MALDI mass spectrometric imaging of cardiac tissue following myocardial infarction in a rat coronary artery ligation model

Although acute myocardial infarction (MI) is consistently among the top causes of death in the United States, the spatial distribution of lipids and metabolites following MI remains to be elucidated. This work presents the investigation of an in vivo rat model of MI using mass spectrometric imaging (MSI) and multivariate data analysis. MSI was conducted on cardiac tissue following a 24-h left anterior descending coronary artery ligation to analyze multiple compound classes. First, the spatial distribution of a small metabolite, creatine, was used to identify areas of infarcted myocardium. Second, multivariate data analysis and tandem mass spectrometry were used to identify phospholipid (PL) markers of MI. A number of lysophospholipids demonstrated increased ion signal in areas of infarction. In contrast, select intact PLs demonstrated decreased ion signal in the area of infarction. The complementary nature of these two lipid classes suggests increased activity of phospholipase A(2), an enzyme that has been implicated in coronary heart disease and inflammation.     

High-spatial resolution mass spectrometric imaging of peptide and protein distributions on a surface

For the first time macromolecular ion microscope images have been recorded using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Single-shot, mass-resolved images of the spatial distributions of intact peptide and protein ions over an area of 200 microm in diameter were obtained in less than 1 ms at a repetition rate of 12 Hz. The magnifying ion optics of the ion microscope allowed ion images to be obtained with a lateral resolution of 4 microm. These results prove the concept of high-resolution MALDI-MS imaging in microscope mode without the need for a tight laser focus and the accompanying sensitivity losses. The ion microscopy approach offers an improvement of several orders of magnitude in speed of acquisition compared to the conventional (microprobe) approach to MALDI-MS imaging.     

Robust data processing and normalization strategy for MALDI mass spectrometric imaging

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) provides localized information about the molecular content of a tissue sample. To derive reliable conclusions from MSI data, it is necessary to implement appropriate processing steps in order to compare peak intensities across the different pixels comprising the image. Here, we review commonly used normalization methods, and propose a rational data processing strategy, for robust evaluation and modeling of MSI data. The approach includes newly developed heuristic methods for selecting biologically relevant peaks and pixels to reduce the size of a data set and remove the influence of the applied MALDI matrix. The methods are demonstrated on a MALDI MSI data set of a sagittal section of rat brain (4750 bins, m/z = 50-1000, 111 × 185 pixels) and the proposed preferred normalization method uses the median intensity of selected peaks, which were determined to be independent of the MALDI matrix. This was found to effectively compensate for a range of known limitations associated with the MALDI process and irregularities in MS image sampling routines. This new approach is relevant for processing of all MALDI MSI data sets, and thus likely to have impact in biomarker profiling, preclinical drug distribution studies, and studies addressing underlying molecular mechanisms of tissue pathology.     

Frontal immunoaffinity chromatography with mass spectrometric detection: a method for finding active compounds from traditional Chinese herbs

Frontal affinity chromatography (FAC) using immobilized polyclone antibodies of compound A coupled with mass spectrometry was used for the screening of affinity compounds from an extract of Phyllanthus urinaria L. Mass spectrometry was used as an analyzer of FAC. It can analyze the frontal affinity chromatogram of each compound of the extract in one program. The extract was dissolved in 2 mM NH4OAc at a concentration of 10 microg/ mL, then loaded on the immobilized antibody column, and data were collected from mass spectrometry to get a frontal affinity chromatogram. The screening of extract resulted in brevifolin, brevifolin carboxylic acid, corilagin, ellagic acid, and phyllanthusiin U. Activity analyses give high inhibitory activities to these compounds. This research work afforded us a new approach to find new leading compounds from nature or a man-made combinatorial library that have different structure styles or to find substitutes for the synthetic active compound that has high toxicity.     

Fiducial markers for combined 3-dimensional mass spectrometric and optical tissue imaging

Mass spectrometric imaging (MSI) has become widely used in the analysis of a variety of biological surfaces. Biological samples are spatially, morphologically, and metabolically complex. Multimodal molecular imaging is an emerging approach that is capable of dealing with this complexity. In a multimodal approach, different imaging modalities can provide precise information about the local molecular composition of the surfaces. Images obtained by MSI can be coregistered with images obtained by other molecular imaging techniques such as microscopic images of fluorescent protein expression or histologically stained sections. In order to properly coregister images from different modalities, each tissue section must contain points of reference, which are visible in all data sets. Here, we report a newly developed coregistration technique using fiducial markers such as cresyl violet, Ponceau S, and bromophenol blue that possess a combination of optical and molecular properties that result in a clear mass spectrometric signature. We describe these fiducial markers and demonstrate an application that allows accurate coregistration and 3-dimensional reconstruction of serial histological and fluorescent microscopic images with MSI images of thin tissue sections from a breast tumor model.     

