Matrix-assisted Laser Desorption/ionization Matrices Research Articles

Nitro Indole Derivatives as Novel Dual-Polarity Matrices for MALDI Mass Spectrometry and Imaging with Broad Applications.

A new matrix framework is presented in this study for the improved ionization efficiency of complex mixtures by matrix-assisted laser desorption ionization (MALDI) mass spectrometry/imaging. Five nitro indole (NI) derivatives [3-methyl-4-nitro-1H-indole (3,4-MNI), 3-methyl-6-nitro-1H-indole (3,6-MNI), 2,3-dimethyl-4-nitro-1H-indole (2,3,4-DMNI), 2,3-dimethyl-6-nitro-1H-indole (2,3,6-DMNI), and 4-nitro-1H-indole (4-NI)] were synthesized and shown to produce both positive and negative ions with a broad class of analytes as MALDI matrices. NI matrices were compared to several common matrices, such as 2,5-dihydroxybenzoic acid (DHB), alpha-cyano-4-hydroxylcinnamic acid (CHCA), sinapinic acid (SA), 1,5-diaminonaphthelene (1,5-DAN), and 9-aminoacridine (9-AA), for the analysis of lipid, peptide, protein, glycan, and perfluorooctanesulfonic acid (PFOS) compounds. 3,4-MNI demonstrated the best performance among the NI matrices. This matrix resulted in reduced ion suppression and better detection sensitivity for complex mixtures, for example, egg lipids/milk proteins/PFOS in tap water, while 2,3,6-DMNI was the best matrix for blueberry tissue imaging. Several important aspects of this work are reported: (1) dual-polarity ion production with NI matrices and complex mixtures; (2) quantitative analysis of PFOS with a LOQ of 0.5 ppb in tap water and 0.05 ppb in MQ water (without solid phase extraction enrichment), with accuracy and precision within 5%; (3) MALDI imaging with 2,3,6-DMNI as a matrix for plant metabolite/lipid identification with ionization enhancement in the negative ion mode m/z 600-900 region; and (4) development of a thin film deposition under/above tissue method for MALDI imaging with a vacuum sublimation matrix on a high-vacuum MALDI instrument.

Contribution of LDI and MALDI for the Characterization of a Lignocellulosic-Based Pyrolysis Bio-Oil.

In recent years, various alternatives to fossil fuels have been developed. One of them involves the production of bio-oils from lignocellulosic-based biomass through pyrolysis. However, bio-oils present numerous heteroatoms and, in particular, oxygen atoms that need to be removed by an upgrading process. To optimize these processes, it is necessary to have good knowledge of the composition of the bio-oils at the molecular level. This work aims to establish the usefulness of laser desorption ionization (LDI) and matrix-assisted laser desorption/ionization (MALDI) techniques on lignocellulosic biomass-based bio-oils. Using a Fourier transform ion cyclotron mass spectrometer (FTICR MS), we showed that MALDI gives more information than LDI. The selectivity of a series of MALDI matrices was investigated, showing that some matrices are selective toward compound families and others ionize a wider range of compounds. In this study, nine proton-transfer matrices and three electron-transfer matrices were used and compared to results obtained in LDI. Dithranol, acetosyringone, and graphene oxide were the three promising matrices selected from all matrices, giving an overall characterization of oxygenated classes in a bio-oil. They allowed the ionization of many more species covering a wide range of polarity, aromaticity, and mass with a homogeneous relative intensity for all molecular classes such as lignin-derivative species, sugars, and lipid-derivative species.

Synthesis and Investigation of Novel CHCA-Derived Matrices for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis of Lipids.

