Atmospheric Pressure Matrix-assisted Laser Desorption/ionization Research Articles

Imaging and quantification of neuropeptides in mouse pituitary tissue by atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry.

Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) mass spectrometry has enabled the untargeted analysis and imaging of neuropeptides and proteins in biological tissues under ambient conditions. Sensitivity in AP-MALDI can be improved by using sample-specific preparation methods. A comprehensive and detailed optimization strategy including instrument parameters, matrix spraying and sample tissue washing pretreatment was implemented to enhance the sensitivity and coverage of neuropeptides in mouse pituitary tissues by commercial AP-MALDI mass spectrometry imaging (MSI). The sensitivity of a commercial AP-MALDI system for endogenous neuropeptides in mouse pituitary was enhanced by up to 15.2-fold by shortening the transmission gap from the sample plate to the inlet, attaching copper adhesive tape to an indium tin oxide-coated glass slide, optimizing the matrix spray solvent and using sample tissue washing pretreatment. Following careful optimization, the distributions of nine endogenous neuropeptides were successfully visualized in the pituitary. Furthermore, the quantitative capability of AP-MALDI for neuropeptides was evaluated and the concentrations of neuropeptides oxytocin and vasopressin in the pituitary posterior lobe were increased approximately twofold under hypertonic saline stress. Mouse pituitary neuropeptides have emerged as important signaling molecules due to their role in stress response. This work indicates the potential of modified AP-MALDI as a promising AP MSI method for in situ visualization and quantification of neuropeptides in complex biological tissues.

A Comprehensive Comparison of Tissue Processing Methods for High-Quality MALDI Imaging of Lipids in Reconstructed Human Epidermis.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has become an important tool for skin analysis, as it allows the simultaneous detection and localization of diverse molecular species within a sample. The use of in vivo and ex vivo human skin models is costly and presents ethical issues; therefore, reconstructed human epidermis (RHE) models, which mimic the upper part of native human skin, represent a suitable alternative to investigate adverse effects of chemicals applied to the skin. However, there are few publications investigating the feasibility of using MALDI MSI on RHE models. Therefore, the aim of this study was to investigate the effect of sample preparation techniques, i.e., substrate, sample thickness, washing, and matrix recrystallization, on the quality of MALDI MSI for lipids analysis of the SkinEthic RHE model. Images were generated using an atmospheric pressure MALDI source coupled to a high-resolution mass spectrometer with a pixel size of 5 μm. Masses detected in a defined region of interest were analyzed and annotated using the LipostarMSI platform. The results indicated that the combination of (1) coated metallic substrates, such as APTES-coated stainless-steel plates, (2) tissue sections of 6 μm thickness, and (3) aqueous washing before HCCA matrix spraying (without recrystallization), resulted in images with a significant signal intensity as well as numerous m/z values. This refined methodology using AP-MALDI coupled to a high-resolution mass spectrometer should improve the current sample preparation workflow to evaluate changes in skin composition after application of dermatocosmetics.

A Rapid Screening Method for the Detection of Additives in Electronics and Plastic Consumer Products Using AP-MALDI-qTOF-MS.

A novel method was developed and optimized for the fast-screening analysis of additives in electronics and plastic consumer products using atmospheric pressure matrix-assisted laser desorption ionization (AP-MALDI) coupled with a high-resolution quadrupole time-of-flight (qTOF) mass spectrometer (MS). To simplify sample preparation and increase sample throughput, an innovative 48 well graphene nanoplatelets (GNP) doped AP-MALDI target plate was developed. The GNP incorporated in the target plate fulfilled the role of the MALDI matrix and, therefore, sample extracts could be directly transferred to the AP-MALDI 48 well target plate and analyzed without a subsequent matrix addition. The homogeneously dispersed and immobilized GNP target plates also provided increased signal intensity and reproducibility. Furthermore, analytical standards of various plastic additives and plastic products with known concentrations of additives were studied to assess the AP-MALDI ionization mechanisms and method capability. The analysis time was 15 s per measurement using an automated sequence. The GNP-doped target plates exhibited high desorption/ionization of low molecular weight molecules (<1000 Da) and can be used in both positive and negative ionization modes. The AP-MALDI-qTOF-MS method was applied to screen for additives in various electronics and plastic consumer products. Suspect screening was performed using a database containing 1366 compounds. A total of 56 additives including antioxidants, flame retardants, plasticizers, UV-stabilizers, and UV-filters were identified (confidence level 4). Identification of certain plastic additives in plastic children's toys may indicate that they are recycled from waste electronic and electronic equipment (WEEE).

