Electrospray sample deposition for matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry with attomole detection limits

Electrospray sample deposition was explored for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In this method, nanoliter volumes of matrix/analyte mixture were electrosprayed from a high voltage biased (1-2 kV) fused-silica capillary onto a grounded MALDI plate mounted 100-500 microm from the capillary outlet. Electrospray deposition with these conditions produced sample spots 200-300 microm in diameter thus matching the laser spot size. Varying spray voltage and distance resulted in different crystal sizes and volatilization rates for alpha-cyano-4-hydroxycinnamic acid matrix. Best results were obtained when the sample was deposited as wet droplets as opposed to deposition as dried solid. Under 'wet-spray' conditions, 2-4 microm diameter crystals were formed and detection limits for several neuropeptides were 0.7-25 amol. Samples could be pre-concentrated on the plate by spraying continuously and allowing sample to evaporate in a small spot. Sample volumes as large as 580 nL were deposited yielding a detection limit of 35 pM for neurotensin 1-11. Electrospray sample deposition yielded similar results when using atmospheric pressure-MALDI coupled with a quadrupole ion trap mass spectrometer, except that the sensitivity was approximately seven-fold worse.     

Determination of pharmaceutical compounds in skin by imaging matrix-assisted laser desorption/ionisation mass spectrometry

Matrix-assisted laser desorption/ionisation (MALDI) quadrupole time-of-flight mass spectrometry (Q-TOFMS) has been used to detect and image the distribution of a xenobiotic substance in skin. Porcine epidermal tissue was treated with 'Nizoral', a medicated shampoo containing ketoconazole (+/-)-1-acetyl-4-[p-[[(2R,4S)-2-(2,4-dichlorophenyl)-2-(imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine) as active ingredient. Following incubation for 1 h at 37 degrees C all excess formulation was washed from the surface. A cross-section of the drug-treated tissue was then blotted onto a cellulose membrane, precoated in matrix (alpha-cyano-4-hydroxycinnamic acid (CHCA)), by airspray deposition. In separate experiments the tissue surface was treated with Nizoral within a triangular former, and subsequently blotted onto a matrix-coated membrane. Sample membranes were then mounted into the recess of specialised MALDI targets with adhesive tape. All samples were analysed by MALDI-TOFMS using an Applied Biosystem 'Q-star Pulsar i' hybrid Q-TOF mass spectrometer fitted with an orthagonal MALDI ion source and imaging software. Detection of the protonated molecule was readily achievable by this technique. Treatment of the tissue within a template gave rise to images depicting the expected distribution of the drug, demonstrating that this technique is capable of producing spatially useful data. Ion images demonstrating the permeation of the applied compound into the skin were achieved by imaging a cross-sectional imprint of treated tissue. A calibration graph for the determination of ketoconazole was prepared using the sodium adduct of the matrix ion as an internal standard. This enabled construction of a quantitative profile of drug in skin. Conventional haematoxylin and eosin staining and microscopy methods were employed to obtain a histological image of the porcine epidermal tissue. Superimposing the mass spectrometric and histological images appeared to indicate drug permeation into the dermal tissue layer.     

Ionic matrix for matrix-enhanced surface-assisted laser desorption ionization mass spectrometry imaging (ME-SALDI-MSI)

Matrix-enhanced surface-assisted laser desorption ionization mass spectrometry imaging (ME-SALDI MSI) has been previously demonstrated as a viable approach to improving MS imaging sensitivity. We describe here the employment of ionic matrices to replace conventional MALDI matrices as the coating layer with the aims of reducing analyte redistribution during sample preparation and improving matrix vacuum stability during imaging. In this study, CHCA/ANI (α-cyano-4-hydroxycinnamic acid/aniline) was deposited atop tissue samples through sublimation to eliminate redistribution of analytes of interest on the tissue surface. The resulting film was visually homogeneous under an optical microscope. Excellent vacuum stability of the ionic matrix was quantitatively compared with the conventional matrix. The subsequently improved ionization efficiency of the analytes over traditional MALDI was demonstrated. The benefits of using the ionic matrix in MS imaging were apparent in the analysis of garlic tissue sections in the ME-SALDI MSI mode.     

