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Charge Distribution between Different Classes of Glycerophospolipids in MALDI-MS Imaging
Abstract
Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is widely used to visualize and analyze the distribution of membrane lipids in an increasingly large number of applications. In this context, different lipoforms of glycerophospholipids (GPL) are among the prime targets of interest. For this group of analytes, however, ion suppression effects have been described to strongly favor the detection of certain GPL classes over others, thereby hampering the analysis of suppressed species and greatly restraining quantitative analysis. These effects are generally attributed to the distribution of available charge carriers during the MALDI process. Here we present a systematic investigation of charge distribution between different classes of GPL under MALDI-MSI conditions. For this, we constructed arrays of artificial tissues with different formulated lipid composition that contained predefined amounts of only two specific GPL-classes and analyzed them with MALDI-MSI in positive and negative ion modes. Next to a characterization of expected ion suppression effects, analysis of these binary systems revealed yet undescribed signal intensity enhancement for the combinations of certain GPL classes. Furthermore, the comprehensive data allowed us to compile a hierarchy of charge affinities for the investigated GPL classes in both polarities. Additional experiments revealed that laser postionization (MALDI-2) has great potential to overwrite changes in signal intensity caused by charge distribution among different GPL classes observed in standard MALDI-MSI.
... 5 In addition to macromolecules, some phospholipid classes have been shown to cause the so-called ion suppression of less basic phospholipids in binary mixtures. 6 To overcome ion suppressive effects (ISEs) in quantitative mass spectrometry of liquid samples, liquid chromatography (LC) is used as a separation tool before ionization to separate interfering substances from analytes. [7][8][9][10][11] Despite the advanced chromatographic separation methods for biological samples, interferences between sample components still occur during the ionization process. ...
... In positive-ion mode, one important factor influencing ion suppression is the high gas-phase basicitiy (GB) of phospholipids relative to values for other endogenous and exogenous metabolites, possibly hampering the protonation of analytes. 22 The relation between GB and signal intensity of binary mixtures in particle-induced ionization methods was first investigated in 1986 by Sunner et al. 23 Lately, matrix effects in ToF-SIMS of amino acid mixtures have been related to GB. 24 Previous studies about charge distribution in phospholipid samples have shown severe ion suppression due to the presence of phosphatidylcholine. 6 The theoretical basis of the correlation of GB and ion suppression stems from the hypothesis that a competition between two bases in the gas phase after desorption/ionization will determine the charge carrier distribution. As already stated, the dwell time of molecules and especially of ions in the selvedge of the sample is comparatively low. ...
... Apart from GB, other properties of phospholipids are additionally responsible for ISEs, as those are also encountered in negative-ion mode. 6 Still, the linear correlation observed in our measurements between GB and signal intensity implies a significant role for GB in the occurrence of ISEs. ...
In mass spectrometry imaging (MSI), ion suppression can lead to a misinterpretation of results. Particularly phospholipids, most of which exhibit high gas-phase basicity (GB), are known to suppress the detection of metabolites and drugs. This study was initiated by the observation that the signal of an herbicide, i.e., atrazine, was suppressed in MSI investigations of earthworm tissue sections. Herbicide accumulation in earthworms was investigated by time-of-flight secondary ion mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Additionally, earthworm tissue sections without accumulation of atrazine but with a homogeneous spray deposition of the herbicide were analyzed to highlight region-specific ion suppression. Furthermore, the relationship of signal intensity and GB in binary mixtures of lipids, amino acids, and atrazine was investigated in both MSI techniques. The GB of atrazine was determined experimentally through a linear plot of the obtained intensity ratios of the binary amino acid mixtures, as well as theoretically. The GBs values for atrazine of 896 and 906 kJ/mol in ToF-SIMS and 933 and 987 kJ/mol in MALDI-MSI were determined experimentally and that of 913 kJ/mol by quantum mechanical calculations. Compared with the GB of a major lipid component, phosphatidylcholine (GB PC = 1044.7 kJ/mol), atrazine's experimentally and computationally determined GBs in this work are significantly lower, making it prone to ion suppression in biological samples containing polar lipids.
... A common example for ion suppression is presented by phosphatidylcholines (PC), which in the positive ion mode notoriously hamper the detection of the many other lipid classes present in complex tissues. 9,10 To meet these challenges, various postionization (PI) techniques have been developed, fostering the analytical depths of an experiment. 3,4 In this regard, a number of methods based on charge distribution reactions have been introduced recently that enable increased ionization e ciencies for a much broader range of analytes. ...
... To stop the sublimation process, the chamber was ushed with nitrogen. 10 emission wavelength: 349 nm; pulse width: 7-10 ns; pulse repetition rate f rep, adjustable from 1 up to a maximum of 5 kHz) was used as the MALDI laser and for material ablation in the MALDI-SPICI experiments. In the presented spectra, the ablation laser was operated with a repetition rate of 300 ...
... After removal from the freezer, tissue sections and standards were thawed under a stream of nitrogen before coating with the MALDI matrix in a home-build matrix sublimation chamber. 10 Mass spectrometer. A Q Exactive Plus Orbitrap (Thermo Fisher Scienti c, Bremen, Germany), coupled with a dual-ion funnel/dual MALDI/ESI Injector (Spectroglyph, Kennewick, WA), was used as the mass analyzer. ...
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a rapidly growing method in many fields of the life sciences. For many analyte classes, however, its sensitivity is limited due to poor ionization efficiencies. To mitigate this problem, we here introduce a novel and cost-effective postionization scheme at high repetition rates based on the interplay of single-photon photoionization and subsequent charge transfer reactions. Importantly, the fine vacuum conditions of a dual ion-funnel ion source effectively thermalize the evolving MALDI plume and enable ample gas-phase reactions as well as the addition of chemical dopants that crucially support chemical ionization. Supported by acetone dopant, [M + H] ⁺ /[M-H] ⁻ signals of numerous glycerophospho-, sphingo-, and further lipids, registered from mammal brain and kidney sections, were boosted by up to three orders of magnitude, similar to results obtained with laser-based postionization (MALDI-2). Experiments utilizing deuterated matrix and dopant, however, indicate complex ionization pathways different from MALDI2.
... fraction of desorbed molecules is ionized [6][7][8][9][10][11][12][13]. Ion yields strongly depend on the chemical properties of the matrix and the analyte itself but also on the overall composition of the sample [12,14,15]. For glycerophospholipids (GPL), for example, it has been shown that the presence of small quantities of phosphatidylcholine (PC) can effectively quench ion yields for other GPL classes like phosphatidylethanolamine (PE) [15,16]. ...
... Ion yields strongly depend on the chemical properties of the matrix and the analyte itself but also on the overall composition of the sample [12,14,15]. For glycerophospholipids (GPL), for example, it has been shown that the presence of small quantities of phosphatidylcholine (PC) can effectively quench ion yields for other GPL classes like phosphatidylethanolamine (PE) [15,16]. This phenomenon is often called ion suppression effect (ISE) and can be observed for a wide variety of analyte classes [17][18][19][20]. ...
... In order to reduce ISE for the targeted analysis of certain heavily suppressed analyte classes, on tissue enzymatic digestion to deplete suppressing substances and on tissue derivatization to increase sensitivity for a certain analyte class have been successfully employed [22][23][24]. In a different approach, laser-induced postionization in a fine vacuum MALDI ion source (MALDI-2) has shown promising first results pointing towards a reduction in ISE in the analysis of membrane lipids [15,[25][26][27]. In this technique, a second laser pulse hits the evolving plume 10 μs after the initial MALDI event ca. ...
MALDI mass spectrometry imaging (MALDI-MSI) is a widely used technique to map the spatial distribution of molecules in sectioned tissue. The technique is based on the systematic generation and analysis of ions from small sample volumes, each representing a single pixel of the investigated sample surface. Subsequently, mass spectrometric images for any recorded ion species can be generated by displaying the signal intensity at the coordinate of origin for each of these pixels. Although easily equalized, these recorded signal intensities, however, are not necessarily a good measure for the underlying amount of analyte and care has to be taken in the interpretation of MALDI-MSI data. Physical and chemical properties that define the analyte molecules’ adjacencies in the tissue largely influence the local extraction and ionization efficiencies, possibly leading to strong variations in signal intensity response. Here, we inspect the validity of signal intensity distributions recorded from murine cerebellum as a measure for the underlying molar distributions. Based on segmentation derived from MALDI-MSI measurements, laser microdissection (LMD) was used to cut out regions of interest with a homogenous signal intensity. The molar concentration of six exemplary selected membrane lipids from different lipid classes in these tissue regions was determined using quantitative nano-HPLC-ESI-MS. Comparison of molar concentrations and signal intensity revealed strong deviations between underlying concentration and the distribution suggested by MSI data. Determined signal intensity response factors strongly depend on tissue type and lipid species.
Graphical abstract
... We propose three avenues to increase the performance of the future generation of MALDI-MSI in-source Baquer et al. Journal of Cheminformatics (2023) 15 [11]. In positive polarity, PC species display stronger signals than other lipids (PE, PS, PG, or PI). ...
... In negative polarity, the effect is reversed and PC species show lower signals than other lipids. These interactions have been characterized in the past [11] and could be leveraged to define a new ranking score to filter out unlikely lipid annotations. ...
Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI) spatially resolves the chemical composition of tissues. Lipids are of particular interest, as they influence important biological processes in health and disease. However, the identification of lipids in MALDI-MSI remains a challenge due to the lack of chromatographic separation or untargeted tandem mass spectrometry. Recent studies have proposed the use of MALDI in-source fragmentation to infer structural information and aid identification. Here we present rMSIfragment, an open-source R package that exploits known adducts and fragmentation pathways to confidently annotate lipids in MALDI-MSI. The annotations are ranked using a novel score that demonstrates an area under the curve of 0.7 in ROC analyses using HPLC–MS and Target-Decoy validations. rMSIfragment applies to multiple MALDI-MSI sample types and experimental setups. Finally, we demonstrate that overlooking in-source fragments increases the number of incorrect annotations. Annotation workflows should consider in-source fragmentation tools such as rMSIfragment to increase annotation confidence and reduce the number of false positives.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13321-023-00756-2.
... This is where standard MALDI, however, has two notable weak points: First, for many compound classes less than one out of a million of the desorbed molecules is concomitantly ionized [6,7]. Second, particularly well-ionizing compounds (e.g., phosphatidylcholines (PC)) can scavenge charges from other compounds, a scenario that is referred to as ion suppression [8]. Several instrumental advancements have recently significantly extended the analytical abilities of MALDI-MS imaging [9,10]. ...
... 4 Notes 1. If tissues contain a high number of fat cells and are, therefore, rich in triacylglycerols, the mass spectral quality obtained from the sections can be severely reduced because of ion suppression [8] as well as "smearing" effects. To mitigate this problem, apply a washing step with heptane (3 Â 1 mL) before matrix application [19]. ...
Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) combined with laser-induced postionization for MALDI-2 enables the simultaneous registration of numerous classes of small molecules (e.g., secondary metabolites including sterols) as well as phospholipids, glycolipids, and glycans from tissue sections and from cell cultures with strongly boosted ion yields. Here, we describe methodological aspects that are key for optimizing the analytical sensitivity and spatial resolution of a MALDI-2 imaging experiment. We will include both top-illumination MALDI-2 as well as the recently introduced transmission (t-) mode MALDI-2 approach.
... MSI is not linked to a chromatogram and therefore individual metabolites are not separated. Since each laser spot will contain a complex mixture of analytes in varying abundances with differences in ionization, MSI can suffer from ion suppression effects [3]. Though MSI can be performed on a high-resolution instrument to distinguish chemical compositions, species that are isobaric, or nearly so, will require additional validation experiments to support compound identification and relative quantification [4]. ...
... However, labeled samples are usually harvested via extraction-based methods requiring tissue disruption which results in a loss of spatial metabolic organization, and thus may mask cell-type-or tissue-specific differences in metabolism. Recognizing that isotopic signatures in metabolites reflect the pathways of use, methods to resolve subcellular or some cellular features to guide flux analysis have been established [3,4,15]. Combining MSI and steady state labeling approaches can provide a comprehensive description of metabolism capable of distinguishing activities in different cell types or heterogeneity within tissues. ...