MS imaging and absorption methods visualizing sun filter skin spatial distribution and penetration.

Sunscreens must now be effective in protecting skin from ultraviolet, as well as visible/infrared radiation. Here, TriAsorB, a new broad-spectrum sun filter, was formulated with three other sunscreens and their distribution on human skin was studied using a standard penetration protocol and two novel mass spectrometry imaging techniques: atmospheric pressure matrix assisted laser desorption ionization (AP-MALDI) coupled to high resolution mass spectrometry and time of flight - secondary ion mass spectrometry (ToF-SIMS). The standard penetration protocol showed that sun filters absorption was very low, with most of the dose recovered at the surface (none entered the receptor fluid). Absorption was not increased in damaged skin. The results were confirmed by AP-MALDI and ToF-SIMS imaging of the spatial distribution of molecular species in cross-section samples of human skin. Each sun filter was detected on or in the stratum corneum, with a good homogenous coverage over the valleys and peaks of the skin, and correlated well with the distribution of endogenous biomarkers. In conclusion, conventional and novel imaging analysis methods showed that the sun filters remained mainly on the skin surface after topical application. Mass spectrometry imaging is a promising complementary approach to traditional skin penetration studies to visualize penetration of compounds.

Spatially revealed perfluorooctane sulfonate-induced nephrotoxicity in mouse kidney using atmospheric pressure MALDI mass spectrometry imaging

Perfluorooctane sulfonate (PFOS), an emerging environmental persistent pollutant, has attracted extensive attention due to its potential nephrotoxicity. However, little is known about the spatial variations of lipid metabolism associated with PFOS exposure. In this study, atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-MALDI MSI) was used to reveal the spatial distributions of PFOS and its adverse effect on lipid metabolism directly in mouse kidney sections. We have observed that PFOS accumulated in the renal pelvis and outer cortex regions, with some found in the medulla and inner cortex regions. Hematoxylin and eosin (H&E) staining results also demonstrated that the accumulation of PFOS caused damage to the mouse kidney, which was consistent with AP-MALDI MSI results. Furthermore, a total of 42 lipids were shown to be significantly different in the spatial distribution patterns and variations between control and PFOS exposure mice groups, including the significant down-regulation of lyso-glycerophospholipids (Lyso-GPs), phosphatidic acids (PA), phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylserines (PS) sphingomyelins (SM) and sulfatides (ST) in renal medulla or cortex region of mouse kidney sections, and remarkable up-regulation of cholesterol and phosphatidylinositols (PI) in the cortex regions of mouse kidney sections. The AP-MALDI MSI provides a new tool to explore spatial distributions and variations of the endogenous metabolites for the risk assessment of environmental pollutants.

