On the use of DHB/aniline and DHB/<Emphasis Type="Italic">N,N</Emphasis>-dimethylaniline matrices for improved detection of carbohydrates: Automated identification of oligosaccharides and quantitative analysis of sialylated glycans by MALDI-TOF mass spectrometry

This study demonstrates the application of 2,5-dihydrohybenzoic acid/aniline (DHB/An) and 2,5-dihydroxybenzoic acid/N,N-dimethylaniline (DHB/DMA) matrices for automated identification and quantitative analysis of native oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Both matrices are shown to be superior to pure DHB for native glycans in terms of signal intensities of analytes and homogeneity of sample distribution throughout the crystal layer. On-target formation of stable aniline Schiff base derivatives of glycans in DHB/An and the complete absence of such products in the mass spectra acquired in DHB/DMA matrix provide a platform for automated identification of reducing oligosaccharides in the MALDI mass spectra of complex samples. The study also shows how enhanced sensitivity is achieved with the use of these matrices and how the homogeneity of deposited sample material may be exploited for quick and accurate quantitative analysis of native glycan mixtures containing neutral and sialylated oligosaccharides in the low-nanogram to mid-picogram range.     

Design considerations for high speed quantitative mass spectrometry with MALDI ionization

A MALDI ion source on a triple quadrupole mass spectrometer constructed for the purpose of obtaining high speed quantitative measurements on drugs and other low molecular weight compounds is described. Particular attention is given to the ion generation and transport phenomena that affect analysis speed, throughput, and practical instrument robustness. In this regard parameters that affect desorption speed, beam spreading, ion flight times, sensitivity, signal-to-noise, ion fragmentation, sample carry-over, and instrument contamination are examined and experimental results are provided. MALDI and electrospray sensitivity is compared, to provide a practical frame of reference.     

Liquid supports for ultraviolet atmospheric pressure matrix-assisted laser desorption/ionization

Atmospheric pressure (AP) liquid matrices for ultraviolet (UV) matrix-assisted laser desorption/ionization (MALDI) are presented. Doping a known organic chromophore, alpha-cyano-4-hydroxycinnamic acid (CHCA), into liquid media yielded a homogenous sample system with simplified sample preparation, increased sample lifetime, and added utility for APMALDI ion sources. Compared with vacuum situations, AP matrices are not as limited by vapor pressure, so liquid matrix formulations can focus on desorption and ionization versus vacuum stability and source contamination. The parameters studied include chromophore concentration, liquid support variations, and quantitation capability. Chromophore concentration adjustments provided insight into the necessary absorbance for UV-APMALDI and demonstrated the importance of laser penetration depth. Liquid support variations allowed adjustments of sample lifetime and analyte solvents. Extended sample lifetime is beneficial for instrument tuning and source optimization; however, increased liquid viscosity lowers signal intensity. The shot-to-shot reproducibility, as examined with individual ion packets, suggests that the liquid matrix can alleviate some inconsistencies seen with solid MALDI, suggesting a possibility for better quantitation. The measurements for laser penetration depth, solution viscosity, and solvent additives could add to the information on MALDI mechanisms. The liquid matrix offers advantages that complement current MALDI methods.     

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.     

Comparison of mass spectra of peptides in different matrices using matrix-assisted laser desorption/ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG.

The mass spectra of peptides obtained with different matrices were compared using a matrix-assisted laser desorption/ionization (MALDI) ion source and a multi-turn time-of-flight (TOF) mass spectrometer, MULTUM-IMG, which has been developed at Osaka University. Two types of solid matrices, alpha-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), and a liquid matrix made from a mixture of 3-aminoquinoline and CHCA were used. When measuring the peak signal intensity of human angiotensin II [M+H]+ from a fixed sample position, the liquid matrix produced a stable signal over 1000 laser shots, while the signal obtained with CHCA and DHB decayed after about 300 and 100 shots, respectively. Significant differences in the mass resolving power were not observed between the spectra obtained with the three matrices. Signal peak areas were measured as a function of the cycle number in a multi-turn ion trajectory, i.e., the total flight time over a millisecond time scale. For both [M+H]+ of human angiotensin II and bovine insulin, the decay of the signal peak area was the most significant with CHCA, while that measured with DHB was the smallest. The results of the mean initial ion velocity measurements suggested that the extent of metastable decomposition of the analyte ions increased in order of DHB, the liquid matrix, and CHCA, which is consistent with the difference in the decay of the signal peak area as the total flight time increased.     