Novel approach to enhance sensitivity in surface‐assisted laser desorption/ionization mass spectrometry imaging using deposited organic‐inorganic hybrid matrices

Sample pretreatment is key to obtaining good data in matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI). Although sublimation is one of the best methods for obtaining homogenously fine organic matrix crystals, its sensitivity can be low due to the lack of a solvent extraction effect. We investigated the effect of incorporating a thin film of metal formed by zirconium (Zr) sputtering into the sublimation process for MALDI matrix deposition for improving the detection sensitivity in mouse liver tissue sections treated with olanzapine. The matrix‐enhanced surface‐assisted laser desorption/ionization (ME‐SALDI) method, where a matrix was formed by sputtering Zr to form a thin nanoparticle layer before depositing MALDI organic matrix comprising α‐cyano‐4‐hydroxycinnamic acid (CHCA) by sublimation, resulted in a significant improvement in sensitivity, with the ion intensity of olanzapine being about 1800 times that observed using the MALDI method, comprising CHCA sublimation alone. When Zr sputtering was performed after CHCA deposition, however, no such enhancement in sensitivity was observed. The enhanced sensitivity due to Zr sputtering was also observed when the CHCA solution was applied by spraying, being about twice as high as that observed by CHCA spraying alone. In addition, the detection sensitivity of these various pretreatment methods was similar for endogenous glutathione. Given that sample preparation using the ME‐SALDI‐MSI method, which combines Zr sputtering with the sublimation method for depositing an organic matrix, does not involve a solvent, delocalization problems such as migration of analytes observed after matrix spraying and washing with aqueous solutions as sample pretreatment are not expected. Therefore, ME‐Zr‐SALDI‐MSI is a novel sample pretreatment method that can improve the sensitivity of analytes while maintaining high spatial resolution in MALDI‐MSI.     

Compressed matrix thin film (CMTF)-assisted laser desorption ionization mass spectrometric analysis

The inhomogeneous re-crystallization process of matrix materials is the major concerns associated with matrix assisted laser desorption/ionization (MALDI) analysis. We describe here the approach termed compressed matrix thin film (CMTF) in order to make a uniform matrix deposition. In this approach, solid matrix particles are compressed under 10 MPa of pressure by a compressor that is regularly used in infrared spectroscopic analysis. Then aqueous samples can be deposited on the surface of the matrix film. Major advantages of the CMTF approach are summarized as follows. (1) Reproducible sample preparation procedure. Size and thickness of matrix thin films can be controlled by using a fixed mold.force and known amount of matrix materials. (2) Significantly decreased shot-to-shot variations and enhanced reproducibility. (3) Tolerance for in situ salt washing. Because matrix materials are hydrophobic, salts can be washed away while proteins or peptides are retained on the surface of matrix thin films through hydrophobic interactions. (4) Improved sensitivity. The hydrophobic coating of MALDI sample plate by matrix thin films prevents the spreading of samples across the plate and confines analytes to a small area, leading to increased local concentration. (5) A new means for tissue analysis. Tissue sections can be directly transferred to the uniform surface of matrix materials for reproducible and quantitative comparison of different molecules in different localization. The proposed CMTF should be an enabling technique for mass spectrometric analysis with improved correlations between signal intensities and sample quantities.     

Three-layer matrix/sample preparation method for MALDI MS analysis of low nanomolar protein samples

A robust and sensitive sample preparation method is presented for matrix-assisted laser desorption ionization (MALDI) mass spectrometric analysis of low nanomolar concentrations of proteins containing high amounts of common salts and buffers. This method involves the production of densely packed sub-micrometer matrix crystals by depositing a matrix solution on top of a matrix seed-layer prepared on a MALDI target. A sub-microliter aliquot of analyte solution is then directly added to the top of the matrix crystals to form a thin-layer. alpha-Cyano-4-hydroxycinnamic acid (4-HCCA) is used as matrix and demonstrated to give better performance than other commonly used matrices, such as 2,5-dihydroxybenzoic acid (DHB), 2-(4-hydroxy-phenylazo) benzoic acid (HABA), or sinapinic acid. This three-layer method is shown to be superior to the other MALDI sample preparation methods, particularly for handling low nanomolar protein solutions containing salts and buffers.     

Automated MALDI Matrix Coating System for Multiple Tissue Samples for Imaging Mass Spectrometry

Uniform matrix deposition on tissue samples for matrix-assisted laser desorption/ionization (MALDI) is key for reproducible analyte ion signals. Current methods often result in nonhomogenous matrix deposition, and take time and effort to produce acceptable ion signals. Here we describe a fully-automated method for matrix deposition using an enclosed spray chamber and spray nozzle for matrix solution delivery. A commercial air-atomizing spray nozzle was modified and combined with solenoid controlled valves and a Programmable Logic Controller (PLC) to control and deliver the matrix solution. A spray chamber was employed to contain the nozzle, sample, and atomized matrix solution stream, and to prevent any interference from outside conditions as well as allow complete control of the sample environment. A gravity cup was filled with MALDI matrix solutions, including DHB in chloroform/methanol (50:50) at concentrations up to 60 mg/mL. Various samples (including rat brain tissue sections) were prepared using two deposition methods (spray chamber, inkjet). A linear ion trap equipped with an intermediate-pressure MALDI source was used for analyses. Optical microscopic examination showed a uniform coating of matrix crystals across the sample. Overall, the mass spectral images gathered from tissues coated using the spray chamber system were of better quality and more reproducible than from tissue specimens prepared by the inkjet deposition method.     

