Stretched tissue mounting for MALDI mass spectrometry imaging

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) combines information-rich chemical detection with spatial localization of analytes. For a given instrumental platform and analyte class, the data acquired can represent a compromise between analyte extraction and spatial information. Here, we introduce an improvement to the spatial resolution achievable with MALDI MSI conducted with standard mass spectrometric systems that also reduces analyte migration during matrix application. Tissue is placed directly on a stretchable membrane that, when stretched, fragments the tissue into micrometer-sized pieces. Scanning electron microscopy analysis shows that this process produces fairly homogeneous distributions of small tissue fragments separated and surrounded by areas of hydrophobic membrane surface. MALDI matrix is then applied by either a robotic microspotter or an artist's airbrush. Rat spinal cord samples imaged with an instrumental resolution of 50-250 μm demonstrate lipid distributions with a 5-fold high spatial resolution (a 25-fold increase in pixel density) after stretching compared to tissues that were not stretched.     

Direct molecular analysis of whole-body animal tissue sections by imaging MALDI mass spectrometry

Imaging mass spectrometry (IMS) that utilizes matrix-assisted laser desorption/ionization (MALDI) technology can provide a molecular ex vivo view of resected organs or whole-body sections from an animal, making possible the label-free tracking of both endogenous and exogenous compounds with spatial resolution and molecular specificity. Drug distribution and, for the first time, individual metabolite distributions within whole-body tissue sections can be detected simultaneously at various time points following drug administration. IMS analysis of tissues from 8 mg/kg olanzapine dosed rats revealed temporal distribution of the drug and metabolites that correlate to previous quantitative whole-body autoradiography studies. Whole-body MALDI IMS is further extended to detecting proteins from organs present in a whole-body sagittal tissue section. This technology will significantly help advance the analysis of novel therapeutics and may provide deeper insight into therapeutic and toxicological processes, revealing at the molecular level the cause of efficacy or side effects often associated with drug administration.     

An analytical pipeline for MALDI in-source decay mass spectrometry imaging

In-source decay (ISD) fragmentation as combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allows protein sequencing directly from mass spectra. Acquisition of MALDI-ISD mass spectra from tissue samples is achieved using an appropriate MALDI matrix, such as 1,5-diaminonaphthalene (DAN). Recent efforts have focused on combining MALDI-ISD with mass spectrometry imaging (MSI) to provide simultaneous sequencing and localization of proteins over a thin tissue surface. Successfully coupling these approaches requires the development of new data analysis tools, but first, investigating the properties of MALDI-ISD as applied to mixtures of protein standards reveals a high sensitivity to the relative protein ionization efficiency. This finding translates to the protein mixtures found in tissues and is used to inform the development of an analytical pipeline for data analysis in MALDI-ISD MS imaging, including software to identify the most pertinent spectra, to sequence protein mixtures, and to generate ion images for comparison with tissue morphology. The ability to simultaneously identify and localize proteins is demonstrated by using the analytical pipeline on three tissue sections from porcine eye lens, resulting in localizations for crystallins and cytochrome c. The variety of protein identifications provided by MALDI-ISD-MSI between tissue sections creates a discovery tool, and the analytical pipeline makes this process more efficient.     

Atmospheric pressure infrared MALDI imaging mass spectrometry for plant metabolomics

The utility of atmospheric pressure infrared MALDI mass spectrometry (AP IR-MALDI) was assessed for plant metabolomics studies. Tissue sections from plant organs, including flowers, ovaries, aggregate fruits, fruits, leaves, tubers, bulbs, and seeds were studied in both positive and negative ion modes. For leaves, single laser pulses sampled the cuticle and upper epidermal cells, whereas multiple pulses were demonstrated to ablate some mesophyll layers. Tandem mass spectra were obtained with collision-activated dissociation to aid with the identification of some observed ions. In the positive mode, most ions were produced as potassium, proton, or sometimes sodium ion adducts, whereas proton loss was dominant in the negative ion mode. Over 50 small metabolites and various lipids were detected in the spectra including, for example, 7 of the 10 intermediates in the citric acid cycle. Key components of the glycolysis pathway occurring in the plant cytosol were found along with intermediates of phospholipid biosynthesis and reactants or products of amino acid, nucleotide, oligosaccharide, and flavonoid biosynthesis. AP IR-MALDI mass spectrometry was used to follow the fluid transport driven by transpiration and image the spatial distributions of several metabolites in a white lily (Lilium candidum) flower petal.     