The combination of 13C-isotopic labeling and mass spectrometry imaging (MSI) offers an approach to analyze metabolic flux in situ. However, combining isotopic labeling and MSI presents technical challenges ranging from sample preparation, label incorporation, data collection, and analysis. Isotopic labeling and MSI individually create large, complex data sets, and this is compounded when both methods are combined. Therefore, analyzing isotopically labeled MSI data requires streamlined procedures to support biologically meaningful interpretations. Using currently available software and techniques, here we describe a workflow to analyze 13C-labeled isotopologues of the membrane lipid and storage oil lipid intermediate―phosphatidylcholine (PC). Our results with embryos of the oilseed crops, Camelina sativa and Thlaspi arvense (pennycress), demonstrated greater 13C-isotopic labeling in the cotyledons of developing embryos compared with the embryonic axis. Greater isotopic enrichment in PC molecular species with more saturated and longer chain fatty acids suggest different flux patterns related to fatty acid desaturation and elongation pathways. The ability to evaluate MSI data of isotopically labeled plant embryos will facilitate the potential to investigate spatial aspects of metabolic flux in situ.
... S5 and S6, significant decreases in total lipids (64%, P < 0.001) and total proteins (97%, P < 0.001) were observed after LACT treatment. Several studies have proved that lipids and proteins could cause severe ion suppression of small molecules 29,30 . Therefore, LACT could achieve an in situ extraction and strips out matrix-analyte cocrystals from the tissue to separate endogenous and exogenous components. ...
... Nevertheless, numerous quantitative MALDI-MSI strategies have been implemented, such as in-solution, on-tissue, or in-tissue quantitative approaches. Despite their utility, these approaches have limitations stemming from matrix effects, extraction efficiency, and required sample preparation (Lagarrigue et al. 2016;Rzagalinski and Volmer 2017;Boskamp and Soltwisch 2020). Consequently, LC-MS methodologies are often preferred in environmental studies for the quantification of low-molecular-weight micropollutants due to their ability to detect trace concentrations. ...
In the European circular economy, agricultural practices introduce pharmaceutical (PhAC) residues into the terrestrial environment, posing a potential risk to earthworms. This study aimed to assess earthworm bioaccumulation factors (BAFs), the ecotoxicological effects of PhACs, the impact of biochar on PhAC bioavailability to earthworms, and their persistence in soil and investigate earthworm uptake mechanisms along with the spatial distribution of PhACs. Therefore, earthworms were exposed to contaminated soil for 21 days. The results revealed that BAFs ranged from 0.0216 to 0.329, with no significant ecotoxicological effects on earthworm weight or mortality (p > 0.05). Biochar significantly influenced the uptake of 14 PhACs on the first day (p < 0.05), with diminishing effects over time, and affected significantly the soil-degradation kinetics of 16 PhACs. Moreover, MALDI-MS analysis revealed that PhAC uptake occurs through both the dermal and oral pathways, as pharmaceuticals were distributed throughout the entire earthworm tissue without specific localization. In conclusion, this study suggests ineffective PhAC accumulation in earthworms, highlights the influence of biochar on PhAC degradation rates in soil, and suggests that uptake can occur through both earthworm skin and oral ingestion.
Graphical Abstract
... One answer lies in the quantitative limitations of MALDI (e.g. ionization biases across all ionizable molecules) [45], and the need for intermediate antibodies or probes for other MSI techniques. It is also interesting to note that many MSI multiomic studies rely on consecutive tissue sections to measure different modalities. ...
... For example, MALDI2 was shown to have increased sensitivity for specific glycerophospholipids in complex biological samples with low-abundant analytes or notable ion suppression. 30 Additionally, DESI's sampling approach allows imaging of bacterial colonies on agar without sample destruction, facilitating subsequent subculturing. ...
Spatial metabolomics using imaging mass spectrometry (MS) enables untargeted and label-free metabolite mapping in biological samples. Despite the range of available imaging MS protocols and technologies, our understanding of metabolite detection under specific conditions is limited due to sparse empirical data and predictive theories. Consequently, challenges persist in designing new experiments, and accurately annotating and interpreting data. In this study, we systematically measured the detectability of 172 biologically-relevant metabolites across common imaging MS protocols using custom reference samples. We evaluated 24 MALDI-imaging MS protocols for untargeted metabolomics, and demonstrated the applicability of our findings to complex biological samples through comparison with animal tissue data. We showcased the potential for extending our results to further analytes by predicting metabolite detectability based on molecular properties. Additionally, our interlaboratory comparison of 10 imaging MS technologies, including MALDI, DESI, and IR-MALDESI, showed extensive metabolite coverage and comparable results, underscoring the broad applicability of our findings within the imaging MS community. We share our results and data through a new interactive web application integrated with METASPACE. This resource offers an extensive catalogue of detectable metabolite ions, facilitating protocol selection, supporting data annotation, and benefiting future untargeted spatial metabolomics studies.
... The application of TIMS in the presence of NaCl also seemed to provide a comprehensive coverage for TGs, where 18 TGs were identified in addition to all 11 TGs detected in previous sections (Fig. 5B). Of all lipids, TIMS activation appeared to be the most beneficial for TG detection, suggesting the increased sentivity of MALDI-TIMS-MSI in picking up low-abundant TG signals that were originally suppressed by ionisation of phospholipids (Boskamp & Soltwisch, 2020). A list of all tentatively identified lipids from MALDI-TIMS-MSI of NaCl-doped samples can be found in Table S1. ...
Oat (Avena sativa L.) is an important cereal grain with a unique nutritional profile including a high proportion of lipids. Understanding lipid composition and distribution in oats is valuable for plant, food and nutritional research, and can be achieved using MALDI mass spectrometry imaging (MALDI-MSI). However, this approach presents several challenges for sample preparation (hardness of grains) and analysis (isobaric and isomeric properties of lipids). Here, oat sections were successfully mounted onto gelatin-coated indium tin oxide slides with minimal tearing. Poor detection of triacylglycerols was resolved by applying sodium chloride during mounting, increasing signal intensity. In combination with trapped ion mobility spectrometry (TIMS), lipid identification significantly improved, and we report the separation of several isobaric and isomeric lipids with visualisation of their "true" spatial distributions. This study describes a novel MALDI-TIMS-MSI analytical technique for oat lipids, which may be used to improve the discovery of biomarkers for grain quality.
... libraries, and (3) compiling MALDI-ISD or MALDI-MS/MS libraries.Ion suppression effects strongly favor certain classes of lipids, di culting the analysis of suppressed species(Boskamp and Soltwisch 2020). In positive polarity, PC species display stronger signals than other lipids (PE, PS, PG, or PI). ...
Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI) spatially resolves the chemical composition of tissues. Lipids are of particular interest, as they influence important biological processes in health and disease. However, the identification of lipids in MALDI-MSI remains a challenge due to the lack of chromatographic separation or untargeted tandem mass spectrometry. Recent studies have proposed the use of MALDI in-source fragmentation to infer structural information and aid identification. Here we present rMSIfragment, an open-source R package that exploits known adducts and fragmentation pathways to confidently annotate lipids in MALDI-MSI. The annotations are ranked using a novel score that demonstrates an area under the curve of 0.7 in ROC analyses using HPLC-MS and Target-Decoy validations. rMSIfragment applies to multiple MALDI-MSI sample types and experimental setups. Finally, we demonstrate that overlooking in-source fragments increases the number of incorrect annotations. Annotation tools should consider in-source fragmentation such as rMSIfragment to increase annotation confidence and reduce the number of false positives.
... 25 Further limits to sensitivity include ion suppression effects whereby certain lipid species, such as PC in positive-ion mode analysis, can lower the ionization efficiencies of other lipid classes. 26 These phenomena limit both the achievable sensitivity and breadth of lipid coverage obtained for MALDI-MSI experiments. Technologies that maximize ion yields from smaller sampling regions are a major focus of recent endeavours in the field, where there is a goal to improve the spatial resolution and encroach on the subcellular world while retaining the rich lipidomic and metabolomic data To this goal, various post-ionization technologies have been developed in recent years that involve subjecting analytes to an additional ionization event subsequent to initial desorption/ionization. ...
Here we report the development and optimization of a mass spectrometry imaging (MSI) platform that combines atmospheric-pressure matrix-assisted laser desorption/ionization platform with plasma post-ionization (AP-MALDI-PPI) and trapped ion mobility spectrometry (TIMS). We discuss optimal parameters for operating the source, characterize the behaviour of a variety of lipid classes in positive- and negative-ion modes and explore the capabilities for lipid imaging using murine brain tissue. The instrument generates high signal-to-noise for numerous lipid species, with mass spectra sharing many similarities to those obtained using laser post-ionization (MALDI-2). The system is especially well suited for detecting lipids such as phosphatidylethanolamine (PE) as well as numerous sphingolipid classes and glycerolipids. For the first time, the coupling of plasma-based post-ionization with ion mobility is presented and we show the value of ion mobility for the resolution and and identification of species within rich spectra that contain numerous isobaric/isomeric signals that are not resolved in the m/z dimension alone, including isomeric PE and demethylated phosphatidylcholine lipids produced by in-source fragmentation. The reported instrument provides a powerful and user-friendly approach for MSI of lipids.
... To increase the analytical sensitivity for numerous classes of lipids and further metabolites, a laser-based postionization strategy, named MALDI-2, can be used (40)(41)(42). The technique reduces ion suppression effects, typical in MALDI-MSI of lipids (43), and increases ion yields by up to three orders of magnitude for a number of lipid classes. MALDI-2 thereby enhances molecular coverage and chemical depth significantly, especially for minute sample amounts. ...
Molecular analysis on the single-cell level represents a rapidly growing field in the life sciences. While bulk analysis from a pool of cells provides a general molecular profile, it is blind to heterogeneities between individual cells. This heterogeneity, however, is an inherent property of every cell population. Its analysis is fundamental to understanding the development, function, and role of specific cells of the same genotype that display different phenotypical properties. Single-cell mass spectrometry (MS) aims to provide broad molecular information for a significantly large number of cells to help decipher cellular heterogeneity using statistical analysis. Here, we present a sensitive approach to single-cell MS based on high-resolution MALDI-2-MS imaging in combination with MALDI-compatible staining and use of optical microscopy. Our approach allowed analyzing large amounts of unperturbed cells directly from the growth chamber. Confident coregistration of both modalities enabled a reliable compilation of single-cell mass spectra and a straightforward inclusion of optical as well as mass spectrometric features in the interpretation of data. The resulting multimodal datasets permit the use of various statistical methods like machine learning–driven classification and multivariate analysis based on molecular profile and establish a direct connection of MS data with microscopy information of individual cells. Displaying data in the form of histograms for individual signal intensities helps to investigate heterogeneous expression of specific lipids within the cell culture and to identify subpopulations intuitively. Ultimately, t-MALDI-2-MSI measurements at 2-µm pixel sizes deliver a glimpse of intracellular lipid distributions and reveal molecular profiles for subcellular domains.
... Common examples for ion suppression are phosphatidylcholines (PC), which in the positive ion mode notoriously hamper the detection of the many other lipid classes present in complex tissues. [9,10] To meet these challenges, various post-ionization (PI) techniques have been developed, fostering the analytical depths of an experiment. [11,12] In this regard, several methods based on charge distribution reactions have recently been introduced that enable increased ionization efficiencies for a much broader range of analytes. ...
Matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI) is a rapidly growing method in the life sciences. However, for many analyte classes, its sensitivity is limited due to poor ionization efficiencies. To mitigate this problem, we here introduce a novel post‐ionization scheme based on single‐photon induced chemical ionization using pulsed RF‐Kr lamps. The fine‐vacuum conditions of a dual ion‐funnel ion source effectively thermalize the evolving MALDI plume and enable ample gas‐phase reactions. Injected chemical dopants crucially support fragment‐less ionization to [M+H]⁺/[M−H]⁻ species. Based on this interplay, numerous glycerophospho‐, sphingo‐, and further lipids, registered from mammalian tissue sections, were boosted by up to three orders of magnitude, similar to results obtained with laser‐based post‐ionization (MALDI‐2). Experiments with deuterated matrix and dopant, however, indicated complex chemical ionization pathways different from MALDI‐2.
