of the isobars and isomers. Different ionization techniques and adduct ions were also tested for optimization of the MS detection and the MS(n) fragmentation. A unique fragmentation pathway of ricinoleic acid is proposed, which can be used as a marker of the polymerization process and the topology of ethoxylation in the CASEO. In addition, characteristic neutral losses of ricinoleic acid reveal its (terminal or internal) position in the molecule.

The formation of low charge state non-covalent complexes of high molar mass polymers with primary and tertiary amines and quaternary ammonium salts (Quats) was studied by electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The amines studied were of different degree of substitution and size, and were added to the acidic polymer solution prior to MS analysis. A comparison was made between the non-covalent complexes of high molar mass polyethers with various amines and Quats, based on the ability to form low charge state adduct ions, overall MS response, and presence of fragment ions due to up-front fragmentation. Tertiary amines showed the best performance in forming low charge state adduct ions and produced mass spectra with the lowest degree of up-front fragmentation. In both primary and tertiary amines, more simplified mass spectra are produced, since no cluster ions are formed (as in the case of Quats). The comparison of the overall MS response did not reveal a specific trend. An amine and a Quat with an optimal performance for formation of low charge state adduct ions were applied to the mass spectrometric analysis of a poly(lactic acid)- block -poly(ethylene glycol)- block -poly(lactic acid) triblock copolymer, allowing the detection of low charge state adduct ions and revealing information about the molecular weight distribution. The method demonstrated here shows that addition of primary/tertiary amines and quaternary ammonium salts to the spray solvent facilitates the analysis of high molar mass polyethers by ESI-MS.

Further dimensions: The title mass spectrometric technique offers an extra dimension of separation based on size and conformation of the molecules in addition to m/z separation without the need of time-consuming liquid chromatography separation. Detailed mapping of the mass and drift time separated ions allowed identification of end groups originating from various initiation and termination reactions.

The characteristic collision energy to obtain 50% fragmentation, expressed as the characteristic collision voltage (CCV), was used as a tool to discriminate different classes of polymers. The CCV value of different polymers was determined in a quadrupole ion trap mass spectrometer. Good linear correlation (0.980R(2)0.999) between the CCV values and precursor ion mass was found for all polymers studied. The position of the various linear trend lines varied among the various polymers, which allowed their grouping based on the respective CCV values. The collision energy necessary to drive fragmentation was decreasing in the order of polyetherspolymethacrylatespolyesterspolysaccharides. This suggests that polysaccharides fragment most easily (low CCVs), while polyethers require the highest collision energy among the polymers studied. The effect of end group on the CCV was also studied, showing a minor influence in most cases. In addition, the applicability of CCV as discriminator was studied for a mixture of (1) polylactic acid (PLA), (2) poly(tetramethylene glycol) (PTMEG), and (3) PLA-block-PTMEG-block-PLA block copolymer. Differences between the CCV values of four nominally isobaric polymers (of which two were copolymers and two were homopolymers) were observed. These results demonstrate that the insertion of a "weak" link into a polymer chain significantly affects the energy required for fragmentation.

Factors such as high polymer dispersity and variation in elemental composition (of copolymers) often complicate the electrospray ionization mass spectrometry (ESI-MS) analysis of synthetic polymers with high molar mass. In the experiments described in this study, quaternary ammonium compounds were observed to facilitate the production of low charge state pseudomolecular ions when added to the spray solution for ESI-MS. This approach was then used for the ESI time-of-flight mass spectrometry (TOF-MS) analysis of synthetic polymers. Hexadecyltrimethylammonium chloride permitted the successful analysis of poly(ethylene glycol) of 2-40 kDa, poly(propylene glycol) and poly(tetramethylene glycol) oligomers. Increasing the quaternary ammonium compounds' concentration results in the production of low charge state pseudomolecular ions. A comparison of structurally different quaternary ammonium compounds showed that the best performance is expected from large molecules with specific charge localization, which leaves the charge available for interactions. The applicability of the method for the MS analysis of other polymeric systems was also studied. In the case of poly(tetramethylene glycol), the method not only shifted the distributions to higher m/z values but also allowed the detection of high molecular weight material that was not observed without addition of the modifier to the spray solution.