Fused-core silica column high-performance liquid chromatography/tandem mass spectrometric determination of rimonabant in mouse plasma

A high-performance liquid chromatography (HPLC) method using a fused-core silica particle packing was evaluated to allow fast and efficient separation for the analysis of pharmaceutical compounds. Fused-core particles are produced by "fusing" a porous silica layer onto a solid silica particle. The efficiencies of columns packed with 2.7 microm "fused-core" particles (a 0.5 microm porous shell fused to a solid 1.7 microm silica core particle) and 1.7 microm porous particles were compared in reversed-phase HPLC using rimonabant as an analyte. The fused-core silica materials providing the shorter diffusional mass transfer path for solutes are less affected in resolving power by increases in mobile-phase velocity than the sub-2 microm porous silica packings resulting in faster separations and higher sample throughput. This fast HPLC technology is comparable with ultrahigh-pressure liquid chromatography (UHPLC) in terms of chromatographic performance but demands neither expensive ultra-high-pressure instrumentation nor new laboratory protocols. The column effluent was directly connected to the atmospheric pressure chemical ionization (APCI) source prior to tandem mass spectrometric detection. In this work, the described fast HPLC-MS/MS and UHPLC-MS/MS approaches requiring approximately 1.5 min per sample were applied and compared for the determination of the rimonabant in mouse plasma samples at the low nanograms per milliliter region in support of a pharmacodynamic study.     

Supercritical fluid chromatography and high-performance liquid chromatography/tandem mass spectrometric methods for the determination of cytarabine in mouse plasma

The separation of cytarabine (ara-C) from the endogenous compounds in mouse plasma by packed-column supercritical fluid chromatography (pSFC) was achieved on bare silica stationary phase with an isocratic mobile phase composed of CO2/methanol solvent with addition of ammonium acetate. SFC is commonly assumed to be only applicable to nonpolar and relatively low-polarity compounds. In this work, a broader range of compound polarities amenable to pSFC with appropriate mobile-phase modifiers and additives under normal-phase retention mechanism was demonstrated. The pSFC was integrated with an atmospheric pressure chemical ionization source and a tandem mass spectrometer (MS/MS) to enhance the sensitivity, selectivity, and speed of the assay. The influence of mobile-phase components on chromatographic performance and ionization efficiency of the test compounds was investigated for improving the sensitivity and separation for the analyte and the internal standard. The pSFC-MS/MS approach requiring approximately 2.5 min/sample for the determination of ara-C at nanograms per milliliter in mouse plasma was partially validated with respect to stability, linearity, and reproducibility. The mouse plasma levels of ara-C obtained by the pSFC-MS/MS method were found to be consistent with those determined by various reversed-phase, high-performance liquid chromatography methods using a porous graphite carbon column, a mixed-mode column, or a C18 column in conjunction with an ion-pairing agent coupled to a tandem mass spectrometer.     

Surface-based and mass spectrometric approaches to deciphering sugar-protein interactions in a galactose-specific agglutinin

Interest in powerful, nanosized tools to analyze in detail glycan-protein interactions has increased significantly over recent years. Here, we report two complementary approaches to characterize such interactions with high sensitivity, low sample consumption, and without the need for sample labeling, namely, surface plasmon resonance (SPR) and an approach that combines limited proteolysis and mass spectrometry. Combination of these two approaches to investigate glycan-protein interactions allows (1) to characterize interactions through kinetic and thermodynamic parameters, (2) to capture efficiently the carbohydrate-binding protein, and (3) to identify the interacted protein and its carbohydrate binding site by mass spectrometry. As a proof of principle, the interaction of the galactose-specific legume lectin Erythrina cristagalli agglutinin with several sugars has been characterized in-depth by means of these two approaches.     

Chlorine stable isotopes: a comparison of dual inlet and thermal ionization mass spectrometric measurements

Chlorine stable isotope ratios, 37Cl/35Cl, currently are measured using dual-inlet and thermal-ionization mass spectrometry. These two different analytical techniques, however, have never been cross calibrated. A set of samples with chlorine stable isotope delta values ranging from -4.4 to +0.3 % relative to standard mean ocean water chloride has been analyzed using both of these techniques. Our data show that both techniques can yield similar results within analytical uncertainty. CsCl thermal ionization data are extremely sensitive to the amount of chlorine being measured and cannot be used to determine absolute ratios without an independent means of correcting for machine-induced mass fractionation. As long as standards and samples are of equivalent size, however, the differences between samples measured by thermal ionization remain constant Dual inlet stable isotope mass spectrometry is suited best for samples of > 10 micromol Cl, yielding chlorine stable isotope data with < or =0.1% reproducibilities (2sigma). Thermal ionization mass spectrometry easily accommodates samples of approximately0.1-0.3 micromol Cl, with achievable uncertainties of < or =0.2% (2sigma).     

