Enhanced sensitivity for high spatial resolution lipid analysis by negative ion mode matrix assisted laser desorption ionization imaging mass spectrometry

We have achieved enhanced lipid imaging to a ~10 μm spatial resolution using negative ion mode matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry, sublimation of 2,5-dihydroxybenzoic acid as the MALDI matrix, and a sample preparation protocol that uses aqueous washes. We report on the effect of treating tissue sections by washing with volatile buffers at different pHs prior to negative ion mode lipid imaging. The results show that washing with ammonium formate, pH 6.4, or ammonium acetate, pH 6.7, significantly increases signal intensity and number of analytes recorded from adult mouse brain tissue sections. Major lipid species measured were glycerophosphoinositols, glycerophosphates, glycerolphosphoglycerols, glycerophosphoethanolamines, glycerophospho-serines, sulfatides, and gangliosides. Ion images from adult mouse brain sections that compare washed and unwashed sections are presented and show up to 5-fold increases in ion intensity for washed tissue. The sample preparation protocol has been found to be applicable across numerous organ types and significantly expands the number of lipid species detectable by imaging mass spectrometry at high spatial resolution.     

Nanoparticle-assisted laser desorption/ionization based mass imaging with cellular resolution

Today, two-dimensional mass spectrometry analysis of biological tissues by means of a technique called mass imaging, mass spectrometry imaging (MSI), or imaging mass spectrometry (IMS) has found application in investigating the distribution of moleculesMSI with matrix-assisted laser desorption/ionization (MALDI) and secondary ion MS (SIMS). However, the size of the matrix crystal and the migration of analytes can decrease the spatial resolution in MALDI, and SIMS can only ionize compounds with relatively low molecular weights. To overcome these problems, we developed a nanoparticle-assisted laser desorption/ionization (nano-PALDI)-based MSI. We used nano-PALDI MSI to visualize lipids and peptides at a resolution of 15 microm in mammalian tissues.     

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.     

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.     

Infrared laser ablation sample transfer for MALDI imaging

An infrared laser was used to ablate material from tissue sections under ambient conditions for direct collection on a matrix assisted laser desorption ionization (MALDI) target. A 10 μm thick tissue sample was placed on a microscope slide and was mounted tissue-side down between 70 and 450 μm from a second microscope slide. The two slides were mounted on a translation stage, and the tissue was scanned in two dimensions under a focused mid-infrared (IR) laser beam to transfer material to the target slide via ablation. After the material was transferred to the target slide, it was analyzed using MALDI imaging using a tandem time-of-flight mass spectrometer. Images were obtained from peptide standards for initial optimization of the system and from mouse brain tissue sections using deposition either onto a matrix precoated target or with matrix addition after sample transfer and compared with those from standard MALDI mass spectrometry imaging. The spatial resolution of the transferred material is approximately 400 μm. Laser ablation sample transfer provides several new capabilities not possible with conventional MALDI imaging including (1) ambient sampling for MALDI imaging, (2) area to spot concentration of ablated material, (3) collection of material for multiple imaging analyses, and (4) direct collection onto nanostructure assisted laser desorption ionization (NALDI) targets without blotting or ultrathin sections.     

Integrating histology and imaging mass spectrometry

MALDI (matrix-assisted laser desorption/ionization) imaging mass spectrometry (IMS) is a new technology that generates molecular profiles and two-dimensional ion density maps of peptide and protein signals directly from the surface of thin tissue sections. This allows specific information to be obtained on the relative abundance and spatial distribution of proteins. One important aspect of this is the opportunity to correlate these specific ion images with histological features observed by optical microscopy. To facilitate this, we have developed protocols that allow MALDI mass spectrometry imaging and optical microscopy to be performed on the same section. Key components of these protocols involve the use of conductive glass slides as sample support for the tissue sections and MS-friendly tissue staining protocols. We show the effectiveness of these with protein standards and with several types of tissue sections. Although stain-specific intensity variations occur, the overall protein pattern and spectrum quality remain consistent between stained and control tissue samples. Furthermore, imaging mass spectrometry experiments performed on stained sections showed good image quality with minimal delocalization of proteins resulting from the staining protocols.     