Development of a MALDI two-layer volume sample preparation technique for analysis of colored conidia spores of <Emphasis Type="Italic">Fusarium</Emphasis> by MALDI linear TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) has been proved to be a powerful tool for the identification and characterization of microorganisms based on their surface peptide/protein pattern. Because of the complexity of microorganisms, there are no standardized protocols to acquire reproducible peptide/protein profiles for a broad range of microorganisms and for fungi in particular. Small variations during MALDI MS sample preparation affect the quality of mass spectra quite often. In this study, we were aiming to develop a sample preparation method for the analysis of colored, a quite often observed phenomenon, and mycotoxin-producing Fusarium conidia spores using MALDI–TOF MS. Different washing solvent systems for light- and deep-colored (from slightly orange to red-brown) conidia spores and connected sample deposition techniques were evaluated based on MS reproducibility and number and intensities of peaks. As a method of choice for generation of reproducible and characteristic MALDI–TOF mass spectra, the use of a washing process for colored Fusarium conidia spores with acetonitrile/0.5% formic acid (7/3) was found and subsequently combined with two-layer volume technique (spores/matrix (ferulic acid) solution was deposited onto a MALDI target, and after solvent evaporation, a second matrix layer was deposited). With the application of this sample preparation method, for deep-colored Fusarium species, 19 abundant molecular ions in the m/z range 2,000–10,000 were always detected with an S/N ratio of 3:1 or better. Finally this optimized sample preparation for the first time provided mass spectrometric fingerprints of strongly colored Fusarium conidia spores resulting in the possibility of differentiation of such spores at the species level.      

Solvent-free matrix dry-coating for MALDI imaging of phospholipids

A fast and simple, solvent-free matrix deposition protocol was developed for positive ionization mode phospholipid analysis in tissues. Finely ground 2,5-dihydroxybenzoic acid was deposited onto sagittal mouse brain sections using a dry-coating technique, in which solid matrix particles were filtered directly onto the tissue through a 20-microm stainless steel sieve. Phospholipid signals were obtained directly off these sections, allowing acquisition of high-resolution MS images. These images were compared to those from serial sections that were spray-coated with a thin-layer chromatography (TLC) reagent sprayer. Signals obtained from the dry matrix deposition method were comparable to those from spray-coated sections, producing identical localization patterns with a simpler and faster sample preparation with virtually no analyte delocalization. This approach was found to yield highly reproducible results, eliminating much of the variance caused by operator differences, and making it an attractive alternative to the currently used matrix application methods.     

A solid-phase bioreactor with continuous sample deposition for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We report the development of a solid‐phase proteolytic digestion and continuous deposition microfluidic chip platform for low volume fraction collection and off‐line matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Tryptic peptides were formed in an on‐chip bioreactor and continuously deposited onto a MALDI target plate using a motor‐driven xyz stage. The bioreactor consisted of a 4 cm × 200 µm × 50 µm microfluidic channel with covalently immobilized trypsin on an array of 50 µm diameter micropost structures with a 50 µm edge‐to‐edge inter‐post spacing. A 50 µm i.d. capillary tube was directly attached to the end of the bioreactor for continuous sample deposition. The MALDI target plate was modified by spin‐coating a nitrocellulose solution containing a MALDI matrix on the surface prior to effluent deposition. Protein molecular weight standards were used for evaluating the performance of the digestion and continuous deposition system. Serpentine sample traces 200 µm wide were obtained with a 30 fmol/mm quantity deposition rate and a 3.3 nL/mm volumetric deposition rate. Copyright © 2011 John Wiley & Sons, Ltd.     

