Matrix-assisted Laser Desorption And Ionization Research Articles

A Five-Year Update on Matrix Compounds for MALDI-MS Analysis of Lipids.

Matrix-assisted laser desorption and ionization (MALDI) is a widely used soft-ionization technique of modern mass spectrometry (MS). MALDI enables the analysis of nearly all chemical compounds-including polar and apolar (phospho)lipids-with a minimum extent of fragmentation. MALDI has some particular advantages (such as the possibility to acquire spatially-resolved spectra) and is competitive with the simultaneously developed ESI (electrospray ionization) MS. Although there are still some methodological aspects that need to be elucidated in more detail, it is obvious that the careful selection of an appropriate matrix plays the most important role in (lipid) analysis. Some lipid classes can be detected exclusively if the optimum matrix is used, and the matrix determines the sensitivity by which a particular lipid is detected within a mixture. Since the matrix is, thus, crucial for optimum results, we provide here an update on the progress in the field since our original review in this journal in 2018. Thus, only the development during the last five years is considered, and lipids are sorted according to increasing complexity, starting with free fatty acids and ending with cardiolipins and phosphoinositides.

In situ spatial glycomic imaging of mouse and human Alzheimer's disease brains.

N-linked protein glycosylation in the brain is an understudied facet of glucose utilization that impacts a myriad of cellular processes including resting membrane potential, axon firing, and synaptic vesicle trafficking. Currently, a spatial map of N-linked glycans within the normal and Alzheimer's disease (AD) human brain does not exist. A comprehensive analysis of the spatial N-linked glycome would improve our understanding of brain energy metabolism, linking metabolism to signaling events perturbed during AD progression, and could illuminate new therapeutic strategies. Herein we report an optimized in situ workflow for enzyme-assisted, matrix-assisted laser desorption and ionization (MALDI) mass spectrometry imaging (MSI) of brain N-linked glycans. Using this workflow, we spatially interrogated N-linked glycan heterogeneity in both mouse and human AD brains and their respective age-matched controls. We identified robust regional-specific N-linked glycan changes associated with AD in mice and humans. These data suggest that N-linked glycan dysregulation could be an underpinning of AD pathologies.

Quantitative Approach for Protein Analysis in Small Cell Ensembles by an Integrated Microfluidic Chip with MALDI Mass Spectrometry.

Increasing evidence has demonstrated that cells are individually heterogeneous. Advancing the technologies for single-cell analysis will improve our ability to characterize cells, study cell biology, design and screen drugs, and aid cancer diagnosis and treatment. Most current single-cell protein analysis approaches are based on fluorescent antibody-binding technology. However, this technology is limited by high background and cross-talk of multiple tags introduced by fluorescent labels. Stable isotope labels used in mass cytometry can overcome the spectral overlap of fluorophores. Nevertheless, the specificity of each antibody and heavy-metal-tagged antibody combination must be carefully validated to ensure detection of the intended target. Thus, novel single-cell protein analysis methods without using labels are urgently needed. Moreover, the labeling approach targets already known motifs, hampering the discovery of new biomarkers relevant to single-cell population variation. Here, we report a combined microfluidic and matrix-assisted laser desorption and ionization (MALDI) mass spectrometric approach for the analysis of protein biomarkers suitable for small cell ensembles. All necessary steps for cell analysis including cell lysis, protein capture, and digestion as well as MALDI matrix deposition are integrated on a microfluidic chip prior to the downstream MALDI-time-of-flight (TOF) detection. For proof of principle, this combined method is used to assess the amount of Bcl-2, an apoptosis regulator, in metastatic breast cancer cells (MCF-7) by using an isotope-labeled peptide as an internal standard. The proposed approach will eventually provide a new means for proteome studies in small cell ensembles with the potential for single-cell analysis and improve our ability in disease diagnosis, drug discovery, and personalized therapy.