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.     

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.     

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.     

Colloidal graphite-assisted laser desorption/ionization mass spectrometry and MSn of small molecules. 1. Imaging of cerebrosides directly from rat brain tissue

Graphite-assisted laser desorption/ionization (GALDI) mass spectrometry (MS) was investigated for analysis of cerebrosides in a complex total brain lipid extract. Conventional MALDI MS and GALDI MS were compared regarding lipid analysis by using high-vacuum (HV, <10-6 Torr) LDI time-of-flight mass spectrometry and intermediate-pressure (IP, 0.17 Torr) linear ion trap mass spectrometry. Cerebrosides were not detected or detected with low sensitivity in MALDI MS because of other dominant phospholipids. By using GALDI, cerebrosides were detected as intense mass peaks without prior separation from other lipid species while mass peaks corresponding to phosphatidylcholines (PCs) were weak. The signal increase for cerebrosides and the signal decrease for PCs in GALDI MS were more significant in HV than in IP. MSn experiments of precursor ions corresponding to cerebrosides and PCs in brain lipid extract were performed to identify the detected species and distinguish isobaric ions. Twenty-two cerebroside species were detected by GALDI whereas eight cerebroside species were detected by MALDI. Sulfatides in brain lipid extract were also easily detected by GALDI MS in the negative ion mode. By forming a colloidal graphite thin film on rat brain tissue, direct lipid profiling by imaging mass spectrometry (IMS) was performed. Chemically selective images for cerebrosides and sulfatides were successfully obtained. Imaging tandem mass spectrometry (IMS/MS) was performed to generate images of specific product ions from isobaric species.     

C60 secondary ion mass spectrometry with a hybrid-quadrupole orthogonal time-of-flight mass spectrometer

A hybrid quadrupole orthogonal time-of-flight mass spectrometer optimized for matrix-assisted laser desorption ionization (MALDI) and electrospray ionization has been equipped with a C 60 cluster ion source. This configuration is shown to exhibit a number of characteristics that improve the performance of traditional time-of-flight secondary ion mass spectrometry (TOF-SIMS) experiments for the analysis of complex organic materials and, potentially, for chemical imaging. Specifically, the primary ion beam is operated as a continuous rather than a pulsed beam, resulting in up to 4 orders of magnitude greater ion fluence on the target. The secondary ions are extracted at very low voltage into 8 mTorr of N 2 gas introduced for collisional focusing and cooling purposes. This extraction configuration is shown to yield secondary ions that rapidly lose memory of the mechanism of their birth, yielding tandem mass spectra that are identical for SIMS and MALDI. With implementation of ion trapping, the extraction efficiency is shown to be equivalent to that found in traditional TOF-SIMS machines. Examples are given, for a variety of substrates that illustrate mass resolution of 12,000-15,600 with a mass range for inorganic compounds to m/ z 40,000. Preliminary chemical mapping experiments show that with added sensitivity, imaging in the MS/MS mode of operation is straightforward. In general, the combination of MALDI and SIMS is shown to add capabilities to each technique, providing a robust platform for TOF-SIMS experiments that already exists in a large number of laboratories.     

Tissue imaging using nanospray desorption electrospray ionization mass spectrometry

Ambient ionization imaging mass spectrometry is uniquely suited for detailed spatially resolved chemical characterization of biological samples in their native environment. However, the spatial resolution attainable using existing approaches is limited by the ion transfer efficiency from the ionization region into the mass spectrometer. Here, we present a first study of ambient imaging of biological samples using nanospray desorption ionization (nano-DESI). Nano-DESI is a new ambient pressure ionization technique that uses minute amounts of solvent confined between two capillaries comprising the nano-DESI probe and the solid analyte for controlled desorption of molecules present on the substrate followed by ionization through self-aspirating nanospray. We demonstrate highly sensitive spatially resolved analysis of tissue samples without sample preparation. Our first proof-of-principle experiments indicate the potential of nano-DESI for ambient imaging with a spatial resolution of better than 12 μm. The significant improvement of the spatial resolution offered by nano-DESI imaging combined with high detection efficiency will enable new imaging mass spectrometry applications in clinical diagnostics, drug discovery, molecular biology, and biochemistry.     