A significant area of study and upgrading for increasing sensitivity and general performances of matrix-assisted laser-desorption ionization (MALDI) mass spectrometry (MS) is related to matrix design. Several efforts have been made to address the challenge of low-mass-region interference-free for metabolomics analysis and specifically for lipidomics. To this aim, rationally designed matrices as 4-chloro-α-cyanocinnamic acid (ClCCA) were introduced and reported to provide enhanced analytical performances. We have taken this rational design one step further by developing and optimizing new MALDI matrices with a range of modifications on the CHCA core, involving different functionalities and substituents. Of particular interest was the understanding of the electron-withdrawing (e.g., nitro-) or donating (e.g., methoxy-) effects along with the extent of conjugation on the ionization efficiency. In the present work, ten matrices were designed on a reasonable basis, synthesized, and characterized by NMR and UV spectroscopies and laser desorption ionization. With the assistance of these putative MALDI matrices, samples containing phospholipids (PL), and neutral di-/tri-acylglycerols (DAG, TAG) were investigated using milk, fish, blood, and human plasma extracts. In comparison with CHCA and ClCCA, four of them, viz. [(2E,4E)-2-cyano-5-(4-methoxyphenyl)penta-2,4-dienoic acid] (1), [(2E,4E)-2-cyano-5-(4-nitrophenyl)penta-2,4-dienoic acid] (2), [(E)-2-cyano-3-(6-methoxynaphthalen-2-yl)acrylic acid] (6) and [(E)-2-cyano-3-(naphthalen-2-yl)acrylic acid] (7) displayed good to even excellent performances as MALDI matrices in terms of ionization capability, interference-free spectra, S/N ratio, and reproducibility. Especially compound 7 (cyano naphthyl acrylic acid, CNAA) was the election matrix for PL analysis and matrix 2 (cyano nitrophenyl dienoic acid, CNDA) for neutral lipids such as DAG and TAG in positive ion mode.

Evaluation of 6 MALDI-Matrices for 10 μm Lipid Imaging and On-Tissue MSn with AP-MALDI-Orbitrap.

Mass spectrometry imaging is a technique uniquely suited to localize and identify lipids in a tissue sample. Using an atmospheric pressure (AP-) matrix-assisted laser desorption ionization (MALDI) source coupled to an Orbitrap Elite, numerous lipid locations and structures can be determined in high mass resolution spectra and at cellular spatial resolution, but careful sample preparation is necessary. We tested 11 protocols on serial brain sections for the commonly used MALDI matrices CHCA, norharmane, DHB, DHAP, THAP, and DAN in combination with tissue washing and matrix additives to determine the lipid coverage, signal intensity, and spatial resolution achievable with AP-MALDI. In positive-ion mode, the most lipids could be detected with CHCA and THAP, while THAP and DAN without additional treatment offered the best signal intensities. In negative-ion mode, DAN showed the best lipid coverage and DHAP performed superiorly for gangliosides. DHB produced intense cholesterol signals in the white matter. One hundred fifty-five lipids were assigned in positive-ion mode (THAP) and 137 in negative-ion mode (DAN), and 76 peaks were identified using on-tissue tandem-MS. The spatial resolution achievable with DAN was 10 μm, confirmed with on tissue line-scans. This enabled the association of lipid species to single neurons in AP-MALDI images. The results show that the performance of AP-MALDI is comparable to vacuum MALDI techniques for lipid imaging.

Effect of the Laser Pulse Width in MALDI-2: A Comparative Study of Picosecond versus Nanosecond Wide Pulses for Laser Postionization.

MALDI-2 is a recently introduced technique for postionization (PI) in matrix-assisted laser desorption/ionization (MALDI). It is based on an initial photoionization of neutrally desorbed matrix molecules and subsequent charge-transfer reactions in a fine vacuum or atmospheric pressure ion source. MALDI-2 significantly increases the ion yields for numerous classes of analytes, including lipids, glycans, and a range of pharmaceuticals. To obtain insights into the ionization mechanisms underlying the primary step of PI in MALDI-2, we here conducted a set of experiments with two lasers at 266 nm wavelength and pulse durations of 28 ps and 6 ns, respectively, on a modified orthogonal-extracting time-of-flight mass spectrometer (QTOF, Synapt). 2,5-Dihydroxybenzoic acid (DHB) and 2,5-dihydroxyacetophenone (DHAP) were investigated as MALDI matrices in the positive-ion mode with standardized lipid samples. Analyte- and matrix-derived ion signals were recorded as a function of PI laser pulse energies. The ion signal intensity displays a quadratic dependency on PI-laser pulse energy for low to moderate intensities of up to ∼107 W/cm2. This behavior suggests the involvement of resonance enhanced two-photon ionization (REMPI) of neutral matrix molecules in the ionization pathways. Comparing nanosecond and picosecond pulses at the same PI laser pulse energy, higher photon density produced by the shorter pulses generally produced sizably higher ion signal intensities, also corroborating an involvement of REMPI-like processes. Based on a theoretical description of the MALDI-2 process derived from prevalent REMPI theory, comparative measurements allow us to determine the lifetime of the excited states of the employed matrices. Resulting values for both matrices are in good agreement with the literature and corroborate the REMPI-based approach.