MALDI MS Analysis to Investigate the Lipid Composition of Sperm

Background: The sperm plasma membrane meets the requirements of sperm transit through the female genital tract and subsequent fertilization. Commonly, the (phospho)lipid composition of sperm is characterized by tremendous amounts of highly unsaturated fatty acyl residues such as docosahexaenoic and docosapentaenoic acid. While human sperm contain almost exclusively diacyl lipids, many animal sperm additionally contain significant amounts of ether lipids such as alkylacyl- and alkenyl-acyl lipids (plasmalogens). Hypothesis/Objective: It is suggested that deviations from the typical lipid composition are indicative of pathological changes. Therefore, simple methods to elucidate the sperm lipid composition are essential. Method: Matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS) is a fast and simple method. Since the selection of the most suitable matrix is a crucial step in MALDI MS, this topic will be highlighted. It will also be shown that MALDI MS can be easily combined with thin-layer chromatography to overcome ion suppression effects. Results: The lipid composition of sperm from different species can be elucidated by MALDI MS. However, different matrix compounds have to be used to record positive and negative ion mass spectra. Since some sperm (glyco)lipids are characterized by the presence of sulfate residues which suppress the detection of less acidic lipids in the negative ion mode, previous separation is often necessary. It will be also emphasized that plasmalogens can be easily identified by either enzymatic digestion or treatment with acids. Conclusion: MALDI MS is a reliable method to obtain sperm lipid fingerprints in a simple and convenient way.

Characterization of Extracellular Biosurfactants Expressed by a Pseudomonas putida Strain Isolated from the Interior of Healthy Roots from Sida hermaphrodita Grown in a Heavy Metal Contaminated Soil.

Pseudomonas putida E41 isolated from root interior of Sida hermaphrodita (grown on a field contaminated with heavy metals) showed high biosurfactant activity. In this paper, we describe data from mass spectrometry and genome analysis, to improve our understanding on the phenotypic properties of the strain. Supernatant derived from P. putida E41 liquid culture exhibited a strong decrease in the surface tension accompanied by the ability for emulsion stabilization. We identified extracellular lipopeptides, putisolvin I and II expression but did not detect rhamnolipids. Their presence was confirmed by matrix-assisted laser desorption and ionization (MALDI) TOF/TOF technique. Moreover, ten phospholipids (mainly phosphatidylethanolamines PE 33:1 and PE 32:1) which were excreted by vesicles were also detected. In contrast the bacterial cell pellet was dominated by phosphatidylglycerols (PGs), which were almost absent in the supernatant. It seems that the composition of extracellular (secreted to the environment) and cellular lipids in this strain differs. Long-read sequencing and complete genome reconstruction allowed the identification of a complete putisolvin biosynthesis pathway. In the genome of P. putida E41 were also found all genes involved in glycerophospholipid biosynthesis, and they are likely responsible for the production of detected phospholipids. Overall this is the first report describing the expression of extracellular lipopeptides (identified as putisolvins) and phospholipids by a P. putida strain, which might be explained by the need to adapt to the highly contaminated environment.

Investigating the effects of gamma radiation on selected chemicals for use in biosignature detection instruments on the surface of Jupiter's moon Europa

Jupiter's moon Europa is a prime target for the search for potential signs of life in the solar system. The Europa Lander Science Definition Team Report outlined investigations and measurement requirements on a future Europa Lander and has led us to consider application of powerful techniques such as pyrolysis and derivatization gas chromatography mass spectrometry (GC-MS) and laser desorption mass spectrometry (LD-MS) to elucidate the organic composition of near-surface ice and minerals. Definitive identification of chemical biosignatures using such techniques is strongly enabled by the use of various chemicals, such as perfluorotributylamine (PFTBA) for the MS calibration, α-cyano-hydroxycinnamic acid (CHCA) for matrix-assisted laser desorption and ionization (MALDI) and N,N-dimethylformamide dimethyl acetal (DMF-DMA), N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) and tetramethylammonium hydroxide (TMAH) for wet chemistry GC-MS protocols. The jovian radiation environment is known to represent a uniquely challenging risk to mission performance and lifetime, principally due to high radiation levels. To assess the potential ionizing radiation damage to these important chemicals, we tested their effectiveness following gamma radiation exposure doses up to the anticipated Europa Lander rating requirement of 300 krad(Si). The chemicals were sealed in glass ampules under vacuum (<10 mTorr), to reduce trapped oxygen gas, as the oxidation by O2 may be enhanced in the presence of radiation. We report that all five chemicals exposed to total ionizing doses of 0, 150 and 300 krad(Si) maintained their full effectiveness, and no significant degradation was observed.

Generation and Propagation of MALDI Ion Packets Probed by Sheet-Like Nanosecond UV Laser Light.