... Molecular profiles acquired by MALDI MS can provide powerful insights for clinical studies, yet, the ionization process in MALDI has been described as inherently susceptible to ion suppression effects [16][17][18]. The presence of highly abundant molecules or molecules with higher ionization efficiencies compared to others can yield ions that dominate molecular profiles, thereby hindering detection of suppressed species and the depth of molecular information obtained [19]. ...
Mass spectrometry imaging provides a powerful approach for the direct analysis and spatial visualization of molecules in tissue sections. Using matrix-assisted laser desorption/ionization mass spectrometry, intact protein imaging has been widely investigated for biomarker analysis and diagnosis in a variety of tissue types and diseases. However, blood-rich or highly vascular tissues present a challenge in molecular imaging due to the high ionization efficiency of hemoglobin, which leads to ion suppression of endogenous proteins. Here, we describe a protocol to selectively reduce hemoglobin signal in blood-rich tissues that can easily be integrated into mass spectrometry imaging workflows.
... Based on this method, even the spatial location of biomarkers in the organization can be provided by means of visualization (Shariatgorji et al., 2019). At present, this technique has successfully achieved the spatial analysis of small molecules, such as amino acids, lipids, nucleic bases, and partial macro-molecules, containing peptides and proteins (Moore et al., 2019;Boskamp and Soltwisch, 2020). However, it also has difficulty in the detection of proteins due to their large molecular weight and low content (Ryan et al., 2019). ...
As a matrix metalloproteinase, the abnormal expression of MMP2 is associated with multiple biological processes, including tissue remodeling and cancer progression. Therefore, spatial analysis of MMP2 protein in tissues can be used as an important approach to evaluate the expression distribution of MMP2 in complex tissue environments, which will help the diagnosis and treatment of various diseases, including tissue or organ injuries. Moreover, this analysis will also help the evaluation of prognoses. However, MMP2 is difficult to be spatially determined by MALDI TOF mass spectrometry due to its large molecular weight (over 72 KD) and low content. Therefore, a new method should be developed to help this detection. Here, we have designed a specific MMP2 probe that closely binds to MMP2 protein in tissue. This probe has a Cl on Tyr at the terminal, which can provide two isotope peaks to help the accuracy quantitative of MMP2 protein. Based on this, we used the probe to determine the spatial expression of MMP2 in the tissues based on MALDI TOF mass spectrometry. This approach may help to study the influence of multifunctional proteases on the degree of malignancy in vivo.
... [6] Ionisation suppression effects can also further reduce ionisation efficiencies, further compounding the difficulties of complex sample imaging analysis. [7] These low ionisation efficiencies result in a practical limitation for high spatial-resolution imaging due to the low signal intensity correlated with reduced sampling volumes. ...
Matrix‐assisted laser/desorption ionisation‐mass spectrometry imaging (MALDI‐MSI) enables label‐free imaging of biomolecules in biological tissues. However, many molecules remain undetected due to their poor ionisation efficiencies. These poor ionisation efficiencies practically limit spatial resolution. Herein, we address this challenge for aromatic antioxidants by reporting an innovative approach involving sequential matrix‐assisted laser desorption and two‐photon ionisation of desorbed neutrals. It is shown that ion yields increase with reduced sampling areas obtained using sub‐threshold primarily laser fluence. This counterintuitive observation could arise from a reduction in radical/ion neutralisation reactions within the sparse plume and/or favorable molecular desorption under low fluence conditions. The utility of this approach is demonstrated for imaging tocopherols and ubiquinols in mouse brain and prostate cancer tissue. This can pave the way for improved sensitivity in MSI experiments at cellular and sub‐cellular resolutions.
... S5 and S6, significant decreases in total lipids (64%, P < 0.001) and total proteins (97%, P < 0.001) were observed after LACT treatment. Several studies have proved that lipids and proteins could cause severe ion suppression of small molecules 29,30 . Therefore, LACT could achieve an in situ extraction and strips out matrix-analyte cocrystals from the tissue to separate endogenous and exogenous components. ...
Accurate localization of central nervous system (CNS) drug distribution in the brain is quite challenging to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), owing to the ionization competition/suppression of highly abundant endogenous biomolecules and MALDI matrix. Herein, we developed a highly efficient sample preparation technique, laser-assisted chemical transfer (LACT), to enhance the detection sensitivity of CNS drugs in brain tissues. A focused diode laser source transilluminated the tissue slide coated with α-cyano-4-hydroxycinnamic acid, an optimal matrix to highly absorb the laser radiation at 405 nm, and a very thin-layer chemical film mainly containing drug molecule was transferred to the acceptor glass slide. Subsequently, MALDI MSI was performed on the chemical film without additional sample treatment. One major advantage of LACT is to minimize ionization competition/suppression from the tissue itself by removing abundant endogenous lipid and protein components. The superior performance of LACT led to the successful visualization of regional distribution patterns of 16 CNS drugs in the mouse brain. Furthermore, the dynamic spatial changes of risperidone and its metabolite were visualized over a 24-h period. Also, the brain-to-plasma (B/P) ratio could be obtained according to MALDI MSI results, providing an alternative means to assess brain penetration in drug discovery.
... Although widely used and suitable in the detection of the majority of lipids, MALDI-MSI also suffers from limitations in the lipidomic context. First, some important lipid species (including neutral lipids such as sterols (ST) and triglycerides (TG)) exhibit low ionization efficiency using conventional organic matrices [48,49] and are generally suppressed by the ionization of phospholipids (phosphatidylcholines (PC), in particular) [50][51][52][53]. Second, the ionization and fragmentation of the matrix produce strong interference signals in the low m/z range [54], usually hindering the analysis of small molecules (< 700 Da) [55,56]. ...
Knowing the spatial location of the lipid species present in biological samples is of paramount importance for the elucidation of pathological and physiological processes. In this context, mass spectrometry imaging (MSI) has emerged as a powerful technology allowing the visualization of the spatial distributions of biomolecules, including lipids, in complex biological samples. Among the different ionization methods available, the emerging surface-assisted laser desorption/ionization (SALDI) MSI offers unique capabilities for the study of lipids. This review describes the specific advantages of SALDI-MSI for lipid analysis, including the ability to perform analyses in both ionization modes with the same nanosubstrate, the detection of lipids characterized by low ionization efficiency in MALDI-MS, and the possibilities of surface modification to improve the detection of lipids. The complementarity of SALDI and MALDI-MSI is also discussed. Finally, this review presents data processing strategies applied in SALDI-MSI of lipids, as well as examples of applications of SALDI-MSI in biomedical lipidomics.
... 55,56 The inverse distribution might be explained by ion suppression effects of especially PC species with high ionization rates. 57 ...
Lipids, such for example the multifaceted category of glycerophospholipids (GP), play a major role in many biological processes. High-resolution mass spectrometry is able to identify these highly diverse lipid species in combination with fragmentation experiments (MS/MS) on the basis of the accurate m/z and fragmentation pattern. However, for the differentiation of isomeric lipids or isobaric interferences, more elaborate separation methods are required. Especially for imaging techniques, such as matrix-assisted laser desorption/ionization (MALDI)-MS imaging, the identification is often exclusively based on the accurate m/z. Fragmentation via MS/MS increases the confidence in lipid annotation in imaging approaches. However, this is sometimes not feasible due to insufficient sensitivity and significantly prolonged analysis time. The use of a separation dimension such as trapped ion mobility spectrometry (TIMS) after ionization strengthens the confidence of the identification based on the collision cross section (CCS). Since CCS libraries are limited, a tissue-specific database was initially generated using hydrophilic interaction liquid chromatography-TIMS-MS. Using this database, the identification of isomeric lipid classes as well as isobaric interferences in a lipid class was performed using a mouse spleen sample in a workflow described in this study. Besides a CCS-based identification as an additional identification criterion for GP in general, the focus was on the distinction of the isomeric GP classes phosphatidylglycerol and bis(monoacylglycero)phosphate, as well as the differentiation of possible isobaric interferences based on the formation of adducts by MALDI-TIMS-MS imaging on a molecular level.
... Some of the lipid classes that benefit most from MALDI-2 are the ones whose sensitivity is affected by ion suppression, like glycerophospholipids. 176 Concerned with ion suppression effects and misleading MALDI-MSI heat map images, Eiersbrock et al. sought to quantify lipid content by performing MALDI-2-MSI in conjunction with LCM and quantitative nano-high-pressure liquid chromatography (HPLC) electrospray ionization (ESI) of the same tissue section. 177 The researchers undersampled while performing MALDI-2-MSI by employing a 30 μm lateral step but a laser ablation size of only 15 μm. ...
Molecular imaging has the potential to unearth important spatial and temporal relationships in biological systems, including intercellular signaling as well as environmentally-cued morphological changes. Various chemical imaging techniques show functional groups, molecular weights, etc., but no individual technique to date has the ability to simultaneously access all chemical information. Thus, it is highly attractive to combine information from two or more analytical techniques. Multimodal imaging is a recent, highly effective strategy for acquiring images by combining chemical information from multiplexed platforms.(1) This emerging integrated imaging approach yields information unattainable from a single method, enabling the evaluation of subtle biochemical changes and opening the way for a quantitative molecular overview of the morphological structure in biological tissues or architecture of cells. As a result, qualitative and quantitative multiomics investigations have the capability to revolutionize our understanding of disease progression and the healing process. Such a strategy has received increasing attention as it helps to elucidate the complex spatial distribution of biomolecules from the surface of a biological sample while circumventing the specific limitations of an individual imaging technique.
... Further work is needed to elucidate these pathways for different combinations of MALDI matrices and laser parameters. 22,25,27,28 The application of MALDI-2 to the analysis of oligosaccharides in negative ion mode could potentially simplify sample preparation workflows and enhance ion yields and would therefore improve reproducibility and sensitivity of the analysis. While already indicated by an increase in signal intensity for small disaccharides (Hex2) by Soltwisch et al., beneficial effects for the analysis of larger, more complex oligosaccharides can, however, currently only be suspected. ...
N-glycans are important players in a variety of pathologies including different types of cancer, (auto)immune diseases, and also viral infections. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an important tool for high-throughput N-glycan profiling and, upon use of tandem MS, for structure determination. By use of MALDI-MS imag-ing (MSI) in combination with PNGase F treatment, also spatially-correlated N-glycan profiling from tissue sections becomes possible. Here we coupled laser-induced postionization, or MALDI-2, to a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF fleX MALDI-2, Bruker Daltonics). We demonstrate that with MALDI-2 the sensitivity for the detection of molecular [M - H]- species of N-glycans increased by about three orders of magnitudes. Compared to the cur-rent gold standard, the positive ion mode analysis of [M + Na]+ adducts, a sensitivity increase by about a factor of ten is achieved. By exploiting the advantageous fragmentation behavior of [M - H]- ions, exceedingly rich structural information on the composition of complex N-glycans was moreover obtained directly from thin tissue sections of human cerebellum and upon use of low-energy collision-induced dissociation tandem MS. In another set of experiments - in this case by use of a modified Synapt G2-S QTOF mass spectrometer (Waters) - we investigated the influence of relevant input parameters, in particular pressure of the N2 cooling gas in the ion source, delay between the two laser pulses, and that of their pulse energies. In this way, analytical conditions were identified at which molecular ion abundances were maximized and fragmentation reactions minimized. The use of negative ion mode MALDI-2-MSI could constitute a valuable tool in glycobiology research.
Biological organisms are multifaceted, intricate systems where slight perturbations can result in extensive changes in gene expression, protein abundance and/or activity, and metabolic flux. These changes occur at different timescales, spatially across cells of heterogenous origins, and within single cells. Hence multimodal measurements at the smallest biological scales are necessary to capture dynamic changes in heterogenous biological systems. Of the analytical techniques used to measure biomolecules, mass spectrometry has proven to be a powerful option due to its sensitivity, robustness, and flexibility with regards to breadth of biomolecules that can be analyzed. Recently many studies have coupled mass spectrometry to other analytical techniques with the goal of measuring multiple modalities from the same single-cell. It is with these concepts in mind that we focus this Review on mass spectrometry-enabled multiomic measurements at single-cell or near-single-cell resolution.