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.     

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.     

A combined ion source for fast switching between electrospray and matrix-assisted laser desorption/ionization in Fourier transform ion cyclotron resonance mass spectrometry

A new ion source has been developed for Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) that enables quick changes between matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) modes. When operating as an ESI source, the sample solution is sprayed through an angled nebulizer. The generated ions pass through a glass capillary followed by a skimmer and three sequential hexapole ion guides. Ions can be accumulated in the third hexapole (storage hexapole) before they are injected into the ICR trap. The second hexapole is mounted on a movable platform which also carries the MALDI sample plate. During the switch from ESI to MALDI, this platform moves the second hexapole out of the hexapole series and locates a MALDI sample plate with 384 sample positions into the area directly in front of the storage hexapole. The storage hexapole is in a medium pressure chamber (MPC) which has windows both for the incoming laser beam and for the observation optics, as well as a gas tube for pulsing collision gas into the chamber. During the MALDI operation the focused laser beam enters the MPC, passes between the hexapole rods and irradiates a MALDI sample on the target plate. The sample molecules are desorbed/ionized into the storage hexapole and simultaneously cooled by collisions with the pulsed gas. Ions desorbed from multiple laser shots can be accumulated in this hexapole before they are transferred to the ICR trap. With the combined ion source a computer-controlled switch between MALDI and ESI modes is possible in less than a minute, depending on the position of the MALDI target on the 384-spot plate. Immediate acquisition of mass spectra is possible after mode switching without the need for tuning or re-calibration.     

C60衍生物的MALDI-TOFMS分析

C_(60)衍生物在超导、非线性光学、催化、材料和生物活性等方面有巨大的潜在应用价值。C_(60)衍生物大多为固体,蒸汽压较低,采用需要加热才能够使样品气化电离的质谱或"硬"电离质谱方法进行测定,易造成C_(60)衍生物分解并释放出配体。近年来国内外应用基体辅助激光解吸软电离质谱法成功分析了许多不同类型的C_(60)衍生物如卤化C_(60)酰胺化C_(60)芳基化C_(60)、C_(60)部花菁、煤基C_(60)烟灰萃取产物、金属C_(60)衍生物以及C_(60)乙二胺膜等。本文报道采用MALDI-TOFMS法分析C_(60)酯衍生物和C_(60)吡咯烷衍生物的结果。     

Qualitative and quantitative analysis of low molecular weight compounds by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry using ionic liquid matrices

A major problem hampering the use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for quantitative measurements is the inhomogeneous distribution of analytes and matrices in solid sample preparations. The use of ionic liquids as matrices for the qualitative and quantitative analysis of low molecular weight compounds like amino acids, sugars and vitamins was investigated. The ionic liquid matrices are composed of equimolar combinations of classical MALDI matrices (sinapinic acid, alpha-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid) with organic bases. These matrix systems allow a homogenous sample preparation with a thin ionic liquid layer having negligible vapour pressure. This leads to a facilitated qualitative and quantitative measurement of the analytes compared with classical solid matrices.     

Electron capture negative ion mass spectra of some typical matrix-assisted laser desorption/ionization matrices

A series of seven typical matrix-assisted laser desorption/ionization (MALDI) matrices has been investigated by means of electron capture negative ion mass spectrometry (ECNI-MS). It has been shown that the most effective matrices form deprotonated negative ions predominantly in the low-energy region. Relative dissociative cross sections have been measured for all molecules under investigation. The relative integrated abundance of [M - H](-) ion formation in the series changes by four orders of magnitude. It has been shown that 2,5-DHB (gentisic acid), one of the most effective MALDI matrices, has maximal relative intensity of [M - H](-) formation at the energy approximately equal 0.8 eV. This result is in accordance with a finding of Frankevich and Zenobi [Book of Abstracts, Workshop-school "Mass spectrometry in chemical physics, bio-physics and environmental sciences", Zvenigorod, Russia, April, 25-26, 2002, p. 40] that a probable origin of negative ions in MALDI is the process of low-energy (0.5-1 eV) dissociative electron capture by matrix molecules.     