Gold-TiO2@gallic acid nanospheres for enhanced surface-assisted laser desorption/ionization mass spectrometry imaging

Surface-assisted laser desorption/ionization (SALDI) mass spectrometry imaging (MSI) can effectively reduce the matrix interference and ion suppression that occurred in traditional matrix-assisted laser desorption/ionization (MALDI) MSI. In this work, gold nanoparticles (AuNPs) modified TiO2 nanospheres were synthesized and modified with gallic acid (GA) to fabricate Au-TiO2@GA nanospheres (Au-TiO2@GA NSs). The SALDI substrate was prepared by coating the ITO glass slide with Au-TiO2@GA NSs using a gas-assisted electric sprayer. Compared with often-used MALDI matrices such as 2,5-dihydroxybenzoic acid (DHB), 2-mercaptobenzothiazole (MBT), 1,5-diaminonaphthalene (DAN), 2,5-dihydroxyacetophenone (DHA), and 9-aminoacridine (9-AA), the Au-TiO2@GA NSs provide higher detection sensitivity, lower background interference, dual-polarity detection and enhanced ionization efficiency of various endogenous molecules, particularly to the detection of neutral lipids. SALDI MSI could be straightforwardly performed on biological tissues thaw-mounted on the prepared substrate. The spatial distribution of various molecular species was unraveled in the mouse brain, kidney, and liver tissues. Not limited to animal tissues, Au-TiO2@GA NSs showed a more universal application to SALDI MSI of secondary metabolites in plant tissues. The spatial distribution of various natural products was mapped using SALDI MSI, such as parishins in Gastrodia elata tuber (Tianma) and glycoalkaloids in potato tuber. This work provides a high-efficient and high-compatible nanomaterial for SALDI MSI

ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES BY MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY: AN UPDATE FOR 2015-2016.

This review is the ninth update of the original article published in 1999 on the application of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2016. Also included are papers that describe methods appropriate to analysis by MALDI, such as sample preparation techniques, even though the ionization method is not MALDI. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation and arrays. The second part of the review is devoted to applications to various structural types such as oligo- and poly-saccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals. Much of this material is presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions and applications to chemical synthesis. The reported work shows increasing use of combined new techniques such as ion mobility and the enormous impact that MALDI imaging is having. MALDI, although invented over 30 years ago is still an ideal technique for carbohydrate analysis and advancements in the technique and range of applications show no sign of deminishing. © 2020 Wiley Periodicals, Inc.

Evaluation of Sibling and Twin Fragment Ions Improves the Structural Characterization of Proteins by Top-Down MALDI In-Source Decay Mass Spectrometry.

Comprehensive determination of primary sequence and identification of post-translational modifications (PTMs) are key elements in protein structural analysis. Various mass spectrometry (MS) based fragmentation techniques are powerful approaches for mapping both the amino acid sequence and PTMs; one of these techniques is matrix-assisted laser desorption/ionization (MALDI), combined with in-source decay (ISD) fragmentation and Fourier-transform ion cyclotron resonance (FT-ICR) MS. MALDI-ISD MS protein analysis involves only minimal sample preparation and does not require spectral deconvolution. The resulting MALDI-ISD MS data is complementary to electrospray ionization-based MS/MS sequencing readouts, providing knowledge on the types of fragment ions is available. In this study, we evaluate the isotopic distributions of z′ ions in protein top-down MALDI-ISD FT-ICR mass spectra and show why these distributions can deviate from theoretical profiles as a result of co-occurring and isomeric z and y-NH3 ions. Two synthetic peptides, containing either normal or deuterated alanine residues, were used to confirm the presence and unravel the identity of isomeric z and y-NH3 fragment ions (“twins”). Furthermore, two reducing MALDI matrices, namely 1,5-diaminonaphthalene and N-phenyl-p-phenylenediamine were applied that yield ISD mass spectra with different fragment ion distributions. This study demonstrates that the relative abundance of isomeric z and y-NH3 ions requires consideration for accurate and confident assignments of z′ ions in MALDI-ISD FT-ICR mass spectra.