Facile detection of pesticides using atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry with multi-walled carbon nanotubes-based matrix

A novel atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry (AP-MALDI-MS) method was established for the facile detection of pesticides in ambient environment. Four kinds of multi-walled carbon nanotubes (MWCNTs)-based matrix were synthesized and utilized to enhance the ionization efficiency of pesticides. Organophosphorus, anilinopyrimidine, carbamate, triazine, triazole and benzimidazole pesticides were directly desorbed and ionized from MWCNTs-based matrix in ambient environment, showing clear background and good sensitivity. In a comparison, Fe 3 O 4 -doped MWCNTs improved the intensity of pesticide ions more than the other three matrices. Moreover, MWCNTs-based matrix exhibited better performance than organic matrix. Quantitative analysis of pesticides using AP-MALDI-MS was validated to be adequate linearity, repeatability and sensitivity. Overall, AP-MALDI-MS combined with MWCNTs-based matrix enables the directly qualitative and quantitative analysis of pesticides in ambient environment. In this work, a novel atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry (AP-MALDI-MS) method for the facile detection of pesticides was established.

Atmospheric pressure matrix‐assisted laser desorption/ionization mass spectrometry of engine oil additive components

RationaleThe efficiency of lubricants strongly depends on the content of functional additives. In order to assess the chemical and structural changes taking place in the lubricating oil and its additives during operation, it is essential to develop a method for simple and prompt analysis.MethodsTwo single additives as well as a fully formulated engine oil were analysed using an atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI) source coupled to a linear trap quadrupole Orbitrap XL hybrid tandem mass spectrometer and compared with results obtained by means of electrospray ionization (ESI) including additional low‐energy collision‐induced dissociation (LE‐CID). The identification of additives directly from technical surfaces was simulated by using steel substrates as AP‐MALDI targets with varying roughness.ResultsAfter assessment and selection of the most suited AP‐MALDI matrix it was found that pure additives such as calcium sulfonate and zinc dialkyldithiophosphates (ZDDPs) could well be identified with abundant signal intensity based on their elemental composition. Molecular identification was corroborated by LE‐CID in ESI mode. Additionally, additives present in the fully formulated commercial oil such as ZDDPs and salicylates could be reliably identified based on the elemental composition of the deprotonated molecules by means of the Orbitrap unit on different substrates including steel surfaces with high roughness.ConclusionsAP‐MALDI is an efficient technique for determination of lubricant additives directly from commercial oil blends. Identification of additive components was also achieved on steel surfaces with high roughness as applied in tribological systems and thus it is expected that it will be possible to assess additive degradation in real applications, enabling more effective and timely maintenance measures.

Rapid and Quantitative Analysis of Aflatoxin M1 From Milk Using Atmospheric Pressure-Matrix Assisted Laser Desorption/Ionization (AP-MALDI)-Triple Quadrupole Selected Reaction Monitoring.

Aflatoxin M1 (AFM1) is a carcinogenic hydroxylated metabolite commonly found in milk. It is relatively stable toward decontamination procedures posing a major health risk, and it requires an international regulatory mandate of detection at trace levels. To develop a high-throughput, reliable, and compliant method for the identification of AFM1 in milk samples using atmospheric pressure-matrix assisted laser desorption/ionization (AP-MALDI) selected reaction monitoring (SRM) quantitation. The milk sample was diluted in water and cleaned with immunoaffinity chromatography (IAC), followed by analysis using AP-MALDI hyphenated with a triple quadrupole mass spectrometer for SRM. A fast and reliable AP-MALDI SRM quantitative method was developed for the determination of AFM1 with analysis time of 1 min per sample. The diagnostic product ions of AFM1 at 273.1 u and 229.2 u were monitored during the SRM. The calibration curves yielded excellent linearity (R2 = 0.99) with good recoveries for quality control samples (97-106%). The ion ratios of the qualifier to quantifier displayed excellent RSD (1-7.8%) for n = 3. The developed method provided rapid quantification for AFM1. The fast AP-MALDI SRM method can allow analysis of AFM1 in a large number of milk samples. Given the time required for analysis, cost-effectiveness, and superior analytical performance, this method can be adopted in commercial food testing laboratories. Aflatoxins (AF) are a major health risk. Speedy analysis of large sample sizes from food is a risk mitigation strategy but remains an unmet need. Quantitative, chromatography-free, and internal standard-free AP-MALDI SRM based analysis of AF is a high-throughput and cost-efficient alternative. Satisfactory performance was achieved for quantitative AP-MALDI SRM analysis of AFM1 in milk subsequent to a simple sample clean-up step.