MALDI Matrices for the Analysis of Low Molecular Weight Compounds: Rational Design,Challenges and Perspectives

The analysis of low molecular weight (LMW) compounds is of great interest to detect small pharmaceutical drugs rapidly and sensitively, or to trace and understand metabolic pathways. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) plays a central role in the analysis of high molecular weight (bio)molecules. However, its application for LMW compounds is restricted by spectral interferences in the low m/z region, which are produced by conventional organic matrices. Several strategies regarding sample preparation have been investigated to overcome this problem. A different rationale is centred on developing new matrices which not only meet the fundamental requirements of good absorption and high ionization efficiency, but are also vacuum stable and “MALDI silent”, i. e., do not give matrix-related signals in the LMW area. This review gives an overview on the rational design strategies used to develop matrix systems for the analysis of LMW compounds, focusing on (i) the modification of well-known matrices, (ii) the search for high molecular weight matrices, (iii) the development of binary, hybrid and nanomaterial-based matrices, (iv) the advance of reactive matrices and (v) the progress made regarding matrices for negative or dual polarity mode.     

Cinnamic acid derivatives as matrices for ultraviolet laser desorption mass spectrometry of proteins

The paper reports the discovery of three new matrices for the matrix-assisted laser desorption of proteins. These new matrices (sinapinic, ferulic and caffeic acids) are cinnamic acid derivatives that have several practical advantages over the nicotinic acid matrices previously used. These materials form much less intense photochemically generated adduct peaks in the protein quasimolecular ion signal and the adduct peaks that are present are easier to resolve. These matrices produce intense protonated-molecule ions from all of the proteins (over 50) so far examined. These new matrices are also very stable in a vacuum, allowing for their convenient use in very high vacuum applications (e.g., Fourier transform ion cyclotron resonance mass spectrometry).     

Rapid fingerprinting of red wines by MALDI mass spectrometry

Here we report a simple and fast method for wine fingerprinting based on direct matrix-assisted laser desorption/ionization (MALDI) mass spectrometry analysis of different red wine samples, useful for batch-to-batch analysis and for the detection of key compounds even in trace amounts which may vary from vintage to vintage, and from one treatment to another one. A series of 20 samples from different wines were subjected to MALDI mass spectrometry. We found that 2,5-dihydroxybenzoic acid is far superior with respect to all the matrices tested To the best of our knowledge this is the first application of an effective wine profiling not limited to detection of anthocyanins. More than 80 molecular species were detected. Moreover, qualitative and quantitative differences were observed, owing to the nature and relative abundance of different chemical compounds among the wines.     

Improvements in ion signal reproducibility obtained using a homogeneous laser beam for on-line laser desorption/ionization of single particles

A major factor limiting on-line single particle mass spectrometry techniques from becoming more quantitative is the large shot-to-shot variability in ion intensities observed in the laser desorption/ionization (LDI) mass spectra.1,2 In previous work, lab-generated particles showed fluctuations of up to 152% in the absolute ion intensities in averaged spectra of 200-300 'identical' particles.2 Most of these fluctuations were attributed to inhomogeneities in the laser beam profile, leading to significant differences in the power each particle encountered depending on the position in the LDI laser beam where it underwent analysis. The goal of the work presented herein is to determine whether a fiber optic actually reduces the observed variability in single particle LDI mass spectral data. Initial results are presented for individual single component organic particles composed of 2,4-dihydroxybenzoic acid (2,4-DHB) analyzed using a low-power flat-top laser beam profile created by sending an ultraviolet (266 nm) DI laser through a fiber optic. Relative standard deviations of the total ion intensities for peaks in individual spectra are reduced to 31%. Single particle spectra, compared with and without laser homogenization at the same nominal laser fluence, show a marked enhancement. Specifically, the ion signal patterns of the 2,4-DHB particle spectra obtained using a homogenous LDI beam look identical to one another (i.e. only one particle type was produced with a commonly used neural network grouping algorithm), whereas without beam homogenization 25 different particle types (based on ion intensity patterns) were obtained. Future publications will explore more particle types and matrices but the initial results described herein are quite encouraging.     