A sheet-like ultraviolet (UV) probe laser is used to investigate the ejection and propagation of ion packets of matrix CHCA, which are produced by matrix-assisted laser desorption and ionization (MALDI). Laser irradiation of the expanding MALDI plume induced photodissociation of the CHCA-related ions, which existed in a sheet-like volume, leading to their absence in their MALDI signal profiles. The MALDI spectra were measured under varying conditions: the temporal delay of the lasers and the distance of the sheet-like probe laser from the MALDI sample surface. It was found that the center of the (CHCA)H+ packets were ejected at 46±11 ns after MALDI laser irradiation, while the (CHCA)2H+ packets were ejected at 64±12 ns, regardless of the magnitude of acceleration static high-voltage in 3.5–5.5 kV. This suggests that (CHCA)2H+ is formed by a proton transfer reaction from (CHCA)H+ to (CHCA)2 in the heated condensed phase and/or near the surface. This study represents the first experimental determination of ion ejection time in the MALDI process, which is also applicable to other species in the MALDI plume.

The effect of antigen retrieval buffers on MALDI mass spectrometry imaging of peptide profiles in skin FFPE tissue

Abstract Objectives: The success of novel in-situ immunohistoproteomics approaches that employ Matrix-assisted laser desorption and ionization (MALDI) mass spectrometry imaging (MSI) heavily depends on good antigen retrieval efficacy. Methods: We have optimized the effect of various antigen retrieval buffers on peptide profiles acquired from FFPE traction alopecia scalp tissue. Heat-induced epitope retrieval (HIER) was performed using Citrate, Tris, and Tris-EDTA buffers under various conditions. MALDI-MSI analysis of the tissue was performed using a high-throughput, state-of-the-art RapiFlex MALDI TOF/TOF tissue-typer equipped with a smart beam laser. Results: Background matrix peaks were found under most conditions except under Tris-EDTA buffer. Trypsin digestion periods longer than overnight and possibly higher concentrations are needed for Citrate and Tris. The Tris-EDTA buffer performed best for MALDI-MSI antigen retrieval at an incubation period of overnight at 60°C. Conclusions: We have identified in this study that antigen retrieval conditions need to be optimised prior to MALDI MSI analysis of different tissue types.

Detection of Neutral Species in the MALDI Plume Using Femtosecond Laser Ionization: Quantitative Analysis of MALDI-MS Signals Based on a Semiequilibrium Proton Transfer Model

We investigated neutral species in the matrix-assisted laser desorption and ionization (MALDI) plume using femtosecond laser ionization spectrometry with simultaneous measurement of the standard MALDI spectrum of the identical MALDI event induced by pulsed UV laser irradiation. The ratio of neutral species in the plume [A]p/[M]p (A = phenylalanine (Phe) or alanine (Ala), M = 2,5-dihydroxybenzoic acid (DHB)) was confirmed to be the same as that of the sample mixture in the range of [A]0/[M]0 = 4 × 10-4-1, indicating the validity of the widely adopted approximation [A]p/[M]p = [A]0/[M]0 in the reaction quotient of the proton transfer reaction MH+ + A ⇄ M + AH+. An effective parameter representing the extent of thermal equilibrium in the thermal proton transfer model is introduced for the first time. Numerical simulation based on this semiequilibrium model successfully reproduced variations of MALDI signal intensities AH+ and MH+ with two parameters: the fraction of ionized matrix a ≤ 10-5 and an effective temperature T = 1200 and 1100 K for Phe/DHB and Ala/DHB systems, respectively. These values show good agreement with those determined previously by different experimental approaches. The extent of thermal equilibrium was determined to be 95% and 98% for Phe/DHB and Ala/DHB systems, respectively, suggesting that the proton transfer reactions almost proceed to their thermal equilibrium.

A simple method to generate oxidized phosphatidylcholines in amounts close to one milligram

Oxidized (phospho)lipids are of paramount interest from different reasons: beside their significant in vivo relevance, these products are often needed in the laboratory to study the response of selected cells to oxidized lipids. Unfortunately, the commercial availability of oxidized lipids is limited and scientists interested in studying the physiological impact of oxidized lipids are normally forced to prepare the required compounds by themselves.We will show here that chain-shortened products of oxidized phosphatidylcholines (PCs) such as aldehydes and carboxylic acids can be easily (and in nearly quantitative yields) generated by the Fenton reaction (H2O2+Fe2+) or the KMnO4-induced oxidation of the PC. Using the Fenton reaction and physiological saline, chlorinated oxidation products such as chlorohydrins are also readily available. Additionally, it will be shown that preparative thin-layer chromatography (TLC) is a convenient but simple method to isolate the individual oxidation products in reasonable yields and high purities: all relevant products could be successfully identified by matrix-assisted laser desorption and ionization (MALDI) mass spectrometry and the amounts of the oxidized products determined by a simple colorimetric assay.