Multispectral method for skin imaging: development and validation

A visible wide field multispectral system for comprehensive imaging of skin chromophores and blood vessels has been implemented, and an inhomogeneous Monte Carlo model of photon migration with randomly distributed blood vessels embedded in dermis has been developed. Predetermined nonlinear transforms have been obtained to address the nonlinear interdependent relationship among diffusive reflectance spectra, skin physiology properties, and geometry. For validation, in addition to real skin experiments and phantoms experiments, two alternative methods for blood vessel imaging have been used on the same set of subjects to compensate for the lack of ground truth for skin subsurface imaging.     

Task-based imaging of colon cancer in the Apc(Min/+) mouse model

Optical coherence tomography (OCT), laser-induced fluorescence (LIF), and laser-scanning confocal microscopy (LSCM) were used for the task of multimodal study of healthy and adenomatous mouse colon. The results from each modality were compared with histology, which served as the gold standard. The Apc(Min/+) genetic mouse model of colon cancer was compared with wild-type mice. In addition, a special diet was used for the task of studying the origins of a 680 nm autofluorescent signal that was previously observed in colon. The study found close agreement among each of the modalities and with histology. All four modalities were capable of identifying diseased tissue accurately. The OCT and LSCM images provided complementary structural information about the tissue, while the autofluorescence signal measured by LIF and LSCM provided biochemical information. OCT and LIF were performed in vivo and nondestructively, while the LSCM and histology required extraction of the tissue. The magnitude of the 680 nm signal correlates with chlorophyll content in the mouse diet, suggesting that the autofluorescent compound is a dietary metabolite.     

Immunopurification and mass spectrometric quantification of the active form of a chimeric therapeutic antibody in human serum

In this study, we show that liquid chromatography coupled with tandem mass spectrometry provides a sensitive, specific, and accurate absolute quantification of Erbitux, a human:murine chimeric mAb used for the treatment of colorectal cancer. Micrometric magnetized beads, functionalized with soluble epidermal growth factor receptor (sEGFR), the pharmacological target of Erbitux, were used for specific immunocapture of Erbitux allowing assessment of the antibody's biological potency and sample purification. Following digestion with trypsin, specific peptides from light and heavy chains were monitored in the selected reaction monitoring (SRM) mode. Assay variability below 20% was provided through optimization of the digestion step and rigorous monitoring of the whole analytical process using an appropriate internal standard. The 20 ng/mL lower limit of quantification was similar to that of ELISA methods. These results show that this mass spectrometric approach is a potential alternative for pharmacokinetic evaluation of mAbs during clinical development.     

3D imaging by mass spectrometry: a new frontier

Imaging mass spectrometry can generate three-dimensional volumes showing molecular distributions in an entire organ or animal through registration and stacking of serial tissue sections. Here, we review the current state of 3D imaging mass spectrometry as well as provide insights and perspectives on the process of generating 3D mass spectral data along with a discussion of the process necessary to generate a 3D image volume.     

Implementation and validation of a triaxiality dependent cohesive model: experiments and simulations

A recently formulated triaxiality dependent cohesive model for plane strain is implemented and its versatility is tested in simulation of ductile fracture of mild steel at different states of stress. The triaxiality dependent model was implemented as linear displacement formulation based elements whose constitutive behaviour was dependent on the stress-state of the neighbouring continuum element. By comparing the experimental data and predictions of corresponding plane strain simulations, the model parameters are estimated. The model is shown to be effective in reproducing characteristic features of the macroscopic response of both pre-cracked as well as geometries without a preexisting nominal defect. Since the model parameters are held constant for simulations at different stress-states, they are effectively material constants.     

Instrumental parameters in the MALDI-TOF mass spectrometric analysis of quaternary protein structures

Several former studies have shown that MALDI-TOF-MS can be applied successfully to investigate the quaternary structure of proteins. Whereas most of these reports were focused on MALDI sample preparation, there is little information about the influence of instrumental parameters on the desorption/ionization and gas-phase behavior of protein subunit assemblies. Therefore, in addition of giving short examples of the quaternary structure analysis of a microheterogeneous glycoprotein, a metalloenzyme, and a heme-binding enzyme by MALDI-TOF-MS, we report a systematic study of the effect of some instrumental parameters on the analysis of chicken egg white avidin. From these tested parameters, only the laser pulse energy was found to influence the relative abundance of the intact assembly as well as the formation of nonspecific cluster ions significantly. This finding suggests that the disruption of the noncovalent interactions during the desorption/ionization process takes place at a very short time interval after the laser ablation, whereas those assemblies that survive this step are rather stable afterward in the gas phase. In addition, we present clear evidence that protein cluster ions are not preformed during sample preparation but originate from nonspecific assemblage during desorption/ionization.     