Mass spectrometry imaging (MSI) has accelerated our
understanding of lipid metabolism and spatial distribution in tissues and cells. However, few MSI studies have approached lipid imaging quantitatively and those that have focused on a single lipid class. We overcome this limitation by using a multiclass internal standard (IS) mixture sprayed homogeneously
over the tissue surface with concentrations that reflect those of endogenous lipids. This enabled quantitative MSI (Q-MSI) of 13 lipid classes and subclasses representing almost 200 sum-composition lipid species using both MALDI (negative ion mode) and MALDI-2 (positive ion mode) and pixel-wise normalization of each lipid species in a manner analogous to that widely used in shotgun lipidomics. The Q-MSI approach covered 3 orders of
magnitude in dynamic range (lipid concentrations reported in pmol/mm2) and revealed subtle changes in distribution compared to data without normalization. The robustness of the method was evaluated by repeating experiments in two laboratories using both timsTOF and Orbitrap mass spectrometers with an ∼4-fold difference in mass resolution power. There was a strong overall correlation in the Q-MSI results obtained by using the two approaches. Outliers were mostly rationalized by isobaric interferences or the higher sensitivity of one instrument for a particular lipid species. These data provide insight into how the mass resolving power can affect Q-MSI data. This approach opens up the possibility of performing large-scale Q-MSI studies across numerous lipid classes and subclasses and revealing how absolute lipid concentrations vary throughout and between biological tissues.
Synthetic polymers are ubiquitous in daily life, and their properties offer diverse benefits in numerous applications. However, synthetic polymers also present an increasing environmental burden through their improper disposal and subsequent degradation into secondary micro- and nanoparticles (MNPs). These MNPs accumulate in soil and water environments and can ultimately end up in the food chain, resulting in potential health risks. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has the potential to study localized biological or toxicological changes in organisms exposed to MNPs. Here, we investigate whether MALDI-2 postionization can provide a sensitivity enhancement in polymer analysis that could contribute to the study of MNPs. We evaluated the effect of MALDI-2 by comparing MALDI and MALDI-2 ion yields from polyethyleneglycol (PEG), polypropylene glycol (PPG), polytetrahydrofuran (PTHF), nylon-6, and polystyrene (PS). MALDI-2 caused a signal enhancement of the protonated species for PEG, PPG, PTHF, and nylon-6. PS, by contrast, preferentially formed radical ions, which we attribute to direct resonance-enhanced multiphoton ionization (REMPI). REMPI of PS led to an improvement in sensitivity by several orders of magnitude, even without cationizing salts. The improved sensitivity demonstrated by MALDI-2 for all polymers tested highlights its potential for studying the distribution of certain classes of polymers in biological systems.
The precise fatty acyl chain configuration of cardiolipin (CL), a tetrameric mitochondrial-specific membrane lipid, exhibits dependence on cell and tissue types. A powerful method to map CL profiles in tissue sections in a spatially resolved manner is matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). To build on and explore this potential, we employed a quadrupole time-of-flight mass spectrometer along with optimized sample preparation protocols. We imaged the CL profiles of individual murine retinal cell layers at a pixel size of 10 μm. In combination with tandem MS, we obtained detailed insights into the CL composition of individual retinal cell layers. In particular, we found differential expression of the polyunsaturated fatty acids (PUFA) linoleic, arachidonic, and docosahexaenoic acids. PUFAs are prone to peroxidation and hence regarded as critical factors in development and progression of retinal pathologies, such as age-related macular degeneration (AMD). The ability of MALDI-MSI to provide cues on the CL composition in neuronal tissue with close to single-cell resolution can provide important insights into retinal physiology in health and disease.
Herein, we assess the complementarity and complexity of data that can be detected within mammalian lipidome mass spectrometry imaging (MSI) via matrix-assisted laser desorption ionization (MALDI) and nanospray desorption electrospray ionization (nano-DESI). We do so by employing 21 T Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) with absorption mode FT processing in both cases, allowing unmatched mass resolving power per unit time (≥613k at m/z 760, 1.536 s transients). While our results demonstrated that molecular coverage and dynamic range capabilities were greater in MALDI analysis, nano-DESI provided superior mass error, and all annotations for both modes had sub-ppm error. Taken together, these experiments highlight the coverage of 1676 lipids and serve as a functional guide for expected lipidome complexity within nano-DESI-MSI and MALDI-MSI. To further assess the lipidome complexity, mass splits (i.e., the difference in mass between neighboring peaks) within single pixels were collated across all pixels from each respective MSI experiment. The spatial localization of these mass splits was powerful in informing whether the observed mass splits were biological or artificial (e.g., matrix related). Mass splits down to 2.4 mDa were observed (i.e., sodium adduct ambiguity) in each experiment, and both modalities highlighted comparable degrees of lipidome complexity. Further, we highlight the persistence of certain mass splits (e.g., 8.9 mDa; double bond ambiguity) independent of ionization biases. We also evaluate the need for ultrahigh mass resolving power for mass splits ≤4.6 mDa (potassium adduct ambiguity) at m/z > 1000, which may only be resolved by advanced FTICR-MS instrumentation.
Here we report the development and optimization of a mass spectrometry imaging (MSI) platform that combines an atmospheric-pressure matrix-assisted laser desorption/ionization platform with plasma postionization (AP-MALDI-PPI) and trapped ion mobility spectrometry (TIMS). We discuss optimal parameters for operating the source, characterize the behavior of a variety of lipid classes in positive- and negative-ion modes, and explore the capabilities for lipid imaging using murine brain tissue. The instrument generates high signal-to-noise for numerous lipid species, with mass spectra sharing many similarities to those obtained using laser postionization (MALDI-2). The system is especially well suited for detecting lipids such as phosphatidylethanolamine (PE), as well as numerous sphingolipid classes and glycerolipids. For the first time, the coupling of plasma-based postionization with ion mobility is presented, and we show the value of ion mobility for the resolution and identification of species within rich spectra that contain numerous isobaric/isomeric signals that are not resolved in the m/z dimension alone, including isomeric PE and demethylated phosphatidylcholine lipids produced by in-source fragmentation. The reported instrument provides a powerful and user-friendly approach for MSI of lipids.
Ambient mass spectrometry imaging (MSI) methods come with the advantage of visualizing biomolecules from tissues with no or minimal sample preparation and operation under atmospheric-pressure conditions. Similar to all other MSI methodologies, however, ambient MSI modalities suffer from a pronounced bias toward either polar or nonpolar analytes due to the underlying desorption and ionization mechanisms of the ion source. In this study, we present the design, construction, testing, and application of an in-capillary dielectric barrier discharge (DBD) module for post-ionization of neutrals desorbed by an ambient infrared matrix-assisted laser desorption/ionization (IR-MALDI) MSI source. We demonstrate that the DBD device enhances signal intensities of nonpolar compounds by up to 104 compared to IR-MALDI without affecting transmission of IR-MALDI ions. This allows performing MSI experiments of mouse tissue and Danaus plexippus caterpillar tissue sections, visualizing the distribution of sterols, fatty acids, monoglycerides, and diglycerides that are not detected in IR-MALDI MSI experiments. The pronounced signal enhancement due to IR-MALDI-DBD compared to IR-MALDI MSI enables mapping of nonpolar analytes with pixel resolutions down to 20 μm in mouse brain tissue and to discern the spatial distribution of sterol lipids characteristic for histological regions of D. plexippus.
Unsaturated lipids containing single or more carbon-carbon double bonds (C═C) within tissues are closely associated with various types of diseases. Mass spectrometry imaging (MSI) has been used to study the spatial distribution of lipid C═C location isomers in tissue sections. However, comprehensive characterization of lipid C═C location isomers using MSI remains challenging. Herein, we established an on-tissue charge-switching Paternò-Büchi (PB) derivatization method using 3-acetylpyridine (3-AP) as a reaction reagent, which can be used to detect and assign C═C location of glycerophospholipids (GPLs) as well as neutral lipids, such as fatty acids (FAs), under the same experimental workflow using matrix-assisted laser desorption/ionization (MALDI)-MSI. High coverage of mono- and poly-unsaturated C═C location isomers among various lipid classes including FA, phosphatidylcholine (PC), and sulfatide (SHexCer) in distinct regions of the mouse brain and kidney was visualized using MALDI-MS/MS imaging. This method has also been applied to map the spatial distribution of lipid C═C location isomers in the Alzheimer's disease (AD) mice model for the first time, which provides a new tool to study the relationships between the distribution of lipid structural diversity and neurodegenerative diseases.
Mass spectrometry imaging (MSI) is a powerful label‐free analysis technique that can provide simultaneous spatial distribution of multiple compounds in a single experiment. By combining the sensitive and rapid screening of high‐throughput mass spectrometry with spatial chemical information, metabolite analysis and morphological characteristics are presented in a single image. MSI can be used for qualitative and quantitative analysis of metabolic profiles and it can provide visual analysis of spatial distribution information of complex biological and microbial systems. Matrix assisted laser desorption ionization, laser ablation electrospray ionization and desorption electrospray ionization are commonly used in MSI. Here, we summarize and compare these three technologies, as well as the applications and prospects of MSI in metabolomics.
Matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI) is a rapidly growing method in the life sciences. However, for many analyte classes, its sensitivity is limited due to poor ionization efficiencies. To mitigate this problem, we here introduce a novel postionization scheme based on single‐photon induced chemical ionization using pulsed RF‐Kr lamps. The fine‐vacuum conditions of a dual ion‐funnel ion source effectively thermalize the evolving MALDI plume and enable ample gas‐phase reactions. Injected chemical dopants crucially support fragment‐less ionization to [M+H] + /[M‐H] ‐ species. Based on this interplay, numerous glycerophospho‐, sphingo‐, and further lipids, registered from mammalian tissue sections, were boosted by up to three orders of magnitude, similar to results obtained with laser‐based postionization (MALDI‐2). Experiments with deuterated matrix and dopant, however, indicated complex chemical ionization pathways different from MALDI‐2.
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.
Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, unlabeled mapping of analytes from tissue sections. However, further work is needed to improve the sensitivity and depth of coverage for protein and peptide IMS. We demonstrate signal enhancement of proteolytic peptides from thin tissue sections of human kidney by conventional MALDI (MALDI-1) augmented using a second ionizing laser (termed MALDI-2). Proteins were digested in situ using trypsin prior to IMS analysis. For tentative identification of peptides and proteins, a tissue homogenate from the same organ used for IMS was analyzed by LC–MS/MS, and data are available via ProteomeXchange with identifier PXD023877. These identified proteins were then digested in silico to generate a database of theoretical peptides to then match to MALDI IMS data sets. Peptides were tentatively identified by matching the MALDI peak list to the database peptide list based on mass accuracy (5 ppm mass error). This resulted in 1337 ± 96 (n = 3) peptides and 2076 ± 362 (n = 3) unique peptides matched to IMS peaks from MALDI-1 and MALDI-2, respectively. Protein identifications requiring two or more peptides per protein resulted in 276 ± 20 proteins with MALDI-1 and 401 ± 60 with MALDI-2. These results demonstrate that MALDI-2 provides enhanced sensitivity for the spatial mapping of tryptic peptides and significantly increases the number of proteins identified in IMS experiments.
Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, untargeted detection of many hundreds of analytes from tissue. Recently, laser postionization (MALDI-2) has been developed for increased ion yield and sensitivity for lipid IMS. However, the dependence of MALDI-2 performance on the various lipid classes is largely unknown. To understand the effect of the applied matrix on MALDI-2 analysis of lipids, samples including an equimolar lipid standard mixture, various tissue homogenates, and intact rat kidney tissue sections were analyzed using the following matrices: α-cyano-4-hydroxycinnamic acid, 2',5'-dihydroxyacetophenone, 2',5'-dihydroxybenzoic acid (DHB), and norharmane (NOR). Lipid signal enhancement of protonated species using MALDI-2 technology varied based on the matrix used. Although signal improvements were observed for all matrices, the most dramatic effects using MALDI-2 were observed using NOR and DHB. For lipid standards analyzed by MALDI-2, NOR provided the broadest coverage, enabling the detection of all 13 protonated standards, including nonpolar lipids, whereas DHB gave less coverage but gave the highest signal increase for those lipids recorded. With respect to tissue homogenates and rat kidney tissue, mass spectra were compared and showed that the number and intensity of neutral lipids tentatively identified with MALDI-2 using NOR increased significantly (e.g., fivefold intensity increase for triacylglycerol). In the cases of DHB with MALDI-2, the number of protonated lipids identified from tissue homogenates doubled with 152 on average compared with 76 with MALDI alone. High spatial resolution imaging (~20 μm) of rat kidney tissue showed similar results using DHB with 125 lipids tentatively identified from MALDI-2 spectra versus just 72 using standard MALDI. From the four matrices tested, NOR provided the greatest increase in sensitivity for neutral lipids (triacylglycerol, diacylglycerol, monoacylglycerol, and cholesterol ester), and DHB provided the highest overall number of lipids detected using MALDI-2 technology.