Gold-enhanced biomolecular surface imaging of cells and tissue by SIMS and MALDI mass spectrometry

Surface metallization by plasma coating enhances desorption/ionization of membrane components such as lipids and sterols in imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) of tissues and cells. High-resolution images of cholesterol and other membrane components were obtained for neuroblastoma cells and revealed subcellular details (resolving power 1.5 mum). Alternatively, in matrix-enhanced SIMS, 2,5-dihydroxybenzoic acid electrosprayed on neuroblastoma cells allowed intact molecular ion imaging of phosphatidylcholine and sphingomyelin at the cellular level. Gold deposition on top of matrix-coated rat brain tissue sections strongly enhanced image quality and signal intensity in stigmatic matrix-assisted laser desorption/ionization imaging mass spectrometry. High-quality total ion count images were acquired, and the neuropeptide vasopressin was localized in the rat brain tissue section at the hypothalamic area around the third ventricle. Although the mechanism of signal enhancement by gold deposition is under debate, the results we have obtained for cells and tissue sections illustrate the potential of this sample preparation technique for biomolecular surface imaging by mass spectrometry.     

Atmospheric pressure molecular imaging by infrared MALDI mass spectrometry

An atmospheric pressure (AP) MALDI imaging interface was developed for an orthogonal acceleration time-of-flight mass spectrometer and utilized to analyze peptides, carbohydrates, and other small biomolecules using infrared laser excitation. In molecular imaging experiments, the spatial distribution of mock peptide patterns was recovered with a detection limit of approximately 1 fmol/pixel from a variety of MALDI matrixes. With the use of oversampling for the image acquisition, a spatial resolution of 40 microm, 5 times smaller than the laser spot size, was achieved. This approach, however, required that the analyte was largely removed at the point of analysis before the next point was interrogated. Native water in plant tissue was demonstrated to be an efficient natural matrix for AP infrared laser desorption ionization. In soft fruit tissues from bananas, grapes, and strawberries, potassiated ions of the most abundant metabolites, small carbohydrates, and their clusters produced the strongest peaks in the spectra. Molecular imaging of a strawberry skin sample revealed the distribution of the sucrose, glucose/fructose, and citric acid species around the embedded seeds. Infrared AP MALDI mass spectrometric imaging without the addition of an artificial matrix enables the in vivo investigation of small biomolecules and biological processes (e.g., metabolomics) in their natural environment.     

Sample preparation for MALDI mass spectrometry using an elastomeric device reversibly sealed on the MALDI target

A new method for improving low-concentration sample recovery and reducing sample preparation steps in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is presented. In the conventional approach, samples are typically desalted and/or concentrated with various techniques and deposited on the MALDI target as small droplets. In this work, we describe a new approach in which an elastomeric device is reversibly sealed on the MALDI target to form a multi-well plate with the MALDI target as the base of the plate. The new format allows a larger volume (5-200 microL) of samples to be deposited on each spot and a series of sample handling processes, including desalting and concentrating, to be performed directly on the MALDI target. Several advantages have been observed: (i) multiple sample transferring steps are avoided; (ii) recovery of low-concentration peptides during sample preparation is improved using a novel desalting method that utilizes the hydrophobic surface of the elastomeric device; and (iii) sequence coverage of the peptide mass fingerprinting map is improved using a novel method in which proteins are immobilized on the hydrophobic surface of the elastomeric device for in-well trypsin digestion, followed by desalting and concentrating the digestion products in the same well.     

