Combining MALDI‐2 and transmission geometry laser optics to achieve high sensitivity for ultra‐high spatial resolution surface analysis

A transmission geometry optical configuration allows for smaller laser spot size to facilitate high‐resolution matrix‐assisted laser/desorption ionization (MALDI) mass spectrometry. This increase in spatial resolution (ie, smaller laser spot size) is often associated with a decrease in analyte signal. MALDI‐2 is a post‐ionization technique, which irradiates ions and neutrals generated in the initial MALDI plume with a second orthogonal laser pulse, and has been shown to improve sensitivity. Herein, we have modified a commercial Orbitrap mass spectrometer to incorporate a transmission geometry MALDI source with MALDI‐2 capabilities to improve sensitivity at higher spatial resolutions.     

In situ isobaric lipid mapping by MALDI–ion mobility separation–mass spectrometry imaging

The highly diverse chemical structures of lipids make their analysis directly from biological tissue sections extremely challenging. Here, we report the in situ mapping and identification of lipids in a freshwater crustacean Gammarus fossarum using matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) in combination with an additional separation dimension using ion mobility spectrometry (IMS). The high‐resolution trapped ion mobility spectrometry (TIMS) allowed efficient separation of isobaric/isomeric lipids showing distinct spatial distributions. The structures of the lipids were further characterized by MS/MS analysis. It is demonstrated that MALDI MSI with mobility separation is a powerful tool for distinguishing and localizing isobaric/isomeric lipids.     

Mass spectrometry imaging of triglycerides in biological tissues by laser desorption ionization from silicon nanopost arrays

Mass spectrometry imaging (MSI) is used increasingly to simultaneously detect a broad range of biomolecules while mapping their spatial distributions within biological tissue sections. Matrix‐assisted laser desorption ionization (MALDI) is recognized as the method‐of‐choice for MSI applications due in part to its broad molecular coverage. In spite of the remarkable advantages offered by MALDI, imaging of neutral lipids, such as triglycerides (TGs), from tissue has remained a significant challenge due to ion suppression of TGs by phospholipids, e.g. phosphatidylcholines (PCs). To help overcome this limitation, silicon nanopost array (NAPA) substrates were introduced to selectively ionize TGs from biological tissue sections. This matrix‐free laser desorption ionization (LDI) platform was previously shown to provide enhanced ionization of certain lipid classes, such as hexosylceramides (HexCers) and phosphatidylethanolamines (PEs) from mouse brain tissue. In this work, we present NAPA as an MSI platform offering enhanced ionization efficiency for TGs from biological tissues relative to MALDI, allowing it to serve as a complement to MALDI‐MSI. Analysis of a standard lipid mixture containing PC(18:1/18:1) and TG(16:0/16:0/16:0) by LDI from NAPA provided an ~49 and ~227‐fold higher signal for TG(16:0/16:0/16:0) relative to MALDI, when analyzed without and with the addition of a sodium acetate, respectively. In contrast, MALDI provided an ~757 and ~295‐fold higher signal for PC(18:1/18:1) compared with NAPA, without and with additional Na⁺. Averaged signal intensities for TGs from MSI of mouse lung and human skin tissues exhibited an ~105 and ~49‐fold increase, respectively, with LDI from NAPA compared with MALDI. With respect to PCs, MALDI provided an ~2 and ~19‐fold increase in signal intensity for mouse lung and human skin tissues, respectively, when compared with NAPA. The complementary coverage obtained by the two platforms demonstrates the utility of using both techniques to maximize the information obtained from lipid MS or MSI experiments.     

