Matrix-assisted laser desorption/ionization-ion mobility separation-mass spectrometry imaging of vinblastine in whole body tissue sections

During early-stage drug development, drug and metabolite distribution studies are carried out in animal tissues using a range of techniques, particularly whole body autoradiography (WBA). While widely employed, WBA has a number of limitations, including the following: expensive synthesis of radiolabeled drugs and analyte specificity and identification. WBA only images the radiolabel. MALDI MSI has been shown previously to be advantageous for imaging the distribution of a range of drugs and metabolites in whole body sections. Ion mobility separation (IMS) adds a further separation step to imaging experiments; demonstrated here is MALDI-IMS-MS whole body imaging of rats dosed at 6 mg/kg i.v. with an anticancer drug, vinblastine and shown is the distribution of the precursor ion m/z 811.4 and several product ions including m/z 793, 751, 733, 719, 691, 649, 524, and 355. The distribution of vinblastine within the ventricles of the brain is also depicted. Clearly demonstrated in these data are the removal of interfering isobaric ions within the images of m/z 811.4 and also of the transition m/z 811-751, resulting in a higher confidence in the imaging data. Within this work, IMS has shown to be advantageous in both MS and MS/MS imaging experiments by separating vinblastine from an endogenous isobaric lipid.     

MALDI ion trap mass spectrometer with charge detector for large biomolecule detection

Up to now, all commercial matrix-assisted laser desorption/ionization (MALDI) mass spectrometers still can not efficiently analyze very large biomolecules. In this work, we report the development of a novel MALDI ion trap mass spectrometer which can enrich biomolecular ions to enhance the detection sensitivity. A charge detector was installed to measure the large ions directly. With this design, we report the first measurement of IgM with the mass-to-charge ratio (m/z) at 980?000. In addition, quantitative measurements of the number of ions can be obtained. A step function frequency scan was first developed to get a clear signal in the m/z range from 200,000 to 1,000,000.     

Advancing matrix-assisted laser desorption/ionization-mass spectrometric imaging for capillary electrophoresis analysis of peptides

In this work, the utilization of matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) for capillary electrophoresis (CE) analysis of peptides based on a simple and robust off-line interface has been investigated. The interface involves sliding the CE capillary distal end within a machined groove on a MALDI sample plate, which is precoated with a thin layer of matrix for continuous sample deposition. MALDI-MSI by time of flight (TOF)/TOF along the CE track enables high-resolution and high-sensitivity detection of peptides, allowing the reconstruction of a CE electropherogram while providing accurate mass measurements and structural identification of molecules. Neuropeptide standards and their H/D isotopic formaldehyde-labeled derivatives were analyzed using this new platform. Normalized intensity ratios of individual ions extracted from the CE trace were compared to MALDI-MS direct analysis and the theoretical ratios. The CE-MALDI-MSI results show potential for sensitive and quantitative analysis of peptide mixtures spanning a wide dynamic range.     

Frequency-scanning MALDI linear ion trap mass spectrometer for large biomolecular ion detection

This study presents the first report on the development of a matrix-assisted laser desorption ionization (MALDI) linear ion trap mass spectrometer for large biomolecular ion detection by frequency scan. We designed, installed, and tested this radio frequency (RF) scan linear ion trap mass spectrometer and its associated electronics to dramatically extend the mass region to be detected. The RF circuit can be adjusted from 300 to 10 kHz with a set of operation amplifiers. To trap the ions produced by MALDI, a high pressure of helium buffer gas was employed to quench extra kinetic energy of the heavy ions produced by MALDI. The successful detection of the singly charged secretory immunoglobulin A ions indicates that the detectable mass-to-charge ratio (m/z) of this system can reach ~385 000 or beyond.     

Analysis of intact tissue by intermediate-pressure MALDI on a linear ion trap mass spectrometer

The use of an intermediate-pressure matrix-assisted laser desorption/ionization (IP-MALDI) source working at 0.17 Torr on a linear ion trap (LIT) was investigated for the analysis of tissue specimens, in particular, spinal cord sections. MALDI, with 2,5-dihydroxybenzoic acid (DHB) as the matrix, was employed for the detection of phospholipids. The matrix was applied to the tissue using electrospray to avoid analyte migration. The results indicate that analyzing tissue specimens at nontraditional MALDI vacuum pressures is possible. Coupling MALDI to an LIT permits the use of MSn, which is critical for the ability to identify compounds desorbed directly from tissue specimens. Using MSn, ions detected from m/z 600-1000 were characterized as phosphatidlycholines, PC. Specifically, using tandem MS, PC ions could be classified as either [M + H]+ or [M + Na]+ because the fragmentation patterns of protonated and sodiated phosphatidlycholines follow different pathways.     