Quantitative measurement of pioglitazone in neoplastic and normal tissues by AP-MALDI mass spectrometry imaging

Pioglitazone is a Peroxisome Proliferator-Activated Receptor (PPAR) agonist of the thiazolidinedione class of compounds with promising anticancer activity. An innovative quantitative mass spectrometry imaging (MSI) method and a HPLC-UV method were developed and validated to investigate its distribution in tumor and liver tissues. The MSI method is based on stable isotope normalization and resulted highly specific and sensitive (0.2 pmol/spot). The correct identification of the drug ion signal is confirmed by MS/MS analysis on tissue. The method shows an optimal lateral resolution (25 μm) relying on the ionization efficiency and fine laser diameter of the atmospheric pressure MALDI source. The HPLC-UV method is simple and straightforward involving quick protein precipitation and shows good sensitivity (50ng/sample) using a small starting volume of biological sample. Thus, it is applicable to samples obtained from both preclinical models and clinical surgical procedures. MSI and HPLC-UV assays were validated assessing linearity, intra- and inter-day precision and accuracy, limit of quantification, selectivity and recovery. These are the first methods developed and validated for the analysis of pioglitazone in tissues, and they were applied successfully to myxoid liposarcoma xenograft-bearing mice, which received clinically relevant drug doses. Pioglitazone was measured by either method in sections of tumor and liver 2, 6 and 24 h post-treatment. Drug distribution was relatively homogeneous.

Matrix-Free High-Resolution Atmospheric-Pressure SALDI Mass Spectrometry Imaging of Biological Samples Using Nanostructured DIUTHAME Membranes.

Applications of mass spectrometry imaging (MSI), especially matrix-assisted laser desorption/ionization (MALDI) in the life sciences are becoming increasingly focused on single cell analysis. With the latest instrumental developments, pixel sizes in the micrometer range can be obtained, leading to challenges in matrix application, where imperfections or inhomogeneities in the matrix layer can lead to misinterpretation of MS images. Thereby, the application of premanufactured, homogeneous ionization-assisting devices is a promising approach. Tissue sections were investigated using a matrix-free imaging technique (Desorption Ionization Using Through-Hole Alumina Membrane, DIUTHAME) based on premanufactured nanostructured membranes to be deposited on top of a tissue section, in comparison to the spray-coating of an organic matrix in a MALDI MSI approach. Atmospheric pressure MALDI MSI ion sources were coupled to orbital trapping mass spectrometers. MS signals obtained by the different ionization techniques were annotated using accurate-mass-based database research. Compared to MALDI MSI, DIUTHAME MS images captivated with higher signal homogeneities, higher contrast and reduced background signals, while signal intensities were reduced by about one order of magnitude, independent of analyte class. DIUTHAME membranes, being applicable only on tissue sections thicker than 50 µm, were successfully used for mammal, insect and plant tissue with a high lateral resolution down to 5 µm.

Integration of omics analysis and atmospheric pressure MALDI mass spectrometry imaging reveals the cadmium toxicity on female ICR mouse

Acute cadmium toxicity induces multi-system organ failure. Mass spectrometry (MS)-based omics analyses and atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-MALDI MSI) are powerful tools for characterizing the biomarkers. Many studies on cadmium toxicity by metabolomics have been investigated, whereas the applications of lipidomics and MSI studies are still inadequate. In this study, the systematic metabolomics study on female ICR mice tissues including liver, kidney, heart, stomach, brain as well as spleen under cadmium exposure was firstly conducted and lipidomic characterizations on female ICR mice liver, kidney and heart were further constructed step by step. To deeply understand its toxicological mechanisms, several representative lipids on the mouse liver were visualized by AP-MALDI MSI. The results demonstrated that exposure to cadmium caused significant metabolic alterations in the liver, kidney and heart among all the tissues. Additionally, the toxicological mechanisms of cadmium in the mouse models are closely associated with the inflammation response, energy expenditure, oxidative stress, DNA and mitochondria damage, and lipid homeostasis. These insights could enhance knowledge in acute cadmium toxicity of public health and guide risk assessment in the future.