Investigations of electrospray sample deposition for polymer MALDI mass spectrometry

In the interest of a more thorough understanding of the relationship between sample deposition technique and the quality of data obtained using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, details of the electrospray (ES) process of sample deposition are investigated using a number of techniques. Sample morphology was observed with scanning electron microscopy (SEM) and atomic force microscopy (AFM), while matrix-enhanced secondary ion mass spectrometry (MESIMS) monitored surface coverage. Electrospray deposition reduces the analyte segregation that can occur during traditional dried droplet deposition for MALDI. We attribute statistically significant improvements in the reproducibility of signal intensity and MALDI average molecular mass measurements to the ES sample deposition technique.     

Compelling Evidence for Lucky Survivor and Gas Phase Protonation: The Unified MALDI Analyte Protonation Mechanism

This work experimentally verifies and proves the two long since postulated matrix-assisted laser desorption/ionization (MALDI) analyte protonation pathways known as the Lucky Survivor and the gas phase protonation model. Experimental differentiation between the predicted mechanisms becomes possible by the use of deuterated matrix esters as MALDI matrices, which are stable under typical sample preparation conditions and generate deuteronated reagent ions, including the deuterated and deuteronated free matrix acid, only upon laser irradiation in the MALDI process. While the generation of deuteronated analyte ions proves the gas phase protonation model, the detection of protonated analytes by application of deuterated matrix compounds without acidic hydrogens proves the survival of analytes precharged from solution in accordance with the predictions from the Lucky Survivor model. The observed ratio of the two analyte ionization processes depends on the applied experimental parameters as well as the nature of analyte and matrix. Increasing laser fluences and lower matrix proton affinities favor gas phase protonation, whereas more quantitative analyte protonation in solution and intramolecular ion stabilization leads to more Lucky Survivors. The presented results allow for a deeper understanding of the fundamental processes causing analyte ionization in MALDI and may alleviate future efforts for increasing the analyte ion yield.     

High-Speed MALDI-TOF Imaging Mass Spectrometry: Rapid Ion Image Acquisition and Considerations for Next Generation Instrumentation

A prototype matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer has been used for high-speed ion image acquisition. The instrument incorporates a Nd:YLF solid state laser capable of pulse repetition rates up to 5 kHz and continuous laser raster sampling for high-throughput data collection. Lipid ion images of a sagittal rat brain tissue section were collected in 10 min with an effective acquisition rate of roughly 30 pixels/s. These results represent more than a 10-fold increase in throughput compared with current commercially available instrumentation. Experiments aimed at improving conditions for continuous laser raster sampling for imaging are reported, highlighting proper laser repetition rates and stage velocities to avoid signal degradation from significant oversampling. As new high spatial resolution and large sample area applications present themselves, the development of high-speed microprobe MALDI imaging mass spectrometry is essential to meet the needs of those seeking new technologies for rapid molecular imaging.     

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.     

Considerations for quantification of lipids in nerve tissue using matrix-assisted laser desorption/ionization mass spectrometric imaging

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging is a technique that provides the ability to identify and characterize endogenous and exogenous compounds spatially within tissue with relatively little sample preparation. While it is a proven methodology for qualitative analysis, little has been reported for its utility in quantitative measurements. In the current work, inherent challenges in MALDI quantification are addressed. Signal response is monitored over successive analyses of a single tissue section to minimize error due to variability in the laser, matrix application, and sample inhomogeneity. Methods for the application of an internal standard to tissue sections are evaluated and used to quantify endogenous lipids in nerve tissue. A precision of 5% or less standard error was achieved, illustrating that MALDI imaging offers a reliable means of in situ quantification for microgram-sized samples and requires minimal sample preparation.