MALDI-MS imaging of features smaller than the size of the laser beam

The feasibility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging of features smaller than the laser beam size has been demonstrated. The method involves the complete ablation of the MALDI matrix coating the sample at each sample position and moving the sample target a distance less than the diameter of the laser beam before repeating the process. In the limit of complete sample ablation, acquiring signal from adjacent positions spaced by distances smaller than the sample probe enhances image resolution as the measured analyte signal only arises from the overlap of the laser beam size and the non-ablated sample surface. Image acquisition of features smaller than the laser beam size has been demonstrated with peptide standards deposited on electron microscopy calibration grids and with neuropeptides originating from single cells. The presented MS imaging technique enables approximately 25 microm imaging spatial resolution using commercial MALDI mass spectrometers having irregular laser beam sizes of several hundred micron diameters. With appropriate sampling, the size of the laser beam is not a strict barrier to the attainable MALDI-MS imaging resolution.     

Molecular Imaging of Biological Samples on Nanophotonic Laser Desorption Ionization Platforms

Mass spectrometry imaging (MSI) is a comprehensive tool for the analysis of a wide range of biomolecules. The mainstream method for molecular MSI is matrix‐assisted laser desorption ionization, however, the presence of a matrix results in spectral interferences and the suppression of some analyte ions. Herein we demonstrate a new matrix‐free MSI technique using nanophotonic ionization based on laser desorption ionization (LDI) from a highly uniform silicon nanopost array (NAPA). In mouse brain and kidney tissue sections, the distributions of over 80 putatively annotated molecular species are determined with 40 μm spatial resolution. Furthermore, NAPA‐LDI‐MS is used to selectively analyze metabolites and lipids from sparsely distributed algal cells and the lamellipodia of human hepatocytes. Our results open the door for matrix‐free MSI of tissue sections and small cell populations by nanophotonic ionization.     

Instrument design and characterization for high resolution MALDI-MS imaging of tissue sections

In previous work, we have reported using a MALDI imaging time-of-flight mass spectrometer for the detection of protein ions from tissue sections with spatial resolution of 25 microm. We present here imaging mass spectrometry results obtained with a high-resolution scanning MALDI time-of-flight mass spectrometer, equipped with a coaxial laser illumination ion source, capable of achieving irradiation areas as small as 40 microm(2) (ca 7 microm diameter). MALDI-generated analyte ion signals from these very small irradiation volumes can be observed in a molecular weight range up to 27,000. High-resolution imaging mass spectrometry images were successfully generated from matrix thin film samples and tissue sections with scanning resolutions at and below 10 microm. This work also provides fundamental characterization of the ion signal dependence as a function of various focus and fluence parameters that will be required for extension to tissue imaging at the subcellular level.     

Correlative mass spectrometry imaging,applying time‐of‐flight secondary ion mass spectrometry and atmospheric pressure matrix‐assisted laser desorption/ionization to a single tissue section

Rationale

Mass spectrometry imaging (MSI) is a powerful tool for mapping the surface of a sample. Time‐of‐flight secondary ion mass spectrometry (TOF‐SIMS) and atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI) offer complementary capabilities. Here, we present a workflow to apply both techniques to a single tissue section and combine the resulting data for the example of human colon cancer tissue.

Methods

Following cryo‐sectioning, images were acquired using the high spatial resolution (1 μm pixel size) provided by TOF‐SIMS. The same section was then coated with a para‐nitroaniline matrix and images were acquired using AP‐MALDI coupled to an Orbitrap mass spectrometer, offering high mass resolution, high mass accuracy and tandem mass spectrometry (MS/MS) capabilities. Datasets provided by both mass spectrometers were converted into the open and vendor‐independent imzML file format and processed with the open‐source software MSiReader.

Results

The TOF‐SIMS and AP‐MALDI mass spectra show strong signals of fatty acids, cholesterol, phosphatidylcholine and sphingomyelin. We showed a high correlation between the fatty acid ions detected with TOF‐SIMS in negative ion mode and the phosphatidylcholine ions detected with AP‐MALDI in positive ion mode using a similar setting for visualization. Histological staining on the same section allowed the identification of the anatomical structures and their correlation with the ion images.

Conclusions

This multimodal approach using two MSI platforms shows an excellent complementarity for the localization and identification of lipids. The spatial resolution of both systems is at or close to cellular dimensions, and thus spatial correlation can only be obtained if the same tissue section is analyzed sequentially. Data processing based on imzML allows a real correlation of the imaging datasets provided by these two technologies and opens the way for a more complete molecular view of the anatomical structures of biological tissues.