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a powerful label-free technique for mapping the spatial distribution of biomolecules directly from tissue. However, like most other MSI techniques, it suffers from low ionization yields and ion suppression effects for biomolecules that might be of interest for a specific application at hand. Recently, a form of laser post-ionization was introduced (coined MALDI-2) that critically boosts the ion yield for many glyco- and phospholipids by several orders of magnitude and makes the detection of further biomolecular species possible. While the MALDI-2 technique is being increasingly applied by the MSI community, it is still only implemented in fine vacuum ion sources in a pressure range of about 1-10 mbar. Here, we show the first implementation of the technique to a custom-built atmospheric pressure ion source coupled to an Orbitrap Elite system. We present results from parameter optimization of MALDI-2 at atmospheric pressure, compare our findings to previously published fine vacuum data and show first imaging results from mouse cerebellum with 20 µm pixel size. Our findings broaden the feasibility of the technique to overall more flexible atmospheric pressure ion sources.
Local lipid variations in tissues are readily revealed with mass spectrometry imaging (MSI) methods, and the resulting lipid distributions serve as bioanalytical signatures to reveal cell- or tissue-specific lipids. Comprehensive MSI lipid mapping requires measurements in both ion polarities. Additionally, structural lipid characterization is necessary to link the lipid structure to lipid function. Whereas some structural elements of lipids are readily derived from high-resolution mass spectrometry (MS) and tandem-MS (MS
n
), the localization of C═C double bonds (DBs) requires specialized fragmentation and/or functionalization methods. In this work, we identify a multifunctional matrix-assisted laser desorption/ionization (MALDI) matrix for spatially resolved lipidomics investigations that reacts with lipids in Paternò-Büchi (PB) reactions during laser irradiation facilitating DB-position assignment and allows dual-polarity high-resolution MALDI-MSI and MALDI MS2I studies. By screening 12 compounds for improved ionization efficiency in positive-/negative-ion mode and the functionalization yield compared to the previously introduced reactive MALDI matrix benzophenone, 2-benzoylpyridine (BzPy) is identified as the best candidate. The new matrix enables DB localization of authentic standards belonging to 12 lipid classes and helps to assign 133/58 lipid features in positive-/negative-ion mode from mouse cerebellum tissue. The analytical capabilities of BzPy as a multifunctional MALDI-MSI matrix are demonstrated by imaging endogenous and PB-functionalized lipids in mouse kidney sections with 7 μm lateral resolution in both ion modes. Tracking diagnostic lipid DB-position fragment ions in mouse pancreatic tissue with down to 10 μm pixel size allows us to identify the islets of Langerhans associated with lipid isomer upregulation and depletion.
A recently introduced technique based on MALDI with laser-induced post-ionization (PI), also named MALDI-2, increases the ion yields for numerous classes of lipids, metabolites, and carbohydrates in MALDI-MS imaging experiments under certain experimental conditions. Here we used a semi-automatic LabVIEW-based protocol to investigate and optimize the efficiency of the PI process in dependence of four relevant input parameters and a dense parameter grid: pulse energies of the two lasers, delay between the laser pulses, and buffer gas pressure in the ion source. All experiments were conducted with a modified MALDI-2 Synapt G2-S mass spectrometer (Waters) and use of a focal spot size on the sample of 15-17 µm. A wavelength-tunable optical parametric oscillator (OPO) laser served for PI at 260 nm or 280 nm. The investigated MALDI matrices were: 2,5-dihydroxybenzoic acid (pos. ion mode, +), 2,5-dihydroxyacetophenone (+), α-cyano-4-hydroxycinnamic acid (+), norharmane (neg. ion mode, -), and 1,5-diaminonapthalene (-). A porcine brain extract served as lipid standard. In the positive ion mode, a maximum boost for the generated [M + H]+ species was found with a N2 buffer gas pressure of ~2 mbar and a delay between the laser emissions of ~10 μs. Higher optimal delay settings of several 10 µs were registered for the two studied matrices in negative ion mode. With regard to the laser fluences, best PI efficiencies were reached using maximum available ablation and PI laser pulse energies of up to 25 µJ and 160 µJ, respectively. For analytes not profiting from MALDI-2, best ion signal yields were recorded for ablation laser pulse energies of around 7 µJ, depending on the MALDI matrix. At higher laser pulse energies, sizeable fragmentation is observed for these ions. The PI laser pulse energy did not have any influence on the ion signals of these species. For optimal ion yield of all analyte species, best results were obtained with an ablation laser pulse energy of ~7 μJ and a PI laser pulse energy of ~160 μJ. Our comprehensive data set provides valuable insight into the mechanisms underlying the MALDI-2 processes and could help to further optimize this emerging technique.
Matrix-assisted laser desorption/ionisation-mass spectrometry imaging (MALDI-MSI) is a powerful technique for visualising the spatial locations of lipids in biological tissues. However, a major challenge in interpreting the biological significance of local lipid compositions and distributions detected using MALDI-MSI is the difficulty in associating spectra with cellular lipid metabolism within the tissue. By-and-large this is due to the typically limited spatial resolution of MALDI-MSI (30–100 μm) meaning individual spectra represent the average spectrum acquired from multiple adjacent cells, each potentially possessing a unique lipid composition and biological function. The use of oversampling is one promising approach to decrease the sampling area and improve the spatial resolution in MALDI-MSI, but it can suffer from a dramatically decreased sensitivity. In this work we overcome these challenges through the coupling of oversampling MALDI-MSI with laser post-ionisation (MALDI-2). We demonstrate the ability to acquire rich lipid spectra from pixels as small as 6 μm, equivalent to or smaller than the size of typical mammalian cells. Coupled with an approach for automated lipid identification, it is shown that MALDI-2 combined with oversampling at 6 μm pixel size can detect up to three times more lipids and many more lipid classes than even conventional MALDI at 20 μm resolution in the positive-ion mode. Applying this to mouse kidney and human brain tissue containing active multiple sclerosis lesions, where 74 and 147 unique lipids are identified, respectively, the localisation of lipid signals to individual tubuli within the kidney and lipid droplets with lesion-specific macrophages is demonstrated.
We report a method that enables automated data-dependent acquisition of lipid tandem mass spectrometry data in parallel with a high-resolution mass spectrometry imaging experiment. The method does not increase the total image acquisition time and is combined with automatic structural assignments. This lipidome-per-pixel approach automatically identified and validated 104 unique molecular lipids and their spatial locations from rat cerebellar tissue.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is widely used for the analysis of large biomolecules in numerous applications. The technique utilizes nanosecond-long laser pulses at various spot sizes to eject and ionize large molecules embedded in a highly absorptive chemical matrix. Despite the methods name, 'molecular desorption' from the matrix crystal surface is not the sole mechanism discussed for material ejection in MALDI, but additional ablation of larger clusters has been reported. Here we present results on the influence of laser fluence and spot size on the mechanisms of the initial material ejection in MALDI and subsequent plume development. We used a laser-based postionization (MALDI-2) as well as a complementary photoacoustic method to monitor the material ejection step. The photoacoustic data reveal a quasi-thermal sublimation process up to a transition fluence. Above this threshold fluence additional ablation processes are observed. Complementary investigations on plume dynamics by MALDI-2 showed an ejection of predominantly fast particles for desorption conditions while ablation produces considerably slower ejecta. Additionally the presented results revealed a peculiar influence of the spot size on analyte fragmentation as well as plume development and allows for new insights into the unexplained spot size effect reported for MALDI.
Tissue lipidomics is one of the latest omics approaches for biomarker discovery in pharmacology, pathology, and the life sciences at large. In this context, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is the most versatile tool to map compounds within tissue sections. However, ion suppression events occurring during MALDI MSI analyses make it impossible to use this method for quantitative investigations without additional validation steps. This is especially true for lipidomics, since different lipid classes are responsible for important ion suppression events. We propose here an improved lipidomics method to assess local ion suppression of phospatidylcholines in tissues. Serial tissue sections were spiked with different amounts of PC(16:0 d31/18:1) using a nebulization device. Settings for standard nebulization were strictly controlled for a detection similar to when using spiked tissue homogenates. The sections were simultaneously analyzed by MALDI MSI using a Fourier transform ion cyclotron resonance analyzer. Such a spray-based approach allows taking into account the biochemical heterogeneity of the tissue for the detection of PC(16:0 d31/18:1). Thus, here we present the perspective to use this method for quantification purposes. The linear regression lines are considered as calibration curves and we calculate PC(16:0/18:1) quantification values for different ROIs. Although those values need to be validated by a using a different independent approach, the workflow offers an insight into new quantitative mass spectrometry imaging (q-MSI) methods. This approach of ion suppression monitoring of phosphocholines in tissues may be highly interesting for a large range of applications in MALDI MSI, particularly for pathology using translational science workflows.
One of the reasons that thermally induced reactions are not considered a crucial mechanism in ultraviolet matrix-assisted laser desorption ionization (UV-MALDI) is the low ion-to-neutral ratios. Large ion-to-neutral ratios (10–4) have been used to justify the unimportance of thermally induced reactions in UV-MALDI. Recent experimental measurements have shown that the upper limit of the total ion-to-neutral ratio is approximately 10–7 at a high laser fluence and less than 10–7 at a low laser fluence. Therefore, reexamining the possible contributions of thermally induced reactions in MALDI may be worthwhile. In this study, the concept of polar fluid was employed to explain the generation of primary ions in MALDI. A simple model, namely thermal proton transfer, was used to estimate the ion-to-neutral ratios in MALDI. We demonstrated that the theoretical calculations of ion-to-neutral ratios exhibit the same trend and similar orders of magnitude compared with those of experimental measurements. Although thermal proton transfer may not generate all of the ions observed in MALDI, the calculations demonstrated that thermally induced reactions play a crucial role in UV-MALDI.
Figure
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Matrix-assisted laser desorption ionisation imaging mass spectrometry (MALDI-MSI) is a rapidly advancing technique for intact tissue analysis that allows simultaneous localisation and quantification of biomolecules in different histological regions of interest. This approach can potentially offer novel insights into tumour microenvironmental (TME) biochemistry. In this study we employed MALDI-MSI to evaluate fresh frozen sections of colorectal cancer (CRC) tissue and adjacent healthy mucosa obtained from 12 consenting patients undergoing surgery for confirmed CRC. Specifically, we sought to address three objectives: (Hogan et al., 2012 Jul 1) To identify biochemical differences between different morphological regions within the CRC TME; (Straussman et al., 2012 Jul 26) To characterise the biochemical differences between cancerous and healthy colorectal tissue using MALDI-MSI; (Engelhardt et al., 2012 Mar 20) To determine whether MALDI-MSI profiling of tumour-adjacent tissue can identify novel metabolic 'field effects' associated with cancer. Our results demonstrate that CRC tissue harbours characteristic phospholipid signatures compared with healthy tissue and additionally, different tissue regions within the CRC TME reveal distinct biochemical profiles. Furthermore we observed biochemical differences between tumour-adjacent and tumour-remote healthy mucosa. We have referred to this 'field effect', exhibited by the tumour locale, as cancer-adjacent metaboplasia (CAM) and this finding builds on the established concept of field cancerisation.
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.
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.
Despite their compositional complexity, lipidomes comprise a large number of isobaric species that cannot be distinguished by conventional low resolution mass spectrometry and therefore in-depth MS/MS analysis was required for their accurate quantification. Here we argue that the progress in high resolution mass spectrometry is changing the concept of lipidome characterization. Because exact masses of isobaric species belonging to different lipid classes are not necessarily identical, they can now be distinguished and directly quantified in total lipid extracts. By streamlining and simplifying the molecular characterization of lipidomes, high resolution mass spectrometry has developed into a generic tool for cell biology and molecular medicine.