Aerosol MALDI with a Reflectron Time-of-Flight Mass Spectrometer

Aerosol matrix-assisted laser desorption/ionization (MALDI) has been combined with a reflectron time-of-flight mass spectrometer for improved mass resolution. A methanol solution of matrix and analyte was sprayed directly into a reflectron time-of-flight mass spectrometer, and the aerosol particles were irradiated and ionized with a frequency-tripled Nd:YAG laser at 355 nm. Mass resolution of over 300 was observed for the peptides bradykinin, angiotensin II, and gramicidin D and for vitamin B(12). This represents a resolution enhancement of approximately 10-fold over that previously reported for aerosol MALDI with a linear time-of-flight instrument.     

Enhanced ionization of phosphorylated peptides during MALDI TOF mass spectrometry

Although alpha-cyano-4-hydroxycinnamic acid functions as an excellent matrix for the analysis of most peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry, the ionization of phosphorylated peptides is usually suppressed by nonphosphorylated peptides. As an alternative matrix, 2',4',6'-trihydroxyacetophenone (THAP) with diammonium citrate was found to overcome this problem for the MALDI TOF mass spectrometric analysis of proteolytic digests of phosphorylated proteins. Specifically, the abundances of phosphorylated peptides in tryptic digests of bovine beta-casein and protein kinase C (PKC)-treated mouse cardiac troponin I were enhanced more than 10-fold using THAP during positive ion MALDI TOF mass spectrometry. The protonated molecules of phosphorylated peptides were sufficiently abundant that postsource decay TOF mass spectrometry was used to confirm the number of phosphate groups in each peptide. Finally, tryptic digestion followed by analysis using MALDI TOF mass spectrometry with THAP as the matrix facilitated the identification of a unique phosphorylation site in PKC-treated troponin I.     

Effect of local matrix crystal variations in matrix-assisted ionization techniques for mass spectrometry

Intense intact molecular ion signals have been obtained from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidyiinositol using matrix-enhanced secondary ion mass spectrometry (ME-SIMS). It was found that the high-mass (m/z >500) regions of the ME-SIMS spectra closely resembled those obtained using matrix-assisted laser desorption/ionization (MALDI). Using high spatial resolution SIMS, a detailed investigation of dried-droplet samples was performed. Based on the detected Na+ and 2,5-DHB matrix signal intensities, different crystal types were distinguished, in addition to different sizes of crystals. Spatially mapping the pseudomolecular and fragment ions of the phospholipids revealed that the nature of the pseudomolecular ions formed, as well as the ratio of intact molecular to fragment ion, was dependent on the type and surface composition of the crystal. The observed chemical bias effects due to crystal heterogeneity and the resulting variation in desorption/ionization efficiency will complicate the interpretation of data obtained from matrix-assisted mass spectrometric (imaging) techniques and is an important factor in the "hot spot" phenomenon frequently encountered in MALDI experiments. In this respect, imaging SIMS was found to be a versatile tool to investigate the effects of the local physicochemical conditions on the detected molecular species.     

Colloidal graphite-assisted laser desorption/ionization MS and MS(n) of small molecules. 2. Direct profiling and MS imaging of small metabolites from fruits

Due to a high background in the low-mass region, conventional MALDI is not as useful for detecting small molecules (molecular masses <500 Da) as it is for large ones. Also, spatial inhomogeneity that is inherent to crystalline matrixes can degrade resolution in imaging mass spectrometry (IMS). In this study, colloidal graphite was investigated as an alternative matrix for laser desorption/ionization (GALDI) in IMS. We demonstrate its advantages over conventional MALDI in the detection of small molecules such as organic acids, flavonoids, and oligosaccharides. GALDI provides good sensitivity for such small molecules. The detection limit of fatty acids and flavonoids in the negative-ion mode are in the low-femtomole range. Molecules were detected directly and identified by comparing the MS and MS/MS spectra with those of standards. Various fruits were chosen to evaluate the practical utility of GALDI since many types of small molecules are present in them. Distribution of these small molecules in the fruit was investigated by using IMS and IMS/MS.     