Automated MALDI matrix deposition method with inkjet printing for imaging mass spectrometry

Careful matrix deposition on tissue samples for matrix-assisted laser desorption/ionization (MALDI) is critical for producing reproducible analyte ion signals. Traditional methods for matrix deposition are often considered an art rather than a science, with significant sample-to-sample variability. Here we report an automated method for matrix deposition, employing a desktop inkjet printer (<$200) with 5760 x 1440 dpi resolution and a six-channel piezoelectric head that delivers 3 pL/drop. The inkjet printer tray, designed to hold CDs and DVDs, was modified to hold microscope slides. Empty ink cartridges were filled with MALDI matrix solutions, including DHB in methanol/water (70:30) at concentrations up to 40 mg/mL. Various samples (including rat brain tissue sections and standards of small drug molecules) were prepared using three deposition methods (electrospray, airbrush, inkjet). A linear ion trap equipped with an intermediate-pressure MALDI source was used for analyses. Optical microscopic examination showed that matrix crystals were formed evenly across the sample. There was minimal background signal after storing the matrix in the cartridges over a 6-month period. Overall, the mass spectral images gathered from inkjet-printed tissue specimens were of better quality and more reproducible than from specimens prepared by the electrospray and airbrush methods.     

Binary matrix for MALDI imaging mass spectrometry of phospholipids in both ion modes

Phospholipids (PLs) are the major building block molecules of cellular membranes. Their composition varies depending on cell types and cellular compartments. Thus, the information regarding PL distribution in tissue has important physiological and pathological significance. Recent developments in imaging mass spectrometry (IMS) have allowed complete mapping of the PL species on tissue. The IMS technique can detect different classes of PLs as well as their location information directly from tissue sections. PL head groups carry either positive and/or negative charges; therefore, IMS experiments must be conducted in both positive- and negative-ion mode to detect all types of phospholipids. Several conventional matrixes were applied on tissue for better identification. This study was conducted to enable appropriate matrix selection and optimized matrix preparation for IMS experiments in both ion modes that maximize PL identification from a single brain tissue section. The optimized matrix 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA) with a mixture of trifluoroacetic acid (TFA) and piperidine as ion pairing agents showed improved stability and consistency during both ion mode experiments and successfully identified >100 peaks of PLs determined by parent ion m/z value. Further tandem mass spectrometric analysis (MS/MS) was performed to those PLs that are anatomically important according to their distribution on rat brain tissue section.     

A microfluidic electrocapture device in sample preparation for protein analysis by MALDI mass spectrometry

The design and operation of a microfluidic device for sample preparation in MALDI mass spectrometry of peptides and proteins is described. It is particularly useful for proteomics applications and for mass determination of proteins in salt- and detergent-containing solutions. The system consists of a flow channel with two conductive areas or electrical junctions where proteins and peptides are retained by means of an electric field. The microfluidic device is made of PEEK tubing, and the junctions are covered with a conductive polymeric membrane. A syringe pump connected to the device produces a flow stream, and injection of sample is carried out manually via hydrodynamic pressure. Proteolytic peptides and intact proteins in salt- and detergent-containing acidic media were captured at the cathode junction followed by exchange of the original solution to a solvent suitable for subsequent mass spectrometry. Using this principle, a significant desalting effect was obtained for tryptic peptides in mass-mapping experiments. Protein sequence coverages were high (up to 40%) at subpicomole levels with results better than those obtained using reversed-phase solid-phase extraction. In contrast to the latter technique, the microfluidic device has the capacity to efficiently remove detergents such as CHAPS before peptide mapping and protein analysis.     

Integrated sample preparation and MALDI mass spectrometry on a microfluidic compact disk

High-throughput microfluidic processing of protein digests integrated with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on a compact disk (CD) is described. Centrifugal force moves liquid through multiple microstructures, each containing a 10-nL reversed-phase chromatography column. The CD enables parallel preparation of 96 samples with volumes ranging from one to several microliters. The peptides in the digests are concentrated, desalted, and subsequently eluted from the columns directly into MALDI target areas (200 x 400 microm) on the CD using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into the MALDI instrument for peptide mass fingerprinting and database identification at a routine sensitivity down to the 200-amol level. Detection of proteolytic peptides down to the 50-amol level is demonstrated. The success rate of the CD technology in protein identification is about twice that of the C(18) ZipTips and standard MALDI steel targets. The CDs are operated using robotics to transfer samples and reagents from microcontainers to the processing inlets on the disposable CD and spinning to control the movement of liquid through the microstructures.     