Determination and characterization of site-specific N-glycosylation using MALDI-Qq-TOF tandem mass spectrometry: case study with a plant protease

MALDI tandem mass spectrometry analysis on a hybrid quadrupole-quadrupole time-of-flight (Qq-TOF) instrument was used in combination with two-dimensional gel electrophoresis, proteolytic digestion, and liquid chromatography for identification and structural characterization of glycosylation in a novel glycoprotein, pathogenesis-related subtilisin-like proteinase P69B from tomato. Glycopeptide fractions from microcolumn reversed-phase HPLC deposited on MALDI targets were identified from MS by their specific m/z spacing patterns (203, 162, 146 u) between glycoforms. In most cases, MS/MS spectra of [M + H]+ ions of glycopeptides featured peaks useful for determining sugar compositions, peptide sequences, and thus probable glycosylation sites. Furthermore, peptide-related product ions could readily be used in database search procedures to identify the glycoprotein. Four out of five predicted glycosylation sites were biologically relevant and occupied by five N-linked glycan side chains each. In addition, the fragmentation efficiency allowed detection of further modification of methionine-containing glycoforms with either oxidized or iodoacetamide alkylated methionine. The high resolution furnished by MALDI-Qq-TOF allowed rapid and sensitive structural characterization of site-specific N-glycosylation from a limited quantity of material and revealed heterogeneity at different levels, including different glycan side-chain modifications, and heterogeneity of oligosaccharide structures on the same glycosylation site.     

High-sensitivity MALDI-MRM-MS imaging of moxifloxacin distribution in tuberculosis-infected rabbit lungs and granulomatous lesions

MALDI-MSI is a powerful technology for localizing drug and metabolite distributions in biological tissues. To enhance our understanding of tuberculosis (TB) drug efficacy and how efficiently certain drugs reach their site of action, MALDI-MSI was applied to image the distribution of the second-line TB drug moxifloxacin at a range of time points after dosing. The ability to perform multiple monitoring of selected ion transitions in the same experiment enabled extremely sensitive imaging of moxifloxacin within tuberculosis-infected rabbit lung biopsies in less than 15 min per tissue section. Homogeneous application of a reference standard during the matrix spraying process enabled the ion-suppressing effects of the inhomogeneous lung tissue to be normalized. The drug was observed to accumulate in granulomatous lesions at levels higher than that in the surrounding lung tissue from 1.5 h postdose until the final time point. MALDI-MSI moxifloxacin distribution data were validated by quantitative LC/MS/MS analysis of lung and granuloma extracts from adjacent biopsies taken from the same animals. Drug distribution within the granulomas was observed to be inhomogeneous, and very low levels were observed in the caseum in comparison to the cellular granuloma regions. In this experiment the MALDI-MRM-MSI method was shown to be a rapid and sensitive method for analyzing the distribution of anti-TB compounds and will be applied to distribution studies of additional drugs in the future.     

Characterizing the phospholipid profiles in mammalian tissues by MALDI FTMS

Discussed here is an analytical method for profiling lipids and phospholipids directly from mammalian tissues excised from Mus musculus (house mouse). Biochemical analysis was accomplished through the use of matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry, where whole tissue sections of mouse brain, heart, and liver were investigated. Lipid and phospholipid ions create complex MALDI mass spectra containing multiple ions with different m/z values corresponding to the same fundamental chemical species. When a computational sorting approach is used to group these ions, the standard deviation for observed relative chemical abundance can be reduced to 6.02%. Relative standard deviations of 10% are commonly accepted for standard chromatographic phospholipid analyses. Average mass measurement accuracy for 232 spectra representing three tissue types from 12 specimens was calculated to be 0.0053 Da. Further it is observed, that the data and the analysis between all the animals have near-identical phospholipid contents in their brain, heart, and liver tissues, respectively. In addition to the need to accurately measure relative abundances of phospholipid species, it is essential to have adequate mass resolution for complete and accurate overall analysis. It is reasonable to make mass composition assignments with spectral resolving power greater than 8000. However, results from the present study reveal 14 instances (C12 carbon isotope) of multiple m/z ions having the same nominal value that require greater resolution in order that overlap will not occur. Spectra measured here have an average resolving power of 12 000. It is established that high mass resolution and mass accuracy coupled with MALDI ionization provide for rapid and accurate phospholipid analysis of mammalian tissue sections.     