     

High‐speed MALDI MS/MS imaging mass spectrometry using continuous raster sampling

A matrix‐assisted laser desorption/ionization time of flight/time of flight tandem mass spectrometer (MALDI TOF/TOF) has been used for high‐speed precursor/fragment ion transition image acquisition. High‐throughput analysis is facilitated by an Nd:YLF solid state laser capable of pulse repetition rates up to 5 kHz, a high digitizer acquisition rate (up to 50 pixels/s), and continuous laser raster sampling. MS/MS experiments are enabled through the use of a precision timed ion selector, second source acceleration, and a dedicated collision cell. Continuous raster sampling is shown here to facilitate rapid MS/MS ion image acquisition from thin tissue sections for the drug rifampicin and for a common kidney lipid, SM4s(d18:1/24:1). The ability to confirm the structural identity of an analyte as part of the MS/MS imaging experiment is an essential part of the analysis. Additionally, the increase in sensitivity and specificity afforded by an MS/MS approach is highly advantageous, especially when interrogating complex chemical environments such as those in biological tissues. Herein, we report continuous laser raster sampling TOF/TOF imaging methodologies which demonstrate 8 to 14‐fold increases in throughput compared with existing MS/MS instrumentation, an important advantage when imaging large areas on tissues. Copyright © 2015 John Wiley & Sons, Ltd.     

The ongoing evolution of laser desorption/ionization mass spectrometry: Some observations on current trends and future directions

The practice of laser desorption/ionization (LDI) mass spectrometry continues to evolve. In the most commonly adopted manifestation of LDI, matrix assisted LDI, attention continues to be directed towards novel sample application strategies and modifications to the sample plate. Specifically, researchers continue to explore adaptations to the conventional, stainless steel sample plate that is the centerpiece of conventional LDI. Numerous variants of LDI‐MS have been reported based on modifications of the plate surface, but none of these is widely adopted, either by end‐users or by instrument manufacturers. Further, at this time, advances in surface engineering have had only modest impact on day‐to‐day operation. In this article, we review and discuss some of the numerous, but scattered reports on novel LDI strategies with an emphasis on modified sample support substrates and plates. We discuss and highlight innovations that have the potential to markedly enhance the utility of LDI‐MS.     

A derivatization and validation strategy for determining the spatial localization of endogenous amine metabolites in tissues using MALDI imaging mass spectrometry

Imaging mass spectrometry (IMS) studies increasingly focus on endogenous small molecular weight metabolites and consequently bring special analytical challenges. Since analytical tissue blanks do not exist for endogenous metabolites, careful consideration must be given to confirm molecular identity. Here, we present approaches for the improvement in detection of endogenous amine metabolites such as amino acids and neurotransmitters in tissues through chemical derivatization and matrix‐assisted laser desorption/ionization (MALDI) IMS. Chemical derivatization with 4‐hydroxy‐3‐methoxycinnamaldehyde (CA) was used to improve sensitivity and specificity. CA was applied to the tissue via MALDI sample targets precoated with a mixture of derivatization reagent and ferulic acid as a MALDI matrix. Spatial distributions of chemically derivatized endogenous metabolites in tissue were determined by high‐mass resolution and MSⁿ IMS. We highlight an analytical strategy for metabolite validation whereby tissue extracts are analyzed by high‐performance liquid chromatography (HPLC)‐MS/MS to unambiguously identify metabolites and distinguish them from isobaric compounds. Copyright © 2014 John Wiley & Sons, Ltd.     