Recent developments in MALDI have enabled direct detection of lipids as intact molecular species present within cellular membranes. Abundant lipid-related ions are produced from the direct analysis of thin tissue slices when sequential spectra are acquired across a tissue surface that has been coated with a MALDI matrix. The lipid-derived ions can often be distinguished from other biomolecules because of the significant mass defect that these ions present due to the large number of covalently bound hydrogen atoms in hydrophobic molecules such as lipids. Collisional activation of the molecular ions can be used to determine the lipid family and often structurally define the molecular species. Specific examples in the detection of phospholipids, sphingolipids, and glycerolipids are presented with images of mouse brain and kidney tissue slices. Regional distribution of many different lipid molecular species and Na+ and K+ attachment ions often define anatomical regions within the tissues.
In the analysis of polymers by matrix-assisted laser desorption/ionization mass spec-trometry (MALDI-MS), a commonly observed ion-ization pathway is cation-adduct formation, as polymers often lack easily ionizable (basic/acidic) functional groups. The mechanism of this process has been hypothesized to involve gas-phase cation attachment. In previous experiments , a split sample plate setup has been introduced , enabling separate deposition of the components on individual MALDI plates. The plates are divided by a small gap of a few micrometers, allowing simultaneous laser irradiation from both plates, while precluding the possibility of any other interactions prior to ablation. Here, we extend on these studies by using different polymer-salt combinations to test the generaliz-ability of a gas-phase ionization process. Clear evidence for in-plume ionization is presented for the model polymers poly (methyl methacrylate) and polystyrene. Furthermore, the contribution of in-plume processes to the overall ion formation by cationization is gauged, providing a first estimate for the importance of this pathway.
The clinical use of mass spectrometry imaging (MSI) is rapidly growing, and applications are expanding. Crucially, the analysis of clinical cohorts requires a combination of higher analytical throughput and higher lateral resolution. Here, we demonstrate the benefits of an improved design of a prototype MALDI source (referred as uMALDI”) mounted on a SYNAPT HDMS G2-Si mass spectrometer – widely used by the MSI community. The uMALDI source allows for imaging of lipids on histologically well documented rat brain sections at a lateral resolution of 15 µm without oversampling. We also investigate the fast rastering capabilities of the platform enabled by a new web-based MS control program (i.e. “WREnS” Waters Research Enabled Software). The combination of the uMALDI and WREnS allows high spatial resolution images at acquisition rates up to 20 pixels per second. Additionally, we demonstrate the capability of this platform to characterize the lipid distribution of osteoarthritic (OA) cartilage at the cellular level, which highlights potential benefit for broader application in clinical context. Finally, we prove that ion mobility separation of isobaric lipid species is maintained and images can be acquired at a rate of 10 scans per seconds. This renders a unique ion mobility mass spectrometric imaging platform with high acquisition rates and high spatial resolution.
Changes in lipid composition of cells or tissue are often linked to various diseases. Studies indicate alterations of bis(monoacylglycero)phosphate (BMP) species in diseases such as cancer. Therefore, an extended phospholipid profiling method based on hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution mass spectrometry (MS) and data-dependent MS/MS acquisition was developed to separate and unambiguously identify BMP species. Lipid species identification was based on retention time, accurate mass and specific MS/MS fragments. The developed method was applied in a proof of concept study to lipid extracts of a cell culture model of conditional oncogene overexpression in MCF-7/NeuT breast cancer cells. Comparison of control and oncogene-induced MCF-7/NeuT breast cancer cells showed changes in BMP species distribution. Thereby, a shift from long-chain to shorter-chain fatty acid composition in BMP species was detected.
In this study we have explored several aspects of regional analyte suppression in mass spectrometry imaging of a heterogeneous sample; transverse cryosections of mouse brain. Olanzapine was homogeneously coated across the section prior to DESI and MALDI mass spectrometry imaging. We employed the concept of a tissue extinction coefficient (TEC) to assess suppression of an analyte on tissue relative to its intensity in an off tissue region. We expanded the use of TEC, by first segmenting anatomical regions using graph-cuts clustering, and calculating a TEC for each cluster. The single ion image of the olanzapine [M+H]+ ion was seen to vary considerably across the image, with anatomical features such as the white matter and hippocampus visible. While trends in regional ion suppression were conserved across MSI modalities, significant changes in the magnitude of relative regional suppression effects between techniques were seen. Notably the intensity of olanzapine was less suppressed in DESI than for MALDI. In MALDI MSI, significant differences in the concentration dependence of regional TECs were seen, with the TEC of white matter clusters exhibiting a notably stronger correlation with concentration than for clusters associated with grey matter regions. We further employed cluster-specific TECs as regional normalisation factors. In comparison to published pixel-by-pixel normalisation methods, regional TEC normalisation exhibited superior reduction ion suppression artefacts. We also considered the usefulness of a segmentation-based approach to compare spectral information obtained from complementary modalities.
Imaging mass spectrometry (IMS) has become a powerful tool to characterise the spatial distribution of biomolecules in thin tissue sections. In the case of matrix-assisted laser desorption ionisation (MALDI) IMS, homogeneous matrix deposition is critical to produce high quality ion images and sublimation in particular has shown to be an excellent matrix deposition method for the imaging of lipids. Matrix deposition by sublimation is, however, a completely solvent-free system, which ought to prevent the mixing of matrix and analytes thought to be necessary for successful MALDI. Using 3D time-of-flight secondary ion imaging mass spectrometry, we have studied the matrix-tissue interface in 3D with high resolution in order to understand the MALDI process of lipids after matrix deposition by sublimation. There is a strong indication that diffusion is the process by which lipids migrate from the tissue to the matrix layer. We show that triacylglycerols and phospholipids have a delayed migratory trend compared to diacylglycerols and monoacylglycerols, which is dependent on time and matrix thickness. Additional experiments show that a pure lipid's capacity to migrate into the matrix is dependent on its fluidity at room temperature. Furthermore, it is shown that cholesterol can only migrate in the presence of a (fluid) lipid and appears to fluidise lipids, which could explain its co-localisation with the diacylglycerols and monoacylglycerols in the matrix.
Mass spectrometry imaging (MSI) is a powerful tool that enables untargeted investigations into the spatial distribution of molecular species in a variety of samples. It has the capability to image thousands of molecules, such as metabolites, lipids, peptides, proteins, and glycans, in a single experiment without labeling. The combination of information gained from mass spectrometry (MS) and visualization of spatial distributions in thin sample sections makes this a valuable chemical analysis tool useful for biological specimen characterization. After minimal but careful sample preparation, the general setup of an MSI experiment involves defining an (x, y) grid over the surface of the sample, with the grid area chosen by the user. The mass spectrometer then ionizes the molecules on the surface of the sample and collects a mass spectrum at each pixel on the section, with the resulting spatial resolution defined by the pixel size. After collecting the spectra, computational software can be used to select an individual mass-to-charge (m/z) value, and the intensity of the m/z is extracted from each pixel’s spectrum. These intensities are then combined into a heat map image depicting the relative distribution of that m/z value throughout the sample’s surface. In order to determine the identity of a specific m/z value, tandem MS (MS/MS) fragmentation can be performed on ions from each pixel, and the fragments can be used to piece together the structure of the unknown molecule. Otherwise, the molecule can be identified based on its intact mass by accurate mass matching to databases of known molecules within a certain mass error range. Overall, the aim of this review is to provide an informative resource for those in the MSI community who are interested in improving MSI data quality and analysis or using MSI for novel applications. Particularly, we discuss advances from the last two years in sample preparation, instrumentation, quantitation, statistics, and multi-modal imaging that have allowed MSI to emerge as a powerful technique in various biomedical applications including clinical settings. Also, several novel biological applications are highlighted to demonstrate the potential for the future of the MSI field.
Coupling laser post-ionisation with a high resolving power MALDI Orbitrap mass spectrometer has realised an up to ∼100-fold increase in the sensitivity and enhanced the chemical coverage for MALDI-MS imaging of lipids relative to conventional MALDI. This could constitute a major breakthrough for biomedical research.
We evaluated the contribution of gas-phase in-plume proton transfer reactions to the formation of protonated and deprotonated molecules in the MALDI process. A split sample holder was used to separately deposit two different samples, which avoids any mixing during sample preparation. The two samples were brought very close to each other and desorbed/ionized by the same laser pulse. By using a combination of deuterated and non-deuterated matrices, it was possible to observe exclusively in-plume proton transfer processes. The hydrogen/deuterium exchange (HDX) kinetics were evaluated by varying the delayed extraction (DE) time, allowing the desorbed ions and neutrals to interact inside the plume for a variable period of time before being extracted and detected. Quantummechanical calculations showed that the HDX energy barriers are relatively low for such reactions, corroborating the importance of gas-phase proton transfer in the MALDI plume. The experimental results, supported by theoretical simulations, confirm that the plumeis a very reactive environment, where HDX reactions could be observed from0 ns up to 400 ns after the laser pulse. These results could be used to evaluate the relevance of previously proposed (and partially conflicting) ionization models for MALDI.
Over the last two decades, lipidomics has evolved into an ‘omics’ technology pari passu with benchmarking ‘omics’ technologies, such as genomics or proteomics. The driving force behind this development was a constant advance in mass spectrometry and related technologies. The aim of this opinion article is to give the interested reader a concise but still comprehensive overview about the technological state of the art in lipidomics, current challenges and perspectives for future development. As such, this article guides through the whole workflow of lipidomics, from sampling to data analysis. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.
Rationale:
Ionization in MALDI MS is a very complicated process. It has been reported that quaternary ammonium salts show extremely strong matrix and analyte suppression effects which cannot satisfactorily be explained by charge transfer reactions. Further investigation of the reasons causing these effects can be useful to improve our understanding of MALDI ionization.
Methods:
The dried-droplet and modified thin-layer methods were used as sample preparation methods. In the dried-droplet method, analytes were co-crystallized with matrix, whereas in the modified thin-layer method analytes were deposited on the surface of matrix crystals. Model compounds, tetrabutylammonium iodide ([N(Bu)4 ]I), cesium iodide (CsI), trihexylamine (THA) and polyethylene glycol 600 (PEG-600) were selected as the test analytes given their ability to generate exclusively pre-formed ions, protonated ions and metal ion adducts respectively in MALDI.
Results:
The strong matrix suppression effect (MSE) observed using the dried-droplet method might disappear using the modified thin-layer method, which suggests that the incorporation of analytes in matrix crystals contributes to the MSE. By depositing analytes on the matrix surface instead of incorporating in the matrix crystals, the competition for evaporation/ionization from charged matrix/analyte clusters could be weakened resulting in reduced MSE. Further supporting evidence for this inference was found by studying the analyte suppression effect using the same two sample deposition methods.
Conclusions:
By comparing differences between the mass spectra obtained via two sample preparation methods, we present evidence suggesting that the generation of gas-phase ions from charged matrix/analyte clusters may induce significant suppression of matrix and analyte ions. The results suggest that the generation of gas-phase ions from charged matrix/analyte clusters is an important ionization step in MALDI MS.
A high resolving power shotgun lipidomics strategy using gas-phase fractionation and data-dependent acquisition (DDA) was applied toward comprehensive characterization of lipids in a hen ovarian tissue in an untargeted fashion. Using this approach, a total of 822 unique lipids across a diverse range of lipid categories and classes were identified based on their MS/MS fragmentation patterns. Classes of glycerophospholipids and glycerolipids, such as glycerophosphocholines (PC), glycerophosphoethanolamines (PE), and triglycerides (TG), are often the most abundant peaks observed in shotgun lipidomics analyses. These ions suppress the signal from low abundance ions and hinder the chances of characterizing low abundant lipids when DDA is used. These issues were circumvented by utilizing gas-phase fractionation, where DDA was performed on narrow m/z ranges instead of a broad m/z range. Employing gas-phase fractionation resulted in an increase in sensitivity by more than an order of magnitude in both positive- and negative-ion modes. Furthermore, the enhanced sensitivity increased the number of lipids identified by a factor of ≈4, and facilitated identification of low abundant lipids from classes such as cardiolipins that are often difficult to observe in untargeted shotgun analyses and require sample-specific preparation steps prior to analysis. This method serves as a resource for comprehensive profiling of lipids from many different categories and classes in an untargeted manner, as well as for targeted and quantitative analyses of individual lipids. Furthermore, this comprehensive analysis of the lipidome can serve as a species- and tissue-specific database for confident identification of other MS-based datasets, such as mass spectrometry imaging.