Extending the laserspray ionization concept to produce highly charged ions at high vacuum on a time-of-flight mass analyzer

A new matrix compound, 2-nitrophloroglucinol, is reported which not only produces highly charged ions similar to electrospray ionization (ESI) under atmospheric pressure (AP) and intermediate pressure (IP) laserspray ionization (LSI) conditions but also the most highly charged ions so far observed for small proteins in mass spectrometry (MS) under high vacuum (HV) conditions. This new matrix extends the compounds that can successfully be employed as matrixes with LSI, as demonstrated on an LTQ Velos (Thermo) at AP, a matrix-assisted laser desorption/ionization (MALDI)-ion mobility spectrometry (IMS) time-of-flight (TOF) SYNAPT G2 (Waters) at IP, and MALDI-TOF Ultraflex, UltrafleXtreme, and Autoflex Speed (Bruker) mass spectrometers at HV. Measurements show that stable multiple charged molecular ions of proteins are formed under all pressure conditions indicating softer ionization than MALDI, which suffers a high degree of metastable fragmentation when multiply charged ions are produced. An important analytical advantage of this new LSI matrix are the potential for high sensitivity equivalent or better than AP-LSI and vacuum MALDI and the potential for enhanced mass selected fragmentation of the abundant highly charged protein ions. A second new LSI matrix, 4,6-dinitropyrogallol, produces abundant multiply charged ions at AP but not under HV conditions. The differences in these similar compounds ability to produce multiply charged ions under HV conditions is believed to be related to their relative ability to evaporate from charged matrix/analyte clusters.     

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.     

High dynamic range bio-molecular ion microscopy with the Timepix detector

Highly parallel, active pixel detectors enable novel detection capabilities for large biomolecules in time-of-flight (TOF) based mass spectrometry imaging (MSI). In this work, a 512 × 512 pixel, bare Timepix assembly combined with chevron microchannel plates (MCP) captures time-resolved images of several m/z species in a single measurement. Mass-resolved ion images from Timepix measurements of peptide and protein standards demonstrate the capability to return both mass-spectral and localization information of biologically relevant analytes from matrix-assisted laser desorption ionization (MALDI) on a commercial ion microscope. The use of a MCP-Timepix assembly delivers an increased dynamic range of several orders of magnitude. The Timepix returns defined mass spectra already at subsaturation MCP gains, which prolongs the MCP lifetime and allows the gain to be optimized for image quality. The Timepix peak resolution is only limited by the resolution of the in-pixel measurement clock. Oligomers of the protein ubiquitin were measured up to 78 kDa.     

Identification of 4-hydroxy-2-nonenal-modified peptides within unfractionated digests using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is generated as a consequence of oxidative stress and can readily react with nucleophilic sites of proteins (e.g., histidine residues), mainly via a Michael addition. The formation of such lipid-protein conjugates can alter protein properties and biological functions, thus leading to highly deleterious effects. The present work describes a rapid (very limited sample preparation) and sensitive (low-femtomole range) procedure to identify HNE-modified peptides (Michael adducts) within unfractionated tryptic digests. The protocol involves the formation of dinitrophenylhydrazones of the Michael adducts, when using 2,4-dinitrophenylhydrazine as reactive matrix, followed by analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The hydrazone derivatives present high desorption/ionization yield and can thus be preferentially detected compared to unmodified peptides. The MALDI mass spectrum obtained is therefore drastically different from the one obtained with the classical 4-hydroxy-alpha-cyanocinnamic acid matrix. Moreover, the presence of HNE, or more generally speaking carbonylated peptides, could be highlighted by 180 mass units differences (corresponding to the dinitrophenylhydrazone moiety) between these two MALDI mass spectra. Further information (e.g., localization/identification of the modified residues, peptide sequences) could be obtained by performing MALDI postsource decay (or electrospray) MS/MS experiments on the ions of interest.