Analysis of protein phosphorylation by hypothesis-driven multiple-stage mass spectrometry

We describe a strategy, which we term hypothesis-driven multiple-stage mass spectrometry (HMS-MS), for the sensitive detection and identification of phosphopeptides derived from enzymatic digests of phosphoproteins. In this strategy, we postulate that any or all of the potential sites of phosphorylation in a given protein may be phosphorylated. Using this assumption, we calculate the m/z values of all the corresponding singly charged phosphopeptide ions that could, in theory, be produced by the enzyme employed for proteolysis. We test ions at these m/z values for the presence of phosphoserine or phosphothreonine residues using tandem mass spectrometry (MS(2)) in a vacuum MALDI ion trap mass spectrometer, where the neutral loss of the elements of H(3)PO(4) (98 Da) provides a sensitive assay for the presence of phosphopeptides. Subsequent MS(3) analysis of the (M + H - 98)(+) peaks allows us to confirm or reject the hypotheses that the putative phosphopeptides are present in the sample. HMS-MS was successfully applied to the detection and identification of phosphopeptides from substrates of the Saccharomyces cerevisiae cyclin-dependent kinase (Cdk) Cdc28, phosphorylated in vitro (Ipl1) and in vivo (Orc6), basing hypothesis formation on the minimal Cdk consensus phosphorylation motif Ser/Thr-Pro. The method was also used to find in vitro phosphopeptides from a domain of the Drosophila melanogaster protein PERIOD, hypothesizing possible phosphorylations of all Ser/Thr residues without assuming a consensus motif. Our results demonstrate that HMS-MS is a sensitive, highly specific tool for systematically surveying proteins for Ser/Thr phosphorylation, and represents a significant step toward our goal of comprehensive phosphorylation mapping.     

Identification of bacterial proteins observed in MALDI TOF mass spectra from whole cells.

Characteristic ions in the MALDI TOF mass spectra from bacterial cells have been associated with four known proteins. The proteins, observed both from cells and in filtered cellular suspensions, were isolated by HPLC and identified on the basis of their mass spectra and their partial amino acid sequence, determined using the Edman method (10-15 residues). The acid resistance proteins HdeA and HdeB give rise to ions near m/z 9735 and 9060 in MALDI TOF mass spectra from cells and from extracts of both Escherichia coli 1090 and Shigella flexneri PHS-1059. However, the proteins associated with proteolytic cleavage by the peptidase Lep, rather than the precursor proteins, were observed, both using cells and from cellular extracts. A cold-shock protein, CspA, was associated with the ion near m/z 7643 from Pseudomonas aeruginosa. Similarly, a cold-acclimation protein, CapB, was identified as the source of the ion near m/z 7684 in P. putida. This last protein was homologous with a known CapB from P. fragi. While these experiments involved the detection of known or homologous proteins from typical bacteria, this same approach could also be applied to the detection of unique proteins or biomarker proteins associated with other bacteria of public health significance.     

Single cell matrix-assisted laser desorption/ionization mass spectrometry imaging

Application of mass spectrometry imaging (MS imaging) analysis to single cells was so far restricted either by spatial resolution in the case of matrix-assisted laser desorption/ionization (MALDI) or by mass resolution/mass range in the case of secondary ion mass spectrometry (SIMS). In this study we demonstrate for the first time the combination of high spatial resolution (7 μm pixel), high mass accuracy (<3 ppm rms), and high mass resolution (R = 100?000 at m/z = 200) in the same MS imaging measurement of single cells. HeLa cells were grown directly on indium tin oxide (ITO) coated glass slides. A dedicated sample preparation protocol was developed including fixation with glutaraldehyde and matrix coating with a pneumatic spraying device. Mass spectrometry imaging measurements with 7 μm pixel size were performed with a high resolution atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) imaging source attached to an Exactive Orbitrap mass spectrometer. Selected ion images were generated with a bin width of Δm/z = ±0.005. Selected ion images and optical fluorescence images of HeLa cells showed excellent correlation. Examples demonstrate that a lower mass resolution and a lower spatial resolution would result in a significant loss of information. High mass accuracy measurements of better than 3 ppm (root-mean-square) under imaging conditions provide confident identification of imaged compounds. Numerous compounds including small metabolites such as adenine, guanine, and cholesterol as well as different lipid classes such as phosphatidylcholine, sphingomyelin, diglycerides, and triglycerides were detected and identified based on a mass spectrum acquired from an individual spot of 7 μm in diameter. These measurements provide molecularly specific images of larger metabolites (phospholipids) in native single cells. The developed method can be used for a wide range of detailed investigations of metabolic changes in single cells.     