Matrix-assisted laser desorption/ionization mass spectrometry of collected bioaerosol particles

A method was developed for collection and analysis of bioaerosols by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry using a modified Andersen N6 bioaerosol collector. The overall goal of the study was to develop methods for obtaining mass spectra with minimal reagents and treatment steps for potential use in remote collection and analysis systems. Test bioaerosol particles were generated from a nebulized E. coli bacterial suspension and collected on MALDI targets placed in an Andersen N6 single-stage aerosol impactor. The bioaerosols were mixed with matrix either by deposition on a bare target with the matrix solution added later, or by deposition on a target pre-coated with matrix. The matrix compounds alpha-cyano-4-hydroxycinnamic acid (CHCA) and sinapic acid (SA) were tested and the SA matrix was found to give the best results in number of peaks, resolution, and signal-to-noise ratio. Deposition of bioaerosol particles onto the matrix pre-coated target did not produce signal in the m/z region above 1000, but the signal could be recovered with the addition of a 1:1 (v/v) acetonitrile/water solvent. Addition of solvent by pipette to the pre-coated targets after particle deposition recovered signal comparable to the dried-droplet sample preparations, whereas solvent sprayed into the impactor recovered fewer peaks. Deposition on pre-coated targets with post-collection solvent addition was superior to deposition on bare target followed by post-collection addition of matrix solution.     

Online deposition of nano‐aerosol for matrix‐assisted laser desorption/ionization mass spectrometry

An online nano‐aerosol sample deposition method for matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry is described in which matrix and analyte particles between 50 and 500 nm are aerodynamically focused onto a tight spot, ca. 200 µm in diameter, on the target plate under vacuum. MALDI analysis of the target is performed without additional sample preparation. The method is evaluated with insulin as the analyte and alpha‐cyano‐4‐hydroxycinnamic acid (CHCA) as the matrix. Two preparation modes are compared with conventional dried‐droplet deposition: mixture deposition where a single layer is deposited consisting of particles that contain both matrix and analyte, and layered deposition where an underlayer of matrix particles and an overlayer of analyte particles are deposited separately. Desalting is performed by adding ammonium sulfate to the solution used to generate the matrix aerosol. With mixture deposition, the optimum matrix‐to‐analyte mole ratio is about 500:1 compared with 5000:1 for the conventional dried‐droplet method. With layered deposition, the thicknesses of the matrix and analyte layers are more important determinants of the analyte signal intensity than the matrix‐to‐analyte mole ratio. Analyte signal intensities are independent of matrix layer thickness above 200 nm, and the optimum analyte signal is obtained with an analyte layer thickness of about 100 nm. Copyright © 2009 John Wiley & Sons, Ltd.     

Improved peptide mass fingerprinting matches via optimized sample preparation in MALDI mass spectrometry

Peptide mass fingerprinting (PMF) is a powerful technique in which experimentally measured m/z values of peptides resulting from a protein digest form the basis for a characteristic fingerprint of the intact protein. Due to its propensity to generate singly charged ions, along with its relative insensitivity to salts and buffers, matrix-assisted laser desorption and ionization (MALDI)-time-of-flight mass spectrometry (TOFMS) is the MS method of choice for PMF. The qualitative features of the mass spectrum can be selectively tuned by employing different methods to prepare the protein digest and matrix for MALDI-TOFMS. The selective tuning of MALDI mass spectra in order to optimize PMF is addressed here. Bovine serum albumin, carbonic anhydrase, cytochrome c, hemoglobin alpha- and beta-chain, and myoglobin were digested with trypsin and then analyzed by MALDI-TOFMS. 2,5-dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) were prepared using six different sample preparation methods: dried droplet, application of protein digest on MALDI plate followed by addition of matrix, dried droplet with vacuum drying, overlayer, sandwich, and dried droplet with heating. Improved results were obtained for the matrix alpha-cyano-4-hydroxycinnamic acid using a modification of the died droplet method in which the MALDI plate was heated to 80 °C prior to matrix application, which is supported by observations from scanning electron microscopy. Although each protein was found to have a different optimum sample preparation method for PMF, in general higher sequence coverage for PMF was obtained using DHB. The best PMF results were obtained when all of the mass spectral data for a particular protein digest was convolved together.     

MALDI post-source decay and LIFT-TOF/TOF investigation of α-cyano-4-hydroxycinnamic acid cluster interferences

Large signals from alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix complexes with sodium and potassium ions were found to interfere with sensitive matrix-assisted laser desorption/ionization (MALDI) analysis of a hydrochloric acid digest of gelatine preparations. The nature of some selected matrix clusters was investigated by conventional post-source decay and LIFT-TOF/TOF experiments. The matrix clusters fragmented readily by neutral evaporation to give smaller sized matrix cluster species without matrix disintegration. Their characterization distinguished them from peptide signals, in particular from those that had the same nominal mass and differed only in the fractional part of the mass as encountered for gelatine-derived peptides. Knowledge of the molecular composition of these cluster species allowed using them for internal calibration of the MALDI mass spectra. The hydrolytic peptides could be analyzed with increased sensitivity when using 2,5-dihydroxy benzoic acid (DHB) as the MALDI matrix.     