The cationization of synthetic polymers in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: Investigations of the salt-to-analyte ratio.

Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique that when used to analyze synthetic polymer analytes often requires the addition of a metal cationization agent (herein termed the "salt"). The choice of both the matrix and the cationization agent needs to be taken into account when considering the polymer under study; different polymers have shown different affinities toward different cationization agents, and their selectivity can change as the matrix changes. Salt-to-analyte ratio (S/A) plots are used in this work to investigate the effect of the quantity of cationization agent employed in the analysis of a poly (methylmethacrylate) (PMMA) analyte with different MALDI matrices. The point at which analyte signal stops increasing with the added cationization agent is termed the "cation saturation point," and it was found to occur around a S/A of 1. When the analyte signal after this point remains constant, it is termed an "ideal case." The "non-ideal case" occurs when the analyte signal decreases after the cation saturation point. The amount of matrix present (measured as the matrix-to-analyte molar ratio, M/A) and the use of different counterions for the salt are also found to affect the intensity of the analyte signal. In non-ideal cases, changes in the counterion or an increase in the M/A are found to increase the analyte signal, often converting an initially observed non-ideal case into an ideal case. Several experiments attempting to uncover the reason for observation of the non-ideal S/A behavior are also described.

Chemical diversity of lignin degradation products revealed by matrix-optimized MALDI mass spectrometry.

Lignin is the most abundant natural resource of aromatic moieties and the second most abundant natural biopolymer. Analytical techniques that obtain as much information as possible on the exact structural content of lignin species are essential for developing efficient processes that transform highly complex lignin wastes into value chemicals and biofuels. For mass spectrometric analysis of lignin samples, usually electrospray ionization, atmospheric pressure chemical ionization, or atmospheric pressure photoionization are used as ionization techniques. Matrix-assisted laser desorption/ionization (MALDI) is less frequently applied but offers a much more rapid screening option for lignin mixtures. In this study, we compared several common MALDI matrices for analysis of alkali lignin and discovered that different chemical matrices exhibited very different ionization efficiencies and selectivity with respect to the structures of the lignin-related compounds as well as the presence of heteroatoms. Importantly, the results highlight that the choice of matrix strongly determines the analytical coverage of molecular species in the complex lignin degradation mixtures. Graphical abstract.

Toward understanding the ionization mechanism of matrix-assisted ionization using mass spectrometry experiment and theory.

Matrix-assisted ionization (MAI) mass spectrometry does not require voltages, a laser beam, or added heat to initiate ionization, but it is strongly dependent on the choice of matrix and the vacuum conditions. High charge state distributions of nonvolatile analyte ions produced by MAI suggest that the ionization mechanism may be similar to that of electrospray ionization (ESI), but different from matrix-assisted laser desorption/ionization (MALDI). While significant information is available for MAI using mass spectrometers operating at atmospheric and intermediate pressure, little is known about the mechanism at high vacuum. Eleven MAI matrices were studied on a high-vacuum time-of-flight (TOF) mass spectrometer using a 266 nm pulsed laser beam under otherwise typical MALDI conditions. Detailed comparisons with the commonly used MALDI matrices and theoretical prediction were made for 3-nitrobenzonitrile (3-NBN), which is the only MAI matrix that works well in high vacuum when irradiated with a laser. Screening of MAI matrices with good absorption at 266 nm but with various degrees of volatility and laser energies suggests that volatility and absorption at the laser wavelength may be necessary, but not sufficient, criteria to explain the formation of multiply charged analyte ions. 3-NBN produces intact, highly charged ions of nonvolatile analytes in high-vacuum TOF with the use of a laser, demonstrating that ESI-like ions can be produced in high vacuum. Theoretical calculations and mass spectra suggest that thermally induced proton transfer, which is the major ionization mechanism in MALDI, is not important with the 3-NBN matrix at 266 nm laser wavelength. 3-NBN:analyte crystal morphology is, however, important in ion generation in high vacuum. The 3-NBN MAI matrix produces intact, highly charged ions of nonvolatile compounds in high-vacuum TOF mass spectrometers with the aid of ablation and/or heating by laser irradiation, and shows a different ionization mechanism from that of typical MALDI matrices.