Multiple ionization mass spectrometry strategy used to reveal the complexity of metabolomics

A multiple ionization mass spectrometry strategy is presented based on the analysis of human serum extracts. Chromatographic separation was interfaced inline with the atmospheric pressure ionization techniques electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in both positive (+) and negative (-) ionization modes. Furthermore, surface-based matrix-assisted laser desorption/ionization (MALDI) and desorption ionization on silicon (DIOS) mass spectrometry were also integrated with the separation through fraction collection and offline mass spectrometry. Processing of raw data using the XCMS software resulted in time-aligned ion features, which are defined as a unique m/z at a unique retention time. The ion feature lists obtained through LC-MS with ESI and APCI interfaces in both +/- ionization modes were compared, and unique ion tables were generated. Nonredundant, unique ion features, were defined as mass numbers for which no mass numbers corresponding to [M + H](+), [M - H](-), or [M + Na](+) were observed in the other ionization methods at the same retention time. Analysis of the extracted serum using ESI for both (+) and (-) ions resulted in >90% additional unique ions being detected in the (-) ESI mode. Complementing the ESI analysis with APCI resulted in an additional approximately 20% increase in unique ions. Finally, ESI/APCI ionization was combined with fraction collection and offline-MALDI and DIOS mass spectrometry. The parts of the total ion current chromatograms in the LC-MS acquired data corresponding to collected fractions were summed, and m/z lists were compiled and compared to the m/z lists obtained from the DIOS/MALDI spectra. It was observed that, for each fraction, DIOS accounted for approximately 50% of the unique ions detected. These results suggest that true global metabolomics will require multiple ionization technologies to address the inherent metabolite diversity and therefore the complexity in and of metabolomics studies.     

MALDI matrices for biomolecular analysis based on functionalized carbon nanomaterials

When used in small molar ratios of matrix to analyte, derivatized fullerenes and single wall nanotubes are shown to be efficient matrices for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The mixing of an acidic functionalized fullerene with a solution of bioanalyte, depositing a dried droplet, and irradiating with a pulsed nitrogen laser yields protonated or cationized molecular ions. Derivatized fullerenes could offer several advantages over conventional MALDI matrices: a high analyte ionization efficiency, a small molar ratios (less than 1) of matrix/analyte, and a broader optical absorption spectrum, which should obviate specific wavelength lasers for MALDI acquisitions. The major disadvantage to the use of fullerenes is the isobaric interference between matrix and analyte ions; however, it is overcome by using MALDI-ion mobility time-of-flight (IM-oTOF) mass spectrometry to preseparate carbon cluster ions from bioanalyte ions prior to TOF mass analysis. However, an alternative to the dried droplet preparation of fullerene MALDI samples is the aerosolization of matrix-analyte solutions (or slurries) followed by impacting the aerosol onto a stainless surface. We also demonstrate that the fullerene matrices can be used to acquire spectra from rat brain tissue.     

Macromolecular ion accelerator

Presented herein are the development of macromolecular ion accelerator (MIA) and the results obtained by MIA. This new instrument utilizes a consecutive series of planar electrodes for the purpose of facilitating stepwise acceleration. Matrix-assisted laser desorption/ionization (MALDI) is employed to generate singly charged macromolecular ions. A regular Z-gap microchannel plate (MCP) detector is mounted at the end of the accelerator to record the ion signals. In this work, we demonstrated the detection of ions with the mass-to-charge (m/z) ratio reaching 30,000,000. Moreover, we showed that singly charged biomolecular ions can be accelerated with the voltage approaching 1 MV, offering the evidence that macromolecular ions can possess much higher kinetic energy than ever before.     

Linking mass spectrometric imaging and traditional peptidomics: a validation in the obese mouse model

MALDI mass spectrometry imaging (MSI) is a promising technique in the field of molecular (immuno)histology but is confronted with the problematic large-scale identification of peptides from thin tissue sections. In this study we present a workflow that significantly increased the number of identified peptides in a given MALDI-MSI data set and we evaluated its power concerning relative peptide quantifications. Fourier transform mass spectrometry (FTMS) profiling on matrix-coated thin tissue sections allowed us to align spectra of different MS sources, matching identical peaks in the process, thus linking MSI data to tandem mass spectrometry (MS/MS) on one hand and semiquantitative liquid chromatography (LC)/MS data on the other. Bonanza clustering was applied in order to group MS/MS spectra of structurally related peptides, making it possible to infer the identity of MSI-detected compounds based on identified members within the same cluster, effectively increasing the number of identifications in a single MSI data set. Out of 136 detected peptides with MALDI-MSI, we were able to identify 46 peptides. For 31 of these, a LC/quadrupole time-of-flight (QTOF) counterpart was detected, and we observed similar obese (ob/ob) to wild-type (wt) peak intensity ratios for 18 peptides. This workflow significantly increased the number of identifications of peptide masses detected with MALDI-MSI and evaluated the power of this imaging method for relative quantification of peptide levels between experimental conditions.     