Repeat MALDI MS imaging of a single tissue section using multiple matrices and tissue washes

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) techniques are continually being assessed with a view to improving the quality of information obtained from a given sample. A single tissue section will typically only be analyzed once by MALDI MSI and is then either used for histological staining or discarded. In this study, we explore the idea of repeat analysis of a single tissue section by MALDI MSI as a route toward improving sensitivity, structural characterization, and diversity of detected analyte classes. Repeat analysis of a single tissue section from a fresh frozen mouse brain is investigated with both α-cyano-4-hydroxycinnamic acid (CHCA) and para-nitroaniline (PNA). Repeat analysis is then applied to the acquisition of MALDI MSI and MALDI tandem mass spectrometry imaging employing collision induced dissociation (MS/MS imaging employing CID) from a formalin-fixed mouse brain section. Finally, both lipid and protein data are acquired from the same tissue section via repeat analysis utilizing CHCA, sinapinic acid (SA), and a tissue wash step. PNA was found to outperform CHCA as a matrix for repeat analysis; multiple lipids were identified using MS/MS imaging; both lipid and protein images were successfully acquired from a single tissue section.

The use of pencil lead as a matrix and calibrant for matrix-assisted laser desorption/ionisation

Pencil lead is shown to be an effective matrix and calibrant in matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry. Various groups of analytes, including peptides, polymers and actinide metals, can be readily ionised using MALDI when deposited onto a pencil lead matrix. The matrix is seen to have advantages in sample preparation relating to its hydrophobic properties and almost complete suppression of the matrix during analysis. Using pencil lead as a matrix is a quick and convenient method of qualitative analysis and has been shown to be quantitative for the isotope ratio analysis of actinide metals.     

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.     

Closely spaced external standard: a universal method of achieving 5 ppm mass accuracy over the entire MALDI plate in axial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Close deposition of the sample and external standard was used in axial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to achieve mass accuracy equivalent to that obtained with an internal standard across the entire MALDI plate. In this work, the sample and external standard were deposited by continuous deposition in separate traces, each approximately 200 micro m wide. The dependence of the mass accuracy on the distance between the sample and standard traces was determined across a MALDI target plate with dimensions of 57.5 mm x 57.0 mm by varying the gap between the traces from 100 micro m to 4 mm. During acquisition, two adjacent traces were alternately irradiated with a 200-Hz laser, such that the peaks in the resulting mass spectra combined the sample and external standard. Ion suppression was not observed even when the peptide concentrations in the two traces differed by more than two orders of magnitude. The five peaks from the external standard trace were used in a four-term mass calibration of the masses of the sample trace. The average accuracy across the whole plate with this method was 5 ppm when peaks of the sample trace had signal-to-noise ratios of at least 30 and the gap between the traces was approximately 100 micro m. This approach was applied to determining peptide masses of a reversed-phase liquid chromatographic (LC) separation of a tryptic digest of beta-galactosidase deposited as a long serpentine trace across the MALDI plate, with accuracy comparable to that obtainable using internal calibration. In addition, the eluent from reversed-phase LC separation of a strong cation-exchange fraction containing tryptic peptides from a yeast lysate along with the closely placed external standard was deposited on the MALDI plate. The data obtained in the MS and MS/MS modes on a MALDI-TOF/TOF mass spectrometer were combined and used in database searching with MASCOT. Since the significant score is a function of mass accuracy in the MS mode, database searching with high mass accuracy reduced the number of false positives and also added peptides which otherwise would have been eliminated at lower mass accuracy (false negatives).     

Ionic (liquid) matrices for matrix-assisted laser desorption/ionization mass spectrometry—applications and perspectives

A large number of matrix substances have been used for various applications in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The majority of matrices applied in ultraviolet-MALDI MS are crystalline, low molecular weight compounds. A problem encountered with many of these matrices is the formation of hot spots, which lead to inhomogeneous samples, thus leading to increased measurement times and hampering the application of MALDI MS for quantitative purposes. Recently, ionic (liquid) matrices (ILM or IM) have been introduced as a potential alternative to the classical crystalline matrices. ILM are equimolar mixtures of conventional MALDI matrix compounds such as 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CCA) or sinapinic acid (SA) together with organic bases [e.g., pyridine (Py), tributylamine (TBA) or N,N-dimethylethylenediamine (DMED)]. The present article presents a first overview of this new class of matrices. Characteristic properties of ILM, their influence on mass spectrometric parameters such as sensitivity, resolution and adduct formation and their application in the fields of proteome analysis, the measurement of low molecular weight compounds, the use of MALDI MS for quantitative purposes and in MALDI imaging will be presented. Scopes and limitations for the application of ILM are discussed.