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.     

Direct profiling of proteins in biological tissue sections by MALDI mass spectrometry

The direct profiling of proteins present in tissue sections for several organs of the mouse has been accomplished using matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Fresh tissue was sectioned and blotted on a conductive polyethylene membrane. The dried membrane blot was coated with matrix, typically sinapinic acid, and directly analyzed in the mass spectrometer. Generally, well over 100 peptide/protein signals in the 2000-30,000 Da range were observed, with 30-50 having relatively high signal intensities. Analysis of different areas of the same tissue gave remarkably similar mass spectra with greater than 90% homology. However, different parts of a segmented tissue, such as the proximal, intermediate, and distal colon, gave some unique protein signals. After treatment of the tissue blot with protease and subsequent MALDI MS analysis using postsource decay methods for peptide sequencing, some of the proteins were identified. The unique protein profiles measured from these tissue blots also showed differences from strain to strain of the mouse, with genetically similar strains having very similar patterns.     

A rotating ball inlet for on-line MALDI mass spectrometry

The rotating ball inlet (ROBIN) is presented in a new design for on-line matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). This method uses a capillary to deliver a matrix and analyte solution to the surface of a rotating ball upon which MALDI is carried out. The ball is in contact with a polymer gasket surrounding the capillary. Sample adhering to the surface of the ball is dragged past the gasket into the vacuum of the mass spectrometer where it is irradiated by a pulsed UV laser, and the resulting ions are mass-separated in a linear time-of-flight mass spectrometer. The mechanical sample introduction prevents clogging of the vacuum interface by matrix crystals or frozen solvent. Preliminary results from flow injection analysis (FIA) suggest that the new interface does not introduce a significant peak-tailing or memory effect. The system is capable of 20-30 h of continuous operation with a flow rate of 2 microL/min before cleaning of the ball is needed. With the prototype inlet, concentration detection limits are at the low micromolar level.     

Combination detergent/MALDI matrix: functional cleavable detergents for mass spectrometry

This study reports the synthesis of the first functional cleavable detergent designed specifically for applications in mass spectrometry. Upon cleavage, two inert compounds and the MALDI matrix are formed, eliminating sources of potential interference originating from traditional cleavable detergents. Analysis of peptides demonstrates that MALDI matrix generated in situ results in MALDI spectra equivalent to those prepared using established protocols. Analysis of the membrane protein diacylglycerol kinase was accomplished using the combination detergent/MALDI matrix. Applications of the functional cleavable detergents to the profiling of whole cell lysates results in increased signal-to-noise ratios of many ions and the detection of additional proteins previously not observed.     

MALDI quadrupole time-of-flight mass spectrometry: a powerful tool for proteomic research

A MALDI QqTOF mass spectrometer has been used to identify proteins separated by one-dimensional or two-dimensional gel electrophoresis at the femtomole level. The high mass resolution and the high mass accuracy of this instrument in both MS and MS/MS modes allow identification of a protein either by peptide mass fingerprinting of the protein digest or from tandem mass spectra acquired by collision-induced dissociation of individual peptide precursors. A peptide mass map of the digest and tandem mass spectra of multiple peptide precursor ions can be acquired from the same sample in the course of a single experiment. Database searching and acquisition of MS and MS/MS spectra can be combined in an interactive fashion, increasing the information value of the analytical data. The approach has demonstrated its usefulness in the comprehensive characterization of protein in-gel digests, in the dissection of complex protein mixtures, and in sequencing of a low molecular weight integral membrane protein. Proteins can be identified in all types of sequence databases, including an EST database. Thus, MALDI QqTOF mass spectrometry promises to have remarkable potential for advancing proteomic research.     