International audience; The collision energy or collision voltage necessary to obtain 50% fragmentation (characteristic collision energy/voltage, CCE or CCV) has been systematically determined for different types of molecules [poly(ethylene glycols) (PEG), poly(tetrahydrofuran) (PTHF), and peptides] over a wide mass (degrees of freedom) range. In the case of lithium-cationized PEGs a clear linear correlation (R(2) > 0.996) has been found between CCE and precursor ion mass on various instrument types up to 4.5 kDa. A similar linear correlation was observed between CCV and the mass-to-charge ratio. For singly and multiply charged polymers studied under a variety of experimental conditions and on several instruments, all data were plotted together and showed correlation coefficient R(2) = 0.991. A prerequisite to observe such a good linear correlation is that the energy and entropy of activation in a class of polymers is likely to remain constant. When compounds of different structure are compared, the CCV will depend not only on the molecular mass but the activation energy and entropy as well. This finding has both theoretical and practical importance. From a theoretical point of view it suggests fast energy randomization up to at least 4.5 kDa so that statistical rate theories are applicable in this range. These results also suggest an easy method for instrument tuning for high-throughput structural characterization through tandem MS: after a standard compound is measured, the optimum excitation voltage is in a simple proportion with the mass of any structurally similar analyte at constant experimental conditions.

Oxidative changes in triacylglycerols and diacylphosphatidylcholines in egg tempera paint strips are used for chemical dosimetry of the quality of the museum environment. High-resolution matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) was used as a rapid method for the determination of the exact elemental composition of the alteration products from diacylphosphatidylcholines and triacylglycerols. Light exposure of the egg tempera paints yields oxygenated diacylphosphatidylcholines and triacylglycerols. In the latter multiple incorporation of oxygen was observed as a recurring mass difference of 15.995, the exact atomic mass of oxygen. Owing to the high resolution of the FTMS data (routinely 20 000 at m/z 1000 in broadband mode), oxidation products with different elemental compositions but identical nominal mass could be distinguished. Products of oxidative cleavage of triacylglycerols were observed in samples exposed for longer times. The relative intensities of the peaks of singly and multiply oxygenated triacylglycerols were used to derive the degree of oxygenation of the egg lipids in the tempera paint dosimeters. The degree of oxygenation was found to be directly related to the light exposure time. Exposure to elevated temperature (60 °C) for a period of 21 days did not lead to oxygenation of the triacylglycerols and diacylphosphatidylcholines. Exposure to NOx and SO2 in the dark greatly increased the degree of oxygenation. Addition of lead- or copper-containing pigments to the egg binding medium (and subsequent storage for 6 months in the dark) led to accelerated conversion of egg lipids to oxidised products. Copyright © 2001 John Wiley & Sons, Ltd.

Quaternary ammonium salts (Quats) and amines are known to facilitate the MS analysis of high molar mass polyethers by forming low charge state adduct ions. The formation, stability, and behavior upon collision-induced dissociation (CID) of adduct ions of polyethers with a variety of Quats and amines were studied by electrospray ionization quadrupole time-of-flight, quadrupole ion trap, and linear ion trap tandem mass spectrometry (MS/MS). The linear ion trap instrument was part of an Orbitrap hybrid mass spectrometer that allowed accurate mass MS/MS measurements. The Quats and amines studied were of different degree of substitution, structure, and size. The stability of the adduct ions was related to the structure of the cation, especially the amine's degree of substitution. CID of singly/doubly charged primary and tertiary ammonium cationized polymers resulted in the neutral loss of the amine followed by fragmentation of the protonated product ions. The latter reveals information about the monomer unit, polymer sequence, and endgroup structure. In addition, the detection of product ions retaining the ammonium ion was observed. The predominant process in the CID of singly charged quaternary ammonium cationized polymers was cation detachment, whereas their doubly charged adduct ions provided the same information as the primary and tertiary ammonium cationized adduct ions. This study shows the potential of specific amines as tools for the structural elucidation of high molar mass polyethers.