Effects of substrate surfaces in matrix‐assisted laser desorption/ionization mass spectrometry

For matrix‐assisted laser desorption/ionization (MALDI) mass spectra, undesirable ion contamination can occur due to the direct laser excitation of substrate materials (i.e., laser desorption/ionization (LDI)) if the samples do not completely cover the substrate surfaces. In this study, comparison is made of LDI processes on substrates of indium and silver, which easily emit their own ions upon laser irradiation, and conventional materials, stainless steel and gold. A simultaneous decrease of ion intensities with the number of laser pulses is observed as a common feature. By the application of an indium substrate to the MALDI mass spectrometry of alkali salts and alkylammonium salts mixed with matrices, 2,5‐dihydroxybenzoic acid (DHB) or N‐(4‐methoxybenzylidene)‐4‐butylaniline (MBBA), the mixing of LDI processes can be detected by the presence of indium ions in the mass spectra. This method has also been found to be useful for investigating the intrinsic properties of the MALDI matrices: DHB samples show an increase in the abundance of fragment ions of matrix molecules and cesium ions with the number of laser pulses irradiating the same sample spot; MBBA samples reveal a decrease in the level of background noise with an increase in the thickness of the sample layer. Copyright © 2009 John Wiley & Sons, Ltd.     

Trypsin and MALDI matrix pre‐coated targets simplify sample preparation for mapping proteomic distributions within biological tissues by imaging mass spectrometry

Prefabricated surfaces containing α‐cyano‐4‐hydroxycinnamic acid and trypsin have been developed to facilitate enzymatic digestion of endogenous tissue proteins prior to matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Tissue sections are placed onto slides that were previously coated with α‐cyano‐4‐hydroxycinnamic acid and trypsin. After incubation to promote enzymatic digestion, the tissue is analyzed by MALDI IMS to determine the spatial distribution of the tryptic fragments. The peptides detected in the MALDI IMS dataset were identified by Liquid chromatography‐tandem mass spectrometry/mass spectrometry. Protein identification was further confirmed by correlating the localization of unique tryptic fragments originating from common parent proteins. Using this procedure, proteins with molecular weights as large as 300 kDa were identified and their distributions were imaged in sections of rat brain. In particular, large proteins such as myristoylated alanine‐rich C‐kinase substrate (29.8 kDa) and spectrin alpha chain, non‐erythrocytic 1 (284 kDa) were detected that are not observed without trypsin. The pre‐coated targets simplify workflow and increase sample throughput by decreasing the sample preparation time. Further, the approach allows imaging at higher spatial resolution compared with robotic spotters that apply one drop at a time. Copyright © 2016 John Wiley & Sons, Ltd.     

Radical cation formation in characterization of novel C3-symmetric disks and their precursors by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Four C3-symmetrical tris(dipeptide) disks and their precursors were characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The C3-symmetrical disks were based on a benzene-1,3,5-triscarboxamide core extended by oligopeptides with trialkoxyanilide tails. The results indicate that MALDI TOF MS is a powerful and straightforward analytical technique for characterizing C3-symmetrical disks and their precursors. Clear (pseudo)-molecular ion peaks could readily be identified. It is remarkable that strong radical ion signals were observed for all the compounds, including the anilines that were expected to be protonated prior to laser irradiation using acidic MALDI matrixes. Possible mechanisms for radical ion formation were investigated with the employment of radical scavengers, with various matrixes and with direct laser desorption/ionization (LDI). Most likely the radicals are formed by losing one electron from the aniline nitrogen and stabilized by conjugation through the phenyl ring. It appears that direct photo/thermal ionization of analytes is an important route for the radical ion formation of the compounds with trialkoxy aniline/anilide groups.     

Quantum dots assisted laser desorption/ionization mass spectrometric detection of carbohydrates: qualitative and quantitative analysis

A quantum dots (QDs) assisted laser desorption/ionization mass spectrometric (QDA‐LDI‐MS) strategy was proposed for qualitative and quantitative analysis of a series of carbohydrates. The adsorption of carbohydrates on the modified surface of different QDs as the matrices depended mainly on the formation of hydrogen bonding, which led to higher MS intensity than those with conventional organic matrix. The effects of QDs concentration and sample preparation method were explored for improving the selective ionization process and the detection sensitivity. The proposed approach offered a new dimension to the application of QDs as matrices for MALDI‐MS research of carbohydrates. It could be used for quantitative measurement of glucose concentration in human serum with good performance. The QDs served as a matrix showed the advantages of low background, higher sensitivity, convenient sample preparation and excellent stability under vacuum. The QDs assisted LDI‐MS approach has promising application to the analysis of carbohydrates in complex biological samples. Copyright © 2016 John Wiley & Sons, Ltd.     