To improve the lateral resolution in matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) beyond the dimensions of the focal laser spot oversampling techniques are employed. However, few data are available on the effect of the laser spot size and its focal beam profile on the ion signals recorded in oversampling mode. To investigate these dependencies, we produced 2 times six spots with dimensions between ~30 and 200 μm. By optional use of a fundamental beam shaper, square flat-top and Gaussian beam profiles were compared. MALDI-MSI data were collected using a fixed pixel size of 20 μm and both pixel-by-pixel and continuous raster oversampling modes on a QSTAR mass spectrometer. Coronal mouse brain sections coated with 2,5-dihydroxybenzoic acid matrix were used as primary test systems. Sizably higher phospholipid ion signals were produced with laser spots exceeding a dimension of ~100 μm, although the same amount of material was essentially ablated from the 20 μm-wide oversampling pixel at all spot size settings. Only on white matter areas of the brain these effects were less apparent to absent. Scanning electron microscopy images showed that these findings can presumably be attributed to different matrix morphologies depending on tissue type. We propose that a transition in the material ejection mechanisms from a molecular desorption at large to ablation at smaller spot sizes and a concomitant reduction in ion yields may be responsible for the observed spot size effects. The combined results indicate a complex interplay between tissue type, matrix crystallization, and laser-derived desorption/ablation and finally analyte ionization. Graphical Abstractᅟ
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to simultaneously visualize the lateral distribution of different lipid classes in tissue sections, but the applicability of the method to real-life samples is often limited by ion suppression effects. In particular, the presence of abundant phosphatidylcholines (PCs) can reduce the ion yields for all other lipid species in positive ion mode measurements. Here we used on-tissue treatment with buffer-free phospholipase C (PLC) to near-quantitatively degrade PCs in fresh-frozen tissue sections. The ion signal intensities of mono-, di-, and oligohexosylceramides were enhanced by up to ten-fold. In addition, visualization of Shiga toxin receptor globotriaosylceramide (Gb3Cer) in the kidneys of wild-type and α-galactosidase A-knock-out (Fabry) mice was possible at about ten micrometer resolution. Importantly, the PLC treatment did not decrease the high lateral resolution of the MS imaging analysis.
The analytical sensitivity in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is largely affected by the specific analyte-matrix interaction, in particular by the possible incorporation of the analytes into crystalline MALDI matrices. Here we used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to visualize the incorporation of three peptides with different hydrophobicities, bradykinin, Substance P, and vasopressin, into two classic MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA). For depth profiling, an Ar cluster ion beam was used to gradually sputter through the matrix crystals without causing significant degradation of matrix or biomolecules. A pulsed Bi3 ion cluster beam was used to image the lateral analyte distribution in the center of the sputter crater. Using this dual beam technique, the 3D distribution of the analytes and spatial segregation effects within the matrix crystals were imaged with sub-μm resolution. The technique could in the future enable matrix-enhanced (ME)-ToF-SIMS imaging of peptides in tissue slices at ultra-high resolution.
Graphical Abstract
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The Coupled Chemical and Physical Dynamics (CPCD) model of matrix assisted laser desorption ionization has been restricted to relative rather than absolute yield comparisons because the rate constant for one step in the model was not accurately known. Recent measurements are used to constrain this constant, leading to good agreement with experimental yield versus fluence data for 2,5-dihydroxybenzoic acid. Parameters for alpha-cyano-4-hydroxycinnamic acid are also estimated, including contributions from a possible triplet state. The results are compared with the polar fluid model, the CPCD is found to give better agreement with the data.
Graphical Abstract
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Matrix-Assisted Laser Desorption Ionization-Imaging Mass Spectrometry (MALDI-IMS) is a rapidly evolving method used for the in situ visualization and localization of molecules such as drugs, lipids, peptides, and proteins in tissue sections. Therefore, molecules such as lipids, for which antibodies and other convenient detection reagents do not exist, can be detected, quantified, and correlated with histopathology and disease mechanisms. Furthermore, MALDI-IMS has the potential to enhance our understanding of disease pathogenesis through the use of "biochemical histopathology". Herein, we review the underlying concepts, basic methods, and practical applications of MALDI-IMS, including post-processing steps such as data analysis and identification of molecules. The potential utility of MALDI-IMS as a companion diagnostic aid for lipid-related pathological states is discussed.
© The Author(s) 2015.
An important recent discovery concerning the fundamentals of matrix-assisted laser desorption/ionization (MALDI) is that the abundance of each ion appearing in a spectrum is fixed, regardless of the experimental condition, when an effective temperature associated with the spectrum is fixed. We describe this phenomenon and the thermal picture for the ion formation in MALDI derived from it. Accepting that matrix-to-analyte proton transfer is in quasi-equilibrium as supported by experimental data, the above thermal determination occurs because the primary (matrix) ion formation processes are thermally governed. We propose that the abundances of the primary ions are limited by the autoprotolysis-recombination process regardless of how they are initially produced. Finally, we note that primary ion formation, secondary (analyte) ion formation, and their dissociations occur sequentially while the effective temperature of the matrix plume falls steadily due to cooling associated with expansion.
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can simultaneously record the lateral distribution of numerous biomolecules in tissue slices, but its sensitivity is restricted by limited ionization. We used a wavelength-tunable postionization laser to initiate secondary MALDI-like ionization processes in the gas phase. In this way we could increase the ion yields for numerous lipid classes, liposoluble vitamins and saccharides, imaged in animal and plant tissue with a 5 micrometer-wide laser spot, by up to two orders of magnitude. Critical parameters for initiation of the secondary ionization processes are pressure of the cooling gas in the ion source, laser wavelength, pulse energy, and delay between the two laser pulses. The technology could enable sensitive MALDI-MS imaging with a lateral resolution in the low micrometer range.
Copyright © 2015, American Association for the Advancement of Science.
Mass spectrometers from the Synapt-G1/G2 family (Waters) are widely employed for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). A lateral resolution of about 50 µm is typically achieved with these instruments, that is, however, below the often desired cellular resolution. Here we show the first MALDI-MSI examples demonstrating a lateral resolution of about ten microns obtained with a Synapt G2-S HDMS mass spectrometer without oversampling. This improvement became possible by laser beam shaping using a 4:1 beam expander, a circular aperture for spatial mode filtering, and by replacement of the default focusing lens. We used dithranol as an effective matrix for imaging of acidic lipids such as sulfatides, gangliosides, and phosphatidylinositols in the negative ion mode. At the same time, the matrix enables MS imaging of more basic lipids in the positive ion mode. Uniform matrix coatings with crystals having average dimensions between 0.5-3 µm were obtained upon spraying a chloroform/methanol matrix solution. Increasing the cooling gas pressure in the MALDI ion source after adding an additional gas line was furthermore found to increase the ion abundances of labile lipids such as gangliosides. The combined characteristics are demonstrated with the MALDI-MSI analysis of fine structures in coronal mouse brain slices.
MALDI imaging mass spectrometry (MALDI-IMS) has been used successfully in mapping different lipids in tissue sections, yet existing protocols fail to detect the diverse species of mitochondria-unique cardiolipins (CLs) in the brain which are essential for cellular and mitochondrial physiology. We have developed methods enabling the imaging of individual CLs in brain tissue. This was achieved by eliminating ion suppressive effects by (i) cross-linking carboxyl/amino containing molecules on tissue with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride and (ii) removing highly abundant phosphatidylcholine head groups via phospholipase C treatment. These treatments allowed the detection of CL species at 100 μm resolution and did not affect the amount or molecular species distribution of brain tissue CLs. When combined with augmented matrix application, these modifications allowed the visualization and mapping of multiple CL species in various regions of the brain including the thalamus, hippocampus, and cortex. Areas such as the dentate and stratum radiatum exhibited higher CL signals than other areas within the hippocampal formation. The habenular nuclear (Hb)/dorsal third ventricle (D3 V) and lateral ventricle (LV) areas were identified as CL "hot spots". Our method also allowed structural MS/MS fragmentation and mapping of CLs with identified fatty acid residues and demonstrated a nonrandom distribution of individual oxidizable (polyunsaturated fatty acid containing) and nonoxidizable (nonpolyunsaturated containing) CLs in different anatomical areas of the brain. To our knowledge, this method is the first label-free approach for molecular mapping of diversified CLs in brain tissue.
RATIONALEThe signal intensity of a given molecule across a tissue section when measured using mass spectrometry imaging (MSI) is prone to changes caused by the molecular heterogeneity across the surface of the tissue. Here we propose a strategy to investigate these effects using electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) on a single high-resolution mass spectrometry (HRMS) platform.METHODSA rat was administered with a single inhaled dose of a compound and sacrificed 1 h after dosing. Sections were prepared from the excised frozen lung and analysed using MALDI, liquid extraction surface analysis (LESA) nano-ESI-MS and nano-ESI liquid chromatography (LC)/MS. The ESI and MALDI ion sources were mounted either side of the ion transfer system of the same Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer.RESULTSMALDI MSI clearly demonstrated widespread distribution of the dosed molecule throughout the lung, with the exception of a non-lung section of tissue on the same sample surface. Comparison of the lipid signals across the sample indicated a change in signal between the lung and the adipose tissue present on the same section. Use of ESI and MALDI, with and without an internal standard, supported the evaluation of changes in the signal of the dosed molecule across the tissue section.CONCLUSIONS
The results demonstrate the successful application of a dual ion source HRMS system to the systematic evaluation of data from MALDI MSI, used to determine the distribution of an inhaled drug in the lung. The system discussed is of great utility in investigating the effects of ion suppression and evaluating the quantitative and qualitative nature of the MSI data. Copyright © 2014 John Wiley & Sons, Ltd.
Mass spectrometry imaging (MSI) has evolved into a valuable tool across many fields of chemistry, biology, and medicine. However, arguably its greatest disadvantage is the difficulty in acquiring quantitative data regarding the surface concentration of the analyte(s) of interest. These difficulties largely arise from the high dependence of the ion signal on the localized chemical and morphological environment and the difficulties associated with calibrating such signals. The development of quantitative MSI approaches would correspond to a giant leap forward for the field, particularly for the biomedical and pharmaceutical fields, and is thus a highly active area of current research. In this review, we outline the current progress being made in the development and application of quantitative MSI workflows with a focus on biomedical applications. Particular emphasis is placed on the various strategies used for both signal calibration and correcting for various ion suppression effects that are invariably present in any MSI study. In addition, the difficulties in validating quantitative-MSI data on a pixel-by-pixel basis are highlighted.
The ionization mechanisms involved in matrix-assisted ultraviolet laser desorption/ionization (MALDI) were studied with a time-of-flight mass spectrometer. When protonated or cationized quasimolecular ions generated by MALDI are not extracted promptly, their abundance is a function of the delay time between laser irradiation and ion extraction, maximizing at an optimum delay time (DTM) of a few hundred nanoseconds. The ion abundance at DTM exceeds that of prompt extraction by a factor of 2 or more. Increasing the cation density near the sample surface reduces the DTM, whereas increasing the desorption laser irradiance has the opposite effect. The enhancement suggests extensive gas-phase ion-molecule reactions after irradiation by the desorption laser has ceased.
As a branch of metabolomics, lipidomics is aimed at full analysis of lipid species and their biological roles with respect to health and diseases and has attracted increasing attention of biological and analytical scientists. As lipidomics has been investigating for almost ten years, there have been several reviews about it. In this review, we focus on the recent advances in analytical methods of lipidomics, especially in past two years. Herein, Mass spectrometry (MS)-based methods, chromatography-based methods and spectroscopy methods are all overviewed. The applications of these analytical methods in several lipidomics fields are also simply discussed based on researches published in latest two years.