The cleaning of paintings using UV lasers is a growing field of interest in the practice of conservation. In this work, we have studied the chemical and physical changes induced by KrF excimer laser at 248 nm of tempera paint dosimeter systems. The changes have been evaluated by using a range of analytical techniques. These include profilometry; colorimetry; optical and vibrational spectroscopies, such as laser-induced fluorescence (LIF), laser-induced breakdown spectroscopy (LIBS), Fourier transform Raman (FTR), and infrared (FT-IR); and analytical mass spectrometric techniques, such as direct-temperature-resolved mass spectrometry (DTMS) and matrix-assisted laser desorption and ionization mass spectrometry (MALDI-MS). Integration of the results obtained by these techniques allowed the investigation of the nature and degree of change of the irradiated paint systems. Direct laser irradiation induces various degrees of discoloration that depend strongly on the nature of the pigment. This effect takes place mainly on the surface layer of the sample. Degradation of the binding medium occurs in the presence of inorganic pigments, and in some cases, evidence of alterations in the molecular composition of the pigment has been obtained. Varnished systems do not display this discoloration when a thin protective layer is left on the paint. A laser cleaning strategy for varnished paintings should be based on the partial removal of the varnish, leaving a residual layer that shields the underlying pigments from direct laser exposure., Funding by the European Commission (Project ENV4-CT98-0787) is gratefully acknowledged.

The Cooperative Research project “Advanced workstation for controlled laser cleaning of artworks” (ENV4-CT98-0787) has yielded important information on the application of UV laser cleaning to paint materials. In the project, in which conservators, researchers and engineers participated, the viability of the laser technique as an additional tool in present conservation practice was investigated. The research was pointed at the definition of the boundary conditions in which laser cleaning can be safely applied. It included a systematic effect study of tempera paint systems. Physical and chemical changes, induced by exposure to UV (248 nm) excimer laser light under various conditions, were evaluated. In parallel, an innovative laser cleaning tool was developed, allowing accurate and controlled removal of superficial layers from paint materials. Both aspects of the project are presented. The presentation of the research focuses on the integration of the results from various analytical techniques, yielding valuable information on the immediate and long-term effects of UV laser radiation on the paint materials. The analytical techniques include colorimetry, spectroscopic techniques, mass spectrometry and profilometry, as well as thermographic and UV transmission measurements. Furthermore, the application of the laser workstation on various painted artworks is shown. This includes the gradual removal of varnish layers and the recovery of original paint colour in fire-damaged paintings., The European Commission is gratefully acknowledged for facilitating the work in the Cooperative Research project “Advanced workstation for controlled laser cleaning of artworks” (ENV4-CT98-0787).

A systematic study of the chemical and physical changes induced by exposure to UV (248 nm) excimer laser light of unvarnished tempera paint samples has been undertaken as a part of the research activities included in the European project "Advanced workstation for controlled laser cleaning of artworks". The direct exposure of the paint to the UV laser configures the worst case scenario of laser cleaning, as a thin protective layer of varnish is normally left to minimize the dose of UV radiation that reaches the paint surface. However, in the practice of laser cleaning, there is a need to characterize and quantify the possible effects of direct UV laser irradiation of unvarnished paints. To this purpose, a broad range of techniques have been used including profilometry, colorimetry, optical and vibrational spectroscopic techniques, such as laser-induced fluorescence (LIF), laser-induced breakdown spectroscopy (LIBS), Fourier transform Raman (FTR) and infrared (FTIR), and analytical mass spectrometric techniques, like direct-temperature-resolved mass spectrometry (DTMS) and laser desorption and ionization time of flight mass spectrometry (LDI-TOF). Integration of the results obtained by these techniques allowed the investigation of the nature and degree of change of the irradiated paint systems. These were observed to strongly depend on the type of paint system. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.