Method development for the analysis of resinous materials with MALDI‐FT‐ICR‐MS: novel internal standards and a new matrix material for negative ion mode

Matrix‐assisted laser desorption/ionization (MALDI) is a mass spectrometry (MS) ionization technique suitable for a wide variety of sample types including highly complex ones such as natural resinous materials. Coupled with Fourier transform ion cyclotron resonance (FT‐ICR) mass analyser, which provides mass spectra with high resolution and accuracy, the method gives a wealth of information about the composition of the sample. One of the key aspects in MALDI‐MS is the right choice of matrix compound. We have previously demonstrated that 2,5‐dihydroxybenzoic acid is suitable for the positive ion mode analysis of resinous samples. However, 2,5‐dihydroxybenzoic acid was found to be unsuitable for the analysis of these samples in the negative ion mode. The second problem addressed was the limited choice of calibration standards offering a flexible selection of m/z values under m/z 1000. This study presents a modified MALDI‐FT‐ICR‐MS method for the analysis of resinous materials, which incorporates a novel matrix compound, 2‐aminoacridine for the negative ion mode analysis and extends the selection of internal standards with m/z <1000 for both positive (15 different phosphazenium cations) and negative (anions of four fluorine‐rich sulpho‐compounds) ion mode. The novel internal calibration compounds and matrix material were tested for the analysis of various natural resins and real‐life varnish samples taken from cultural heritage objects. Copyright © 2017 John Wiley & Sons, Ltd.     

Detection and imaging of chrome yellow (lead chromate) in latent prints,solid residues,and minerals by laser‐desorption/ionization mass spectrometry (LDI‐MS)

In the past, chrome yellow (lead chromate, PbCrO₄), a bright orange‐red substance, has been widely used as an inorganic pigment in the production of paints, coatings, and plastics. Herein, we demonstrate that laser desorption/ionization mass spectrometry (LDI‐MS) is a powerful tool for the detection of lead chromate in solid residues. In fact, lead chromate in trace amounts is easily detectable by LDI‐MS even from residues left as latent prints. For example, a latent print obtained by stamping the exposed laterally cut surface of a pencil over 50 years old on an acetonitrile‐moistened paper, was successfully imaged for both lead and chromate using a Synapt G2 HDMS mass spectrometer. After rastering the print with a 355 nm laser beam and recording positive‐ and negative‐ion mass spectra over the range m/z 50–1200, we generated false‐color ‘heat maps’ (single‐ion images) for ²⁰⁸Pb^+• (m/z 207.98) and Cr₂O₆^−• (m/z 199.85). The heat maps matched closely with the faint visual image of the pencil imprint. Moreover, our results confirmed that lead chromate was used in the pigment coatings of old pencils. Evidently, LDI‐MS imaging is an efficient procedure to survey for the presence of lead and chromate in minerals and other materials. Copyright © 2017 John Wiley & Sons, Ltd.     