Desorption electrospray ionization then MALDI mass spectrometry imaging of lipid and protein distributions in single tissue sections

Imaging mass spectrometry (MS) is a powerful technique for mapping the spatial distributions of a wide range of chemical compounds simultaneously from a tissue section. Co-localization of the distribution of individual molecular species, including particular lipids and proteins, and correlation with the morphological features of a single tissue section are highly desirable for comprehensive tissue analysis and disease diagnosis. We now report on the use, in turn, of desorption electrospray ionization (DESI), matrix assisted laser desorption ionization (MALDI), and then optical microscopy to image lipid and protein distributions in a single tissue section. This is possible through the use of histologically compatible DESI solvent systems, which allow for sequential analyses of the same section by DESI then MALDI. Hematoxylin and eosin (H&E) staining was performed on the same section after removal of the MALDI matrix. This workflow allowed chemical information to be unambiguously matched to histological features in mouse brain tissue sections. The lipid sulfatide (24:1), detected at m/z 888.8 by DESI imaging, was colocalized with the protein MBP isoform 8, detected at m/z 14117 by MALDI imaging, in regions corresponding to the corpus callosum substructure of the mouse brain, as confirmed in the H&E images. Correlation of lipid and protein distributions with histopathological features was also achieved for human brain cancer samples. Higher tumor cell density was observed in regions demonstrating higher relative abundances of oleic acid, detected by DESI imaging at m/z 281.4, and the protein calcyclin, detected by MALDI at m/z 10085, for a human glioma sample. Since correlation between molecular signatures and disease state can be achieved, we expect that this methodology will significantly enhance the value of MS imaging in molecular pathology for diagnosis.     

Mass and composition matrix-assisted laser desorption ionization time of flight analysis enabling inference of the sequence of most peptides where the protein of origin is known

Mass spectrometers combining matrix-assisted laser-desorption ionization (MALDI) and time-of-flight analysis are among the most widely used in peptide analysis. They excel at accurate mass determinations on complex samples but, compared with tandem instruments, have very limited capacity to determine amino acid sequence through daughter ion analysis. Here we have investigated the sequence information that can be inferred from the masses of peptides in the special circumstance in which the peptides are known to be sub-sequences of known parent sequences. We show how sequence can be inferred from the measured m/z of a peptide (mass analysis) and examine the parameters that influence the level of confidence that can be placed in "inferred sequences". We further describe how specific amino acid modifications can be used with MALDI-TOF analysis to obtain partial composition information and demonstrate that combined mass and composition (MAC) analysis enables the sequences of most peptide ions to be inferred with very high confidence.     

In vivo analysis and spatial profiling of phytochemicals in herbal tissue by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was developed for spatial profiling of phytochemicals and secondary metabolites in integrated herbal tissue without solvent extraction. Abundant alkaloid ions, including (+)-menisperine (m/z 356), magnoflorine (m/z 342), stepharanine (m/z 324), protonated sinomenine (m/z 330), protonated sinomendine (m/z 338), and a metabolite at m/z 314, could be directly desorbed from alpha-cyano-4-hydroxycinnamic acid- (CHCA-) coated stem tissue of Sinomenium acutum upon N2 laser (337 nm) ablation, while the ion signals desorbed from sinapinic acid- (SA-) coated and 2,5-dihydroxybenzoic acid- (DHB-) coated stem tissue were at least 10 times weaker. Solvent composition in the matrix solution could have significant effects on the ion intensity of the metabolites. Under optimized conditions that maximize the ion intensity and form homogeneous matrix crystals on the tissue surface, spatial distributions of the metabolites localized in different tissue regions, including cortex, phloem, xylem, rim, and pith, and their relative abundances could be semiquantitatively determined. The three metabolites detected at m/z 356, 342, and 314 showed specific distributions in the herbal samples collected from different growing areas, while others were not. By applying principal component analysis (PCA), the characteristic metabolites in specific tissue regions could be easily determined, allowing unambiguous differentiation of the herbal samples from different geographic locations.     

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.