The 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-positional isomers of dihydroxybenzoic (DHB) acid were investigated with respect to their function as matrix-assisted laser desorption/ionization (MALDI) matrices. Optical absorption spectra of solid matrix samples, recorded in diffuse reflection from samples revealed peak broadening and a red shift of the peaks relative to the solution spectra. Single crystals of all isomers were grown from solution and analyzed by x-ray crystallography. Single crystals, as well as standard dried droplet and thin layer preparations with added cytochrome c, were analyzed by UV- (266, 308, 337, 355 nm) and IR- (2.94 μm) MALDI time-of-flight mass spectrometry (MALDI-TOF-MS). A spectrophotometric measurement of the heme absorption around 400 nm of redissolved single crystals showed a quantitative incorporation of the protein into crystals of 2,5-DHB and a partial protein incorporation with large statistical fluctuations into single crystals of 2,4-DHB. No protein incorporation above the detection limit of 10−5 molar analyte-to-matrix ratio was found for the other isomers. Best MALDI spectra from standard preparations were recorded for dried droplet preparations of 2,5-DHB at 337 and 355 nm and for 2,4-DHB at 266 and 308 nm. Both matrices performed well in the IR, too. 2,6-DHB yielded spectra of comparable quality at 337 nm and 2.94 μm, but only when prepared in a thin layer from an acetone solution. The results suggest that protein incorporation into the crystals of solid MALDI matrices is helpful, but not a prerequisite for MALDI. A large surface-to-volume ratio, typical for microcrystalline thin layer preparations supports protein desorption if no measurable incorporation occurs. Undesirable matrix adduct formation to the protein ions was seen for all DHB isomers at the wavelengths of 308 and 266 nm.
Mass spectrometry is now an indispensable tool for lipid analysis and is arguably the driving force in the renaissance of lipid research. In its various forms, mass spectrometry is uniquely capable of resolving the extensive compositional and structural diversity of lipids in biological systems. Furthermore, it provides the ability to accurately quantify molecular-level changes in lipid populations associated with changes in metabolism and environment; bringing lipid science to the "omics" age. The recent explosion of mass spectrometry-based surface analysis techniques is fuelling further expansion of the lipidomics field. This is evidenced by the numerous papers published on the subject of mass spectrometric imaging of lipids in recent years. While imaging mass spectrometry provides new and exciting possibilities, it is but one of the many opportunities direct surface analysis offers the lipid researcher. In this review we describe the current state-of-the-art in the direct surface analysis of lipids with a focus on tissue sections, intact cells and thin-layer chromatography substrates. The suitability of these different approaches towards analysis of the major lipid classes along with their current and potential applications in the field of lipid analysis are evaluated.
A normal-phase HPLC-MS method was established to analyze mitochondrial phospholipids quantitatively as well as qualitatively. An efficient extraction procedure and chromatographic conditions were developed using twelve standardized phospholipids and lysophospholipids. The chromatographic conditions provided physical separation of phospholipids by class, and efficient ionization allowed detection of low abundance phospholipids such as phosphatidylglycerol and monolysocardiolipin. The chromatographic separation of each class of phospholipid permitted qualitative identification of molecular species without interference from other classes. This is advantageous for mitochondrial lipidomics because the composition of mitochondrial phospholipids varies depending on tissue source, pathological condition, and nutrition. Using the method, seven classes of phospholipids (phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, cardiolipin, and monolysocardiolipin) were detected in rat heart and skeletal muscle mitochondria and all but phosphatidylserine were quantified. The concentration was calculated using standard curves with an internal standard generated for each class of phospholipid. The method was validated for intraday and interday variation and showed excellent reproducibility and accuracy. This new method, with each step documented, provides a powerful tool for accurate quantitation of phospholipids, a basic structural component of mitochondrial membranes.
Since its introduction mass spectrometry imaging (MSI) has proven to be a powerful tool for the localization of molecules in biological tissues. In drug discovery and development, understanding the distribution of both drug and its metabolites is of critical importance. Traditional methods suffer from a lack of spatial information (tissue extraction followed by LCMS) or lack of specificity resulting in the inability to resolve parent drug from its metabolites (whole body autoradiography). MSI is a sensitive and label-free approach for imaging drugs and metabolites in tissues. In this article we review the different MSI technologies that have been applied to the imaging of pharmaceuticals. Recent technical advances, applications and current analytical limitations are discussed.
At appropriate matrix:analyte mixing ratios, small to moderate sized analyte ions (1000–20 000 u) can fully suppress positively charged matrix ions in matrix-assisted laser desorption/ionization (MALDI) mass spectra. This is true for all matrix species, including radical cations and adducts with protons or alkali-metal ions. Full matrix suppression is also observed, regardless of the preferred analyte ion form, be it protonated or an alkali adduct. These facts lead us to propose a mechanism for prompt, primary (not secondary gas-phase) MALDI ionization in which excited matrix molecules are the key species. At least two such excited molecules are believed to be necessary for free ion generation. This model is found to be consistent with the available data, as well as making several predictions which are confirmed by new observations. The model also predicts that the matrix suppression effect will not be observable with heavy analytes because their large excluded volume precludes desorption at the necessary mixing ratios.
The laser wavelength constitutes a key parameter in ultraviolet-matrix-assisted laser desorption ionization-mass spectrometry (UV-MALDI-MS). Optimal analytical results are only achieved at laser wavelengths that correspond to a high optical absorption of the matrix. In the presented work, the wavelength dependence and the contribution of matrix proton affinity to the MALDI process were investigated. A tunable dye laser was used to examine the wavelength range between 280 and 355 nm. The peptide and matrix ion signals recorded as a function of these irradiation parameters are displayed in the form of heat maps, a data representation that furnishes multidimensional data interpretation. Matrixes with a range of proton affinities from 809 to 866 kJ/mol were investigated. Among those selected are the standard matrixes 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA) as well as five halogen-substituted cinnamic acid derivatives, including the recently introduced 4-chloro-α-cyanocinnamic acid (ClCCA) and α-cyano-2,4-difluorocinnamic acid (DiFCCA) matrixes. With the exception of DHB, the highest analyte ion signals were obtained toward the red side of the peak optical absorption in the solid state. A stronger decline of the molecular analyte ion signals generated from the matrixes was consistently observed at the low wavelength side of the peak absorption. This effect is mainly the result of increased fragmentation of both analyte and matrix ions. Optimal use of multiply halogenated matrixes requires adjustment of the excitation wavelength to values below that of the standard MALDI lasers emitting at 355 (Nd:YAG) or 337 nm (N(2) laser). The combined data provide new insights into the UV-MALDI desorption/ionization processes and indicate ways to improve the analytical sensitivity.
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has emerged as a novel powerful MS methodology that has the ability to generate both molecular and spatial information within a tissue section. Application of this technology as a new type of biochemical lipid microscopy may lead to new discoveries of the lipid metabolism and biomarkers associated with area-specific alterations or damage under stress/disease conditions such as traumatic brain injury or acute lung injury, among others. However there are limitations in the range of what it can detect as compared with liquid chromatography-MS (LC-MS) of a lipid extract from a tissue section. The goal of the current work was to critically consider remarkable new opportunities along with the limitations and approaches for further improvements of MALDI-MSI. Based on our experimental data and assessments, improvements of the spectral and spatial resolution, sensitivity and specificity towards low abundance species of lipids are proposed. This is followed by a review of the current literature, including methodologies that other laboratories have used to overcome these challenges.
Since the early days of matrix-assisted laser desorption/ionization (MALDI), the definition of a unified theory on the MALDI process is a major challenge. The new results presented in this paper clearly show that the idea of a uniform MALDI mechanism that accounts for the spreading applications has to be given up. Based on different preparation protocols distinct differences in the desorption/ionization process for carbohydrates in contrast to peptides/proteins are elucidated. Although isolated/incorporated and cluster-desorbed peptides/proteins are effectively entrained and cooled within the expanding plume of matrix clusters, as shown by a low degree of metastable analyte-ion fragmentation, a laser desorption and gas-phase cationization mechanism can be confirmed as the dominant part in ionization for neutral oligosaccharides which can be initiated even for particulate analyte material or deposits onto a matrix surface. The previously presented “lucky survivor-model” on cluster desorption of preformed ions thus needs this extension.
The determination of the compound distribution in laboratory animal tissue in early development is a standard process in pharmaceutical research. While this information is traditionally obtained by means of whole-body autoradiography using radiolabeled compounds, this technology does not distinguish between metabolites and parent compound. The technique described in this article, termed matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging, can fill this gap by simultaneously measuring compound and multiple metabolites distributed in whole-body tissue sections, using non-labeled compounds.
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is an established tool for the analysis of proteins, whereas it gained by far less interest in the field of lipid analysis. This method works well with phospholipids as well as organic cell extracts and provides high sensitivity and reproducibility. The aim of the present paper is to extend our previous studies to the analysis of lysophospholipids and phospholipid mixtures. To study the suitability of MALDI-TOF mass spectrometry for the analysis of lysophospholipids, different phospholipids like phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, and phosphatidylinositol as well as their mixtures were digested with phospholipase A2. Positive and negative ion mass spectra of all phospholipids before and after digestion were recorded. In all these cases, the molecular ions of the expected digestion products could be detected and only a very small extent of further fragmentation was observed. On the other hand, spectra of phospholipid mixtures containing phosphatidylcholine were strongly dominated by phosphatidylcholine and lysophosphatidylcholine signals, which prevented the detection of further phospholipids even if those lipids were present in comparable amounts. This is of paramount interest for the analysis of tissue and cell extracts.
MALDI dried droplet preparations using 2,5-dihydroxybenzoic acid as a matrix were investigated by scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry (SMALDI MS). Mass spectrometric images of lateral distributions of sample components and impurities were obtained with a lateral resolution of 1 μm. Results show the crystallization behavior of the matrix as a function of various physico-chemical parameters as well as inhomogeneous separation of components on various scales between millimeters and sub-micrometers. Segregation was found to be a sample-wide, crystal-wide and sub-crystalline, general phenomenon. Effective parameters in this context are hydrophobicity, polarity, and mobility, among others. Peptide ion signals were observed from inside the matrix crystals, while carbohydrate signals and alkali ion signals were observed predominantely from outside the larger matrix crystals.The described investigations shed some light on processes that cause well-known problems of quantification and sample suppression in MALDI MS. They also help to optimize matrix preparation for high-resolution MALDI imaging of biological samples.
Previous studies have shown that matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) is useful for studying the distribution of various small metabolites, particularly lipids. However, in this technique, selective ionization of the target molecules is imperative, particularly when analyzing small molecules. Since the sample clean-up procedures available for the MALDI-IMS of small metabolites are limited, the tissue sample will contain numerous molecular species other than the target molecules. These molecules will compete for ionization resulting in severe ion suppression. Hence, it is necessary to develop and optimize a sample preparation protocol for the target molecules. In this study, through model experiments using reference compounds, we optimized the composition of the matrix solution used for positively charged lipids in terms of the concentration of the organic solvent and presence/absence of alkali metal salts. We demonstrated that a high concentration of organic solvent in the matrix solution favors the preferential detection of lipids over peptides. The presence of alkali metal salts in the matrix solution was favorable for the detection of polar lipids, while a salt-free matrix solution was suitable for the detection of nonpolar lipids. Furthermore, potassium salts added to the matrix solution caused merging of various lipid adducts (adducts with proton, sodium, and potassium) into one single potassiated species. Using the optimized protocols, we selectively analyzed phosphatidylcholine (PC) and triacylglycerol (TG) with different fatty acid compositions in a rat kidney section.
In the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis of some quaternary ammonium salts (QASs), very clean spectra of the quaternary ammonium ions were recorded with a strong matrix suppression effect (MSE). The QASs also showed a considerable analyte suppression effect (ASE). It was demonstrated that the MSE and ASE of the QASs can be explained well by the cluster ionization model. According to this model, MALDI ions are formed from charged matrix/analyte clusters. Various analyte ions and matrix ions might coexist in the cluster, and they will compete for the limited number of net charges available. If enough quaternary ammonium ions are present in the cluster, they will take away the net charges, thus resulting in the MSE and ASE. Our results also suggest that 'the cluster ionization model' is not in conflict with 'the theory of ionization via secondary gas-phase reactions'. The initial MALDI ions produced from charged matrix/analyte clusters will collide with other molecules or ions in the MALDI plume. Depending on the properties of the initial ions and the composition of the MALDI plume, secondary gas-phase reactions might result from these collisions. The final ions observed are the combined results of 'cluster ionization' and 'ionization via secondary gas-phase reactions'.