Laser desorption postionization for imaging MS of biological material

Vacuum ultraviolet single photon ionization (VUV SPI) is a soft ionization technique that has the potential to address many of the limitations of matrix‐assisted laser desorption/ionization (MALDI) for imaging MS. Laser desorption postionization (LDPI) uses VUV SPI for postionization and is experimentally analogous to a MALDI instrument with the addition of a pulsed VUV light source. This review discusses progress in LDPI‐MS over the last decade, with an emphasis on imaging MS of bacterial biofilms, analytes whose high salt environment make them particularly resistant to imaging by MALDI‐MS. This review first considers fundamental aspects of VUV SPI including ionization mechanisms, cross sections, quantum yields of ionization, dissociation and potential mass limits. The most common sources of pulsed VUV radiation are then described along with a newly constructed LDPI‐MS instrument with imaging capabilities. Next, the detection and imaging of small molecules within intact biofilms is demonstrated by LDPI‐MS using 7.87 eV (157.6 nm) VUV photons from a molecular fluorine excimer laser, followed by the use of aromatic tags for detection of selected species within the biofilm. The final section considers the future prospects for imaging intact biological samples by LDPI‐MS. Copyright © 2010 John Wiley & Sons, Ltd.     

Chemical and Topographical Single‐Cell Imaging by Near‐Field Desorption Mass Spectrometry

Simultaneously acquiring chemical and topographical information within a single cell at nanoscale resolutions is vital to cellular biology, yet it remains a great challenge due to limited lateral resolutions and detection sensitivities. Herein, the development of near‐field desorption mass spectrometry for correlated chemical and topographical imaging is reported, thereby bridging the gap between laser‐based mass spectrometry (MS) methods and multimodal single‐cell imaging. Using this integrated platform, an imaging resolution of 250 nm and 3D topographically reconstructed chemical single‐cell imaging were achieved. This technique offers more in‐depth cellular information than micrometer‐range laser‐based MS imaging methods. Considering the simplicity and compact size of the near‐field device, this technique can be introduced to MALDI‐MS, expanding the multimodal abilities of MS at nanoscale resolutions.     

Uncovering matrix effects on lipid analyses in MALDI imaging mass spectrometry experiments

The specific matrix used in matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) can have an effect on the molecules ionized from a tissue sample. The sensitivity for distinct classes of biomolecules can vary when employing different MALDI matrices. Here, we compare the intensities of various lipid subclasses measured by Fourier transform ion cyclotron resonance (FT‐ICR) IMS of murine liver tissue when using 9‐aminoacridine (9AA), 5‐chloro‐2‐mercaptobenzothiazole (CMBT), 1,5‐diaminonaphthalene (DAN), 2,5‐Dihydroxyacetophenone (DHA), and 2,5‐dihydroxybenzoic acid (DHB). Principal component analysis and receiver operating characteristic curve analysis revealed significant matrix effects on the relative signal intensities observed for different lipid subclasses and adducts. Comparison of spectral profiles and quantitative assessment of the number and intensity of species from each lipid subclass showed that each matrix produces unique lipid signals. In positive ion mode, matrix application methods played a role in the MALDI analysis for different cationic species. Comparisons of different methods for the application of DHA showed a significant increase in the intensity of sodiated and potassiated analytes when using an aerosol sprayer. In negative ion mode, lipid profiles generated using DAN were significantly different than all other matrices tested. This difference was found to be driven by modification of phosphatidylcholines during ionization that enables them to be detected in negative ion mode. These modified phosphatidylcholines are isomeric with common phosphatidylethanolamines confounding MALDI IMS analysis when using DAN. These results show an experimental basis of MALDI analyses when analyzing lipids from tissue and allow for more informed selection of MALDI matrices when performing lipid IMS experiments.     

Spatial profiling invertebrate ganglia using MALDI MS

The ability of MALDI TOF MS to spatially map peptides and proteins directly from a tissue is an exciting advance to imaging mass spectrometry. Recent advances in instrumentation for MS have resulted in instruments capable of achieving several micron spatial resolution while acquiring high-resolution mass spectra. Currently, the ability to obtain high quality mass spectrometric images depends on sample preparation protocols that often result in limited spatial resolution. A number of sample preparation and matrix deposition protocols are evaluated for spatial profiling of Aplysia californica exocrine gland and neuronal tissues. Such samples are different from mammalian tissues, but make good targets for method optimization because of the wealth of biochemical information available on neuropeptide processing and distribution. Electrospray matrix deposition and a variety of freezing methods have been found to be optimum for these invertebrate tissues, with the exact protocols being tissue dependent.