Novel two-step laser ablation and ionization mass spectrometry (2S-LAIMS) of actor-spectator ice layers: Probing chemical composition of D2O ice beneath a H2O ice layer

In this work, we report for the first time successful analysis of organic aromatic analytes imbedded in D2O ices by novel infrared (IR) laser ablation of a layered non-absorbing D2O ice (spectator) containing the analytes and an ablation-active IR-absorbing H2O ice layer (actor) without the analyte. With these studies we have opened up a new method for the in situ analysis of solids containing analytes when covered with an IR laser-absorbing layer that can be resonantly ablated. This soft ejection method takes advantage of the tenability of two-step infrared laser ablation and ultraviolet laser ionization mass spectrometry, previously demonstrated in this lab to study chemical reactions of polycyclic aromatic hydrocarbons (PAHs) in cryogenic ices. The IR laser pulse tuned to resonantly excite only the upper H2O ice layer (actor) generates a shockwave upon impact. This shockwave penetrates the lower analyte-containing D2O ice layer (spectator, a non-absorbing ice that cannot be ablated directly with the wavelength of the IR laser employed) and is reflected back, ejecting the contents of the D2O layer into the vacuum where they are intersected by a UV laser for ionization and detection by a time-of-flight mass spectrometer. Thus, energy is transmitted from the laser-absorbing actor layer into the non-absorbing spectator layer resulting its ablation. We found that isotope cross-contamination between layers was negligible. We also did not see any evidence for thermal or collisional chemistry of PAH molecules with H2O molecules in the shockwave. We call this "shockwave mediated surface resonance enhanced subsurface ablation" technique as "two-step laser ablation and ionization mass spectrometry of actor-spectator ice layers." This method has its roots in the well-established MALDI (matrix assisted laser desorption and ionization) method. Our method offers more flexibility to optimize both the processes--ablation and ionization. This new technique can thus be potentially employed to undertake in situ analysis of materials imbedded in diverse media, such as cryogenic ices, biological samples, tissues, minerals, etc., by covered with an IR-absorbing laser ablation medium and study the chemical composition and reaction pathways of the analyte in its natural surroundings.

Mass spectrometry and inflammation—MS methods to study oxidation and enzyme-induced changes of phospholipids

Many diseases such as arthritis or atherosclerosis are accompanied by inflammatory processes. Inflammation is characterized by the infiltration of cells such as neutrophilic granulocytes and (a) the release of phospholipases [particularly phospholipase A2 (PLA2)] and (b) the generation of reactive oxygen as well as nitrogen species (ROS and RNS). While PLA2 leads to defined lyso products (lacking one acyl residue), lipid oxidation is characterized by much more complex product patterns, including lipid peroxides, aldehydes (by double bond cleavage), and many others. Nevertheless, oxidation processes are highly important under in vivo conditions because molecules with regulatory functions are generated by the oxidation of lipids and/or free fatty acids. Therefore, lipid oxidation products as well as lysolipids are increasingly assumed to represent important disease (bio)markers. Consequently, there is also increasing interest in methods to characterize these products qualitatively and quantitatively.Mass spectrometry (MS) seems to be the method of choice to study (phospho)lipids changed under inflammatory conditions: nowadays, soft ionization MS methods are regularly used to study oxidative lipid modifications because of their high sensitivities and the tremendous mass resolutions that are achievable by using modern mass spectrometers. However, experimental care is required to be able to detect all relevant products. Although electrospray ionization (ESI) MS is so far most popular, applications of matrix-assisted laser desorption and ionization (MALDI) MS are continuously increasing. This review aims to summarize the so far available data on MS analyses of oxidized lipids as well as lysolipids. In addition to model systems, special attention will be paid to the monitoring of oxidized lipids and lysolipids under in vivo conditions. It is the aim of this review to provide a critical survey of the advantages and drawbacks of the different MS methods, with the focus on MALDI and ESI.

MALDI-TOF MS: Much More than only Protein Analysis!