Optical and mass spectrometric study of the pyrolysis gas of wood particles

A detailed experimental investigation has been made of the pyrolysis--the first step in biomass combustion--of single birchwood particles. In addition to mass spectrometric and gravimetric analysis, the pyrolysis volatiles were characterized by different optical techniques. Absorption measurements showed a nearly featureless absorption in the ultraviolet spectral region with a continuously stronger absorption for shorter wavelengths. Using different excitation wavelengths, laser-induced fluorescence measurements revealed generally broad spectra in the spectral region from 300-500 nm, which are characteristic spectral signatures for larger hydrocarbons. The optical data were monitored at different times in the pyrolysis process of the particles and compared with the results from the mass spectrometric and gravimetric analysis. The sensitivity of the optical techniques for differentiation between specific molecules was rather low, although formaldehyde could be observed both in absorption and fluorescence spectra. Laser-induced fluorescence measurements were also made for two-dimensional visualization of the pyrolysis volatiles emitted from heated birchwood particles, indicating much higher flows along the fiber direction than across.     

STEP (Statistical Test of Equivalent Pathways) analysis: a mass spectrometric method for carbohydrates and peptides

We have recently developed a new mass spectrometry method, the STEP (statistical test of equivalent pathways) analysis that uses ion abundances in two tandem mass spectrometry experiments to obtain genealogy information about product ions present in mass spectra. The method requires minimal sample, and it can be performed using a conventional quadrupole ion trap mass spectrometer. To obtain genealogy information, STEP ratios are calculated by comparing the relative abundances of product ions in two MS/MS experiments. These ratios are directly related to the origin of the product ions. Product ions that result directly from the precursor ion always have STEP ratios that are 相似文献   

Phakometry and lens tilt and decentration using a custom-developed Purkinje imaging apparatus: validation and measurements

We present a Purkinje imaging system for phakometry and measurement of tilt and decentration of crystalline and intraocular lenses (IOLs). Crystalline lens radii of curvature were estimated by using both a merit function and the equivalent mirror approaches. Tilts and decentrations were estimated by using Phillips's linear analysis. We present a complete validation of the technique through exhaustive computer simulations and control experiments, and measurements in 17 normal eyes (mean age 26.67 +/- 2.31) and nine postcataract surgery eyes (mean age 74 +/- 2.3). Crystalline lens radii ranged from 12.7 to 8.81 mm and from -5.64 to -7.09 mm for anterior and posterior surfaces, respectively. Crystalline lens tilt ranged from 2.8 to -2.87 deg horizontally and from 2.58 to -1 deg vertically. Crystalline lens decentration ranged from 0.09 to 0.45 mm horizontally and from 0.09 to -0.22 mm vertically. IOL tilt ranged from 3.6 to -1.51 deg horizontally and from 5.97 to -1.85 deg vertically. IOL decentration ranged from 0.53 to -0.31 mm horizontally and from 0.13 to -0.96 mm vertically.     

Determination of serum cholesterol by a modification of the isotope dilution mass spectrometric definitive method

An isotope dilution mass spectrometric (ID/MS) method for cholesterol is described that uses capillary gas chromatography with cholesterol-13C3 as the labeled internal standard. Labeled and unlabeled cholesterol are converted to the trimethylsilyl ether. Combined capillary column gas chromatography and electron impact mass spectrometry are used to obtain the abundance ratio of the unlabeled and labeled [M+.] ions from the derivative. Quantitation is achieved by measurement of each sample between measurements of two standards whose unlabeled/labeled ratios bracket that of the sample. Seven pools were analyzed by this method: standard reference material (SRM) 1951, which consists of three frozen serum pools with low, medium, and high levels of cholesterol; SRM 1952, which consists of three freeze-dried serum pools with low, medium, and high levels of cholesterol; and SRM 909, a freeze-dried serum pool. The method is a modification of our original definitive method for cholesterol. The modified method uses much better chromatographic separations to assure specificity and a new method of implementing selected ion monitoring on a magnetic mass spectrometer to obtain high-precision measurements of ion intensity ratios on narrow gas chromatographic peaks. The modified method has a coefficient of variation (CV) of 0.22%, which is an improvement over the original method's CV of 0.36%. The measurements were found to be free of interference. The high precision and absence of bias qualify this method as a candidate definitive method.