Recent Developments of Useful MALDI Matrices for the Mass Spectrometric Characterization of Lipids.

Matrix-assisted laser desorption/ionization (MALDI) is one of the most successful “soft” ionization methods in the field of mass spectrometry and enables the analysis of a broad range of molecules, including lipids. Although the details of the ionization process are still unknown, the importance of the matrix is commonly accepted. Both, the development of and the search for useful matrices was, and still is, an empirical process, since properties like vacuum stability, high absorption at the laser wavelength, etc. have to be fulfilled by a compound to become a useful matrix. This review provides a survey of successfully used MALDI matrices for the lipid analyses of complex biological samples. The advantages and drawbacks of the established organic matrix molecules (cinnamic or benzoic acid derivatives), liquid crystalline matrices, and mixtures of common matrices will be discussed. Furthermore, we will deal with nanocrystalline matrices, which are most suitable to analyze small molecules, such as free fatty acids. It will be shown that the analysis of mixtures and the quantitative analysis of small molecules can be easily performed if the matrix is carefully selected. Finally, some basic principles of how useful matrix compounds can be “designed” de novo will be introduced.

Matrix-free mass spectrometry imaging of mouse brain tissue sections on silicon nanopost arrays.

Mass spectrometry imaging (MSI) is capable of detection and identification of diverse classes of compounds in brain tissue sections, whereas simultaneously mapping their spatial distributions. Given the vast array of chemical components present in neurological systems, as well as the innate diversity within molecular classes, MSI platforms capable of detecting a wide array of species are useful for achieving a more comprehensive understanding of their biological roles and significance. Currently, matrix-assisted laser desorption ionization (MALDI) is the method of choice for the molecular imaging of brain samples by mass spectrometry. However, nanostructured laser desorption ionization platforms, such as silicon nanopost arrays (NAPA), are emerging as alternative MSI techniques that can provide complementary insight into molecular distributions in the central nervous system. In this work, the molecular coverage of mouse brain lipids afforded by NAPA-MSI is compared to that of MALDI-MSI using two common MALDI matrices. In positive ion mode, MALDI spectra were dominated by phosphatidylcholines and phosphatidic acids. NAPA favored the ionization of phosphatidylethanolamines and glycosylated ceramides, which were poorly detected in MALDI-MSI. In negative ion mode, MALDI favored sulfatides and free fatty acids, whereas NAPA spectra were dominated by signal from phosphatidylethanolamines. The complementarity in lipid coverages between the NAPA- and MALDI-MSI platforms presents the possibility of selective lipid analysis and imaging dependent upon which platform is used. Nanofabrication of the NAPA platform offers better uniformity compared to MALDI, and the wider dynamic range offered by NAPA promises improved quantitation in imaging.

Ionic liquid matrices for MALDI mass spectrometry of lignin.

The use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for the study of lignin is still extremely limited due to its low ionization efficiency. We have developed an approach for obtaining high-intensity MALDI mass spectra of lignin, based on the use of ionic liquids as matrices. Thirty-two ionic liquids consisting of large nitrogen-containing cations and anions of aromatic acids, traditionally used as crystalline matrices, were tested. It was established that ionic liquids based on N,N-diisopropyl-N-ethylammonium, N-isopropyl-N-methyl-N-tert-butylammonium, 3-aminoquinolinium, pyridinium, and 1-methylimidazolium cations and anions of ferulic, α-cyanohydroxycinnamic, and 2,5-dihydroxybenzoic acids as MALDI matrices provided high efficiency of lignin desorption/ionization with generation of singly charged protonated molecules of its oligomers. The use of such matrices in combination with the MALDI quadrupole ion trap-time-of-flight technique allows high-intensity mass spectra of lignin to be obtained without interferences from the matrix in the molecular weight range up to 3kDa, adequately reflecting the molecular mass characteristics of lignin preparations. Using ionic liquid matrices, MS2 and MS3 MALDI mass spectra of lignins for various precursor ions were first obtained, including in the region of large (> 2kDa) molecular weights. Differences in tandem mass spectra of coniferous and deciduous lignins, reflecting the structural features of corresponding oligomers were demonstrated. Graphical abstract ᅟ.

Carbon Nanoparticles and Graphene Nanosheets as MALDI Matrices in Glycomics: a New Approach to Improve Glycan Profiling in Biological Samples.