Carbohydrates and particularly lipids represent molecules with significant structural variabilities: this is stemming from (a) the headgroups, (b) the different fatty acyl residues and (c) the linkage types (acyl-acyl-, alkyl-acyl-, and alkenyl-acyl-). Therefore, analytical methods to clarify these aspects are of immense significance. As the amount of the available biological material is normally limited, suitable methods should also exhibit reasonable sensitivities. It is our aim to introduce MALDI (matrix-assisted laser desorption and ionization) MS (mass spectrometry) [1] (often but not necessarily with a time-offlight (TOF) mass analyzer) as a simple and sensitive analytical method that helps to overcome many problems related to carbohydrate and particularly lipid analysis [2]. Although the history of MS dates back to the end of the nineteenth century, applications of MS to the structural analysis of biomolecules are rather new: the MS analysis of such complex molecules (often in combination with high polarity and low volatility) became only possible with the invention of “soft ionization” methods such as electrospray ionization (ESI) and “MALDI” [3]. Both techniques are nowadays widely used, whereby the focus is often on the investigation of proteins - normally subsequent to enzymatic digestion of the (unknown) protein into characteristic peptides, the MS of which enables the elucidation of the protein sequence and further structural properties [4]. It is a characteristic property of “soft ionization” MS that there is only a small extent of analyte fragmentation and, thus, the intact ions of the analyte of interest can be observed. This is a pronounced difference in comparison to the classical “electron ionization” (EI) technique. Although MALDI MS has many advantages it should be noted that the analysis of small molecules (for instance, free fatty acids) is still a problem - despite many improvements that could be recently achieved [5]. This problem with small molecules can be easily understood when the principle of MALDI MS is considered: MALDI MS is based on laser ionization, whereby normally UV lasers (emitting often at λ=337 nm) are used. Since not all potential analytes exhibit a sufficient absorption at this wavelength, a “matrix” must be used [1]. This matrix is normally a small organic molecule (such as 2,5-dihydroxybenzoic acid (DHB)) [6] that absorbs the laser energy, is evaporated and simultaneously carries the analyte into the gas phase (high vacuum). In the gas phase collisions between the analyte and cations (such as H + or Na + ), that are either already present in the lipid extract of interest or artificially added, occur leading to ion generation. As the mass of the observed (positive) ions is slightly higher than the mass of the analyte, these are normally termed “adducts” or “quasimolecular” ions. Of course, analytes with acidic groups are also detectable as negative ions. Since the matrix is normally in considerable excess over the analyte (at least 100:1), it is not surprising that the smaller mass range is dominated by matrix ions [7]. This is the reason why the small mass range is normally not shown when MALDI spectra are discussed. Despite this drawback, however, there are a lot of advantages: 1. The necessary sample preparation is simple and fast - and comprises often only the mixing of the analyte with the matrix and the deposition of this mixture onto the sample carrier, the “target”.

New methods to study the composition and structure of the extracellular matrix in natural and bioengineered tissues

The extracellular matrix (ECM) comprises a gel of numerous biopolymers that occurs in a multitude of biological tissues. The ECM provides the basic support and mechanical strength of skeletal tissue and is responsible for shape retention. At the same time, the ECM is responsible for the viscoelastic properties and the elasticity of soft tissues. As expected, there are several important diseases that affect and degenerate the ECM with severe consequences for its properties. Bioengineering is a promising approach to support the regenerative capacity of the body. Unfortunately, the biomechanical properties of bioengineered ECM often only poorly meet the standards of their native counterparts. Many bioengineered tissues are characterized by an increased glycosaminoglycan (GAG) but decreased collagen content. This leads to an enhanced water content that strongly alters the viscoelastic and thus the biomechanical properties. Therefore, compositional analysis is important to estimate the tissue quality. We will show that nuclear magnetic resonance (NMR) spectroscopy and soft-ionization mass spectrometry (MS) represent useful techniques for ECM research both in natural and bioengineered tissues. Both methods are strongly complimentary: while MS techniques such as matrix-assisted laser desorption and ionization (MALDI) are excellent and very sensitive analytical tools to determine the collagen and the GAG contents of tissues, NMR spectroscopy provides insight into the molecular architecture of the ECM, its dynamics and other important parameters such as the water content of the tissue as well as the diffusion of molecules within the ECM.