Metabolomic analysis of eukaryotic tissue and prokaryotes using negative mode MALDI time-of-flight mass spectrometry

Metabolites in islets of Langerhans and Escherichia coli strain DH5-alpha were analyzed using negative-mode, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For analysis of anionic metabolites by MALDI, 9-aminoacridine as the matrix yielded a far superior signal in comparison to alpha-cyano-4-hydroxycinnamic acid, 2,5-dihydrobenzoic acid, 2,4,6,-trihydroxyacetophenone, and 3-hydroxypicolinic acid. Limits of detection for metabolite standards were as low as 15 nM for GDP, GTP, ADP, and ATP and as high as 1 muM for succinate in 1-muL samples. Analysis of islet extracts allowed detection of 44 metabolites, 29 of which were tentatively identified by matching molecular weight to compounds in METLIN and KEGG databases. Relative quantification was demonstrated by comparing the ratio of selected di- and triphosphorylated nucleotides for islets incubated with different concentrations of glucose. For islets at 3 mM glucose, concentration ratios of ATP/ADP, GTP/GDP, and UTP/UDP were 1.9 +/- 1.39, 1.12 +/- 0.50, and 0.79 +/- 0.35 respectively, and at 20 mM glucose stimulation, the ratios increased to 4.13 +/- 1.89, 5.62 +/-4.48, and 4.30 +/- 4.07 (n = 3). Analysis was also performed by placing individual, intact islets on a MALDI target plate with matrix and impinging the laser directly on the dried islet. Direct analysis of single islets allowed detection of 43 metabolites, 28 of which were database identifiable. A total of 43% of detected metabolites from direct islet analysis were different from those detected in islet extracts. The method was extended to prokaryotic cells by analysis of extracts from E. coli. Sixty metabolites were detected, 39 of which matched compounds in the MetaCyc database. A total of 27% of the metabolites detected from prokaryotes overlapped those found in islets. These results show that MALDI can be used for detection of metabolites in complex biological samples.     

Selective sampling of phosphopeptides for detection by MALDI mass spectrometry

The analysis of phosphopeptides by mass spectrometry (MS) is one of the most challenging tasks in proteomics. This is due to the lower isoelectric point (pI) of phosphopeptides, which leads to inefficient sample ionization in MS, particularly when competing with other peptides. The problem is compounded by the typical low abundance of phosphopeptides in biological samples. We describe here a simple nonsorptive method to isolate phosphopeptides based on their pI. A voltage is applied to selectively migrate the phosphopeptides into a capillary, which are negatively charged at acidic pH. The selectively sampled fraction is directly deposited onto MALDI sample target in nanoliter volumes (7-35 nL) for highly sensitive MS detection. No significant sample loss is evident in this procedure; hence, the MS was able to detect the isolated phosphopeptides at trace quantity. In this case, attomole-level detection limit is achieved for synthetic phosphopeptides (nM concentration and nL volume), from a mixture containing other peptides at up to 1 million times higher in concentration. Selective sampling was also applied to the tryptic digest of beta- and alpha-caseins to reveal the multiple phosphorylated peptides at the low-femtomole level using MALDI MS. Knowledge of pI based on the rejection/injection of peptides was found to be useful in peak assignment. To confirm the sequence of the selectively sampled peptides, fraction collection was performed for offline ESI MS/MS analysis.     

Atmospheric pressure MALDI/ion trap mass spectrometry

A new sample ionization technique, atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI), was coupled with a commercial ion trap mass spectrometer. This configuration enables the application-specific selection of external atmospheric ionization sources: the electrospray/APCI (commercially available) and AP MALDI (built in-house), which can be readily interchanged within minutes. The detection limit of the novel AP MALDI/ion trap is 10-50 fmol of analyte deposited on the target surface for a four-component mixture of peptides with 800-1700 molecular weight. The possibility of peptide structural analysis by MS/MS and MS3 experiments for AP MALDI-generated ions was demonstrated for the first time.     

High-throughput method for N-terminal sequencing of proteins by MALDI mass spectrometry

A high-throughput method for sequencing of N termini of proteins by using postsource decay (PSD) of matrix-assisted laser desorption/ionization mass spectrometry has been developed. After a protein blotted on the PVDF membrane was successively reduced, S-alkylated, and guanidinated, its N-amino group was coupled to biotinylcysteic acid. The protein was then extracted from the membrane and digested with trypsin. The derivatized N-terminal fragment was then specifically isolated from the tryptic digest with avidin resins, and its de novo sequencing was successfully performed by PSD utilizing a sulfonic acid group introduced to the N terminus.