Glycomics continues to be a highly dynamic and interesting research area due to the need to comprehensively understand the biological attributes of glycosylation in many important biological functions such as the immune response, cell development, cell differentiation/adhesion, and host-pathogen interactions. Although matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) has proven to be suitable for glycomic profiling studies, there is a need for improved sensitivity in the detection of native glycans, which ionize inefficiently. In this study, we investigated the efficiencies of graphene nanosheets (GNs) and carbon nanoparticles (CNPs) as MALDI matrices and co-matrices in glycan profiling. Our results indicated an enhancement of signal intensity by several orders of magnitude upon using GNs and CNPs in MALDI analysis of N-glycans derived from a variety of biological samples. Interestingly, increasing the amounts of CNPs and GNs improved not only the signal intensities but also prompted in-source decay (ISD) fragmentations, which produced extensive glycosidic and cross-ring cleavages. Our results indicated that the extent of ISD fragmentation could be modulated by CNP and GN concentrations, to obtain MS2 and pseudo-MS3 spectra. The results for glycan profiling in high salt solutions confirmed high salt-tolerance capacities for both CNPs and GNs. Finally, the results showed that by using CNPs and GNs as co-matrices, DHB crystal formation was more homogeneous which improved shot-to-shot reproducibility and sensitivity. Graphical Abstract ᅟ.

Structure-performance relationships of phenyl cinnamic acid derivatives as MALDI-MS matrices for sulfatide detection.

A key aspect for the further development of matrix-assisted laser desorption ionization (MALDI)-mass spectrometry (MS) is a better understanding of the working principles of MALDI matrices. To address this issue, a chemical compound library of 59 structurally related cinnamic acid derivatives was synthesized. Potential MALDI matrices were evaluated with sulfatides, a class of anionic lipids which are abundant in complex brain lipid mixtures. For each matrix relative mean S/N ratios of sulfatides were determined against 9-aminoacridine as a reference matrix using negative ion mass spectrometry with 355 and 337nm laser systems. The comparison of matrix features with their corresponding relative mean S/N ratios for sulfatide detection identified correlations between matrix substitution patterns, their chemical functionality, and their MALDI-MS performance. Crystal structures of six selected matrices provided structural insight in hydrogen bond interactions in the solid state. Principal component analysis allowed the additional identification of correlation trends between structural and physical matrix properties like number of exchangeable protons at the head group, MW, logP, UV-Vis, and sulfatide detection sensitivity. Graphical abstract Design, synthesis and mass spectrometric evaluation of MALDI-MS matrix compound libraries allows the identification of matrix structure - MALDI-MS performance relationships using multivariate statistics as a tool.

Liquid MALDI MS Analysis of Complex Peptide and Proteome Samples.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is well-known to be a powerful technique for the analysis of biological samples. By using glycerol-based liquid support matrices (LSMs) instead of conventional MALDI matrices the power of this technique can be extended further. In this study, we exploited LSMs for the identification of complex samples, that is, the Lactobacillus proteome and a bovine serum albumin (BSA) digest. Liquid and solid MALDI samples were manually and robotically prepared by coupling a nanoflow high-performance liquid chromatography (nanoHPLC) system to an automated MALDI sample spotting device. MS and MS/MS data were successfully acquired at the femtomole level using TOF/TOF as well as Q-TOF instrumentation and used for protein identification searching sequence databases. For the BSA digest analysis, liquid MALDI samples resulted in peptide mass fingerprints, which led to a higher confidence in protein identification compared with solid (crystalline) MALDI samples; however, postsource decay (PSD) MS/MS analysis of both the proteome of Lactobacillus plantarum WCFS1 cells and BSA digest showed that further optimization of the formation and detection of peptide fragment ions is still needed for liquid MALDI samples, as the MS/MS ion search score was lower than that for the solid MALDI samples, reflecting the poorer quality of the liquid MALDI-PSD spectra, which can be attributed to the differences in PSD parameters and their optimization that is currently achievable.

Microscale Solubility Measurements of Matrix-Assisted Laser Desorption-Ionization (MALDI) Matrices Using Attenuated Total Reflection (ATR) Fourier Transform Infrared Spectroscopy (FT-IR) Coupled with Partial Least Squares (PLS) Analysis.