TOFwave: reproducibility in biomarker discovery from time-of-flight mass spectrometry data

Many are the sources of variability that can affect reproducibility of disease biomarkers from time-of-flight (TOF) Mass Spectrometry (MS) data. Here we present TOFwave, a complete software pipeline for TOF-MS biomarker identification, that limits the impact of parameter tuning along the whole chain of preprocessing and model selection modules. Peak profiles are obtained by a preprocessing based on Continuous Wavelet Transform (CWT), coupled with a machine learning protocol aimed at avoiding selection bias effects. Only two parameters (minimum peak width and a signal to noise cutoff) have to be explicitly set. The TOFwave pipeline is built on top of the mlpy Python package. Examples on Matrix-Assisted Laser Desorption and Ionization (MALDI) TOF datasets are presented. Software prototype, datasets and details to replicate results in this paper can be found at http://mlpy.sf.net/tofwave/.

High-Reactivity Matrices Increase the Sensitivity of Matrix Enhanced Secondary Ion Mass Spectrometry

Secondary ion mass spectrometry (SIMS) is a desorption/ionization method in which ions are generated by the impact of a primary ion beam on a sample. Classic matrix assisted laser desorption and ionization (MALDI) matrices can be used to increase secondary ion yields and decrease fragmentation in a SIMS experiment, which is referred to as matrix enhanced SIMS (ME-SIMS). Contrary to MALDI, the choice of matrices for ME-SIMS is not constrained by their photon absorption characteristics. This implies that matrix compounds that exhibit an insufficient photon absorption coefficient have the potential of working well with ME-SIMS. Here, we evaluate a set of novel derivatives of the classical MALDI matrices α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) for usability in ME-SIMS. This evaluation was carried out using peptide mixtures of different complexity and demonstrates significant improvements in signal intensity for several compounds with insufficient UV absorption at the standard MALDI laser wavelengths. Our study confirms that the gas-phase proton affinity of a matrix compound is a key physicochemical characteristic that determines its performance in a ME-SIMS experiment. As a result, these novel matrices improve the performance of matrix enhanced secondary ion mass spectrometry experiments on complex peptide mixtures.

Effect of Nanoparticles in Protein Separation by Capillary Electrophoresis

Consequently, there is a critical need for analytical techniques that can provide func-tion and identification of numerous and diversified proteins. Determination of molecular weight is one of the most im-portant aspects of protein characterization. Mass spectrometry (MS) with either matrix-assisted laser desorption and ionization (MALDI)

Purification, Activity and Sequence of Angiotensin I Converting Enzyme Inhibitory Peptide from Alcalase Hydrolysate of Peanut Flour

Peanut hydrolysate obtained after 6 h of digestion by Alcalase was used to isolate angiotensin I converting enzyme (ACE) inhibitory peptides. After centrifugation and ultrafiltration through a 0.2 microm nylon filter, the hydrolysate was filtered through the polyethersulfone membrane with a molecular weight cutoff (MWCO) of 10 kDa. The resulting permeate was then separated by primary reverse-phase high performance liquid chromatography (RP-HPLC). Eluate was divided into six major fractions according to eluation time. The fraction with eluting time 50-60 min showed the most potent ACE inhibition and was subjected to further purification by the secondary RP-HPLC. Four peaks were found to have strong ACE inhibitory activities, and their IC(50) values were determined. Peptide mass for the most potent peak was obtained by matrix-assisted laser desorption and ionization (MALDI), and sequence was determined by MALDI tandem TOF-TOF (time-of-flight) mass spectrometer (MS/MS) to be Lys-Ala-Phe-Arg.

Recent Developments of Useful MALDI Matrices for the Mass Spectrometric Characterization of Apolar Compounds

Matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS) - often but not exclusively combined with time-of-flight (TOF) mass analyzers - is one of the most successful “soft” ionization methods that is applicable to a very broad range of analytes. Although the ionization process is still widely unknown and a matter of intense research, it is commonly accepted that the matrix plays a crucial role. The development of and the search for useful matrix compounds, however, was - and still is - an empirical process because only some obvious properties (vacuum stability, high absorption at the laser wavelength, etc.) that must be fulfilled by a compound to become a useful MALDI matrix are known so far. Although MALDI MS is primarily used for the analysis of polar biopolymers, such as proteins and nucleic acids, the focus of this review is on the analysis of apolar molecules with special emphasis on lipids and phospholipids. Beside established organic matrix molecules, such as cinnamic or benzoic acid derivatives, the advantages and drawbacks of liquid crystalline matrices and mixtures of common matrices will be also shortly discussed. It will be shown that only the careful selection of the matrix enables the detection of the compounds of interest. It will also be demonstrated that mixture analysis and the quantitative analysis of small molecules can be accurately performed only if the matrix is carefully selected. Finally, some basic principles how useful matrix compounds can be de novo “designed” will be introduced.

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.