In this work an attenuated total reflection Fourier transform infrared (FT-IR) absorption based method is used to measure the solubility of two matrix-assisted laser desorption-ionization (MALDI) matrices in a few pure solvents and mixtures of acetonitrile and water using low microliter amounts of solution. Results from a method that averages the values obtained from multiple calibration curves created by manual peak picking are compared to those predicted using a partial least squares (PLS) chemometrics approach. The PLS method provided solubility values that were in good agreement with the manual method with significantly greater ease of analysis. As a test, the solubility of adipic acid in acetone was measured using the two methods of analysis, and the values are in good agreement with solubility values reported in literature. The solubilities of the MALDI matrices α-cyano-4-hydroxy cinnamic acid (CHCA) and sinapinic acid (SA) were measured in a series of mixtures made from acetonitrile (ACN) and water; surprisingly, the results show a highly nonlinear trend. While both CHCA and SA show solubility values of less than 10 mg/mL in the pure solvents, the solubility value for SA increases to 56.3 mg/mL in a 75:25 v/v ACN:water mixture. This can have a significant effect on the matrix-to-analyte ratios in the MALDI experiment when sample protocols call for preparation of a saturated solution of the matrix in the chosen solvent system.

Changes in the molecular ion yield and fragmentation of peptides under various primary ions in ToF-SIMS and matrix-enhanced ToF-SIMS.

Time of flight secondary ion mass spectrometry (ToF-SIMS) is a powerful technique for the nanoanalysis of biological samples, but improvements in sensitivity are needed in order to detect large biomolecules, such as peptides, on the individual cell level at physiological concentrations. Two promising options to improve the sensitivity of SIMS to large peptides are the use of cluster primary ions to increase desorption of intact molecules or the use of matrix-assisted laser desorption/ionization (MALDI) matrices to increase the ionization probability. In this paper, the authors have combined these two approaches in order to improve understanding of the interaction between ionization and fragmentation processes. The peptides bradykinin and melittin were prepared as neat monolayers on silicon, in a Dextran-40 matrix and in two common MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxy cinnamic acid (HCCA). ToF-SIMS spectra of these samples were collected using a range of small Bi cluster primary ions and large Ar cluster primary ions. The trends observed in the molecular ion yield and the [M+H](+)/C4H8N(+) ratio with primary ion cluster size were sample system dependent. The molecular ion yield of the bradykinin was maximized by using 30 keV Bi3 (+) primary ions in a DHB matrix but in the HCCA matrix, the maximum molecular ion yield was obtained by using 30 keV Bi7 (+) primary ions. In contrast, the molecular ion yield for melittin in both matrices was greatest using 20 keV Ar2000 (+) primary ions. Improvements in the molecular ion yield were only loosely correlated with a decrease in small fragment ions. The data indicate a complex interplay between desorption processes and ion formation processes which mean that the optimal analytical conditions depend on both the target analyte and the matrix.

Development and Application of a High-Resolution Imaging Mass Spectrometer for the Study of Plant Tissues.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) or imaging mass spectrometry (imaging MS) has been a powerful tool to map the spatial distribution of molecules on the surface of biological materials. This technique has frequently been applied to animal tissue slices for the purpose of mapping proteins, peptides, lipids, sugars or small metabolites to find disease-specific biomarkers or to study drug metabolism. Recently, it has also been applied to intact plant tissues or thin slices thereof using commercial mass spectrometers. The present work is concerned with the refinement of MALDI/laser desorption/ionization (LDI)-Fourier transform ion cyclotron resonance (FTICR)-MS incorporating certain specific features namely, ultra-high mass resolution (>100,000), ultra-high molecular mass accuracy (<1 p.p.m.) and high spatial resolution (<10 µm) for imaging MS of plant tissues. Employing an in-house built mass spectrometer, the imaging MS analysis of intact Arabidopsis thaliana tissues, namely etiolated seedlings and roots of seedlings, glued to a small transparent ITO (indium tin oxide)-coated conductive glass was performed. A matrix substance was applied to the vacuum-dried intact tissues by sublimation prior to the imaging MS analysis. The images of various small metabolites representing their two-dimensional distribution on the dried intact tissues were obtained with or without different matrix substances. The effects of MALDI matrices on the ionization of small metabolites during imaging MS acquisition are discussed.