Metal Ionization in Sub-atmospheric Pressure MALDI Interface: A New Tool for Mass Spectrometry of Volatile Organic Compounds.

A novel approach for the analysis of volatile organic compounds (VOCs) based on chemical ionization by Au+ ions has been proposed. The ionization is carried out in a commercially available dual sub-atmospheric pressure MALDI/ESI interface without any modifications. The Au+ ions are generated by laser ablation of a gold nanolayer with the MALDI laser, and VOCs are infused via the ESI capillary. The ultrahigh resolving power and sub-ppm mass accuracy of the employed mass spectrometer allow straightforward identification of the formed ion-molecule complexes and other products of Au+ interactions with VOCs in the gas phase. The performance of the technique is demonstrated on the analysis of various classes of organic molecules, namely, alkanes, alkenes, alcohols, aldehydes, ketones, aromatic compounds, carboxylic acids, ethers, or organosulfur compounds, expanding the portfolio of currently available methods for the analysis of VOCs such as secondary electrospray ionization, proton-transfer reaction, and selected ion flow tube mass spectrometry.

Microscopic Mass Spectrometry Imaging Reveals the Distribution of Phytochemicals in the Dried Root of Isatis tinctoria.

The dried root of Isatis tinctoria L. (Brassicaceae) is one of the most popular traditional Chinese medicines with well-recognized prevention and treatment effects against viral infections. Above 300 components have been isolated from this herb, but their spatial distribution in the root tissue remains unknown. In recent years, mass spectrometry imaging (MSI) has become a booming technology for capturing the spatial accumulation and localization of molecules in fresh plants, animal, or human tissues. However, few studies were conducted on the dried herbal materials due to the obstacles in cryosectioning. In this study, distribution of phytochemicals in the dried root of Isatis tinctoria was revealed by microscopic mass spectrometry imaging, with application of atmospheric pressure–matrix-assisted laser desorption/ionization (AP-MALDI) and ion trap–time-of-flight mass spectrometry (IT-TOF/MS). After optimization of the slice preparation and matrix application, 118 ions were identified without extraction and isolation, and the locations of some metabolites in the dried root of Isatis tinctoria were comprehensively visualized for the first time. Combining with partial least square (PLS) regression, samples collected from four habitats were differentiated unambiguously based on their mass spectrometry imaging.

Atmospheric Pressure MALDI Mass Spectrometry Imaging Using In-Line Plasma Induced Postionization.

Atmospheric pressure ionization methods confer a number of advantages over more traditional vacuum based techniques, in particular ease of hyphenation to a range of mass spectrometers. For atmospheric pressure matrix assisted desorption/ionization (AP-MALDI), several ion sources, operating in a range of geometries have been reported. Most of these platforms have, to date, generally demonstrated relatively low ion yields and/or poor ion transmission compared to vacuum sources. To improve the detection of certain ions, we have developed a second-generation transmission mode (TM) AP-MALDI imaging platform with in-line plasma postionization using the commercially available SICRIT device, replacing the previously used low temperature plasma probe from our developmental AP-TM-MALDI stage. Both plasma devices produce a significant ionization enhancement for a range of compounds, but the overall higher enhancement obtained by the SICRIT device in addition to the ease of installation and the minimal need for optimization presents this commercially available tool as an attractive method for simple postionization in AP-MALDI MSI.

Advancing Liquid Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Toward Ultrahigh-Throughput Analysis.

Label-free high-throughput screening using mass spectrometry has the potential to provide rapid large-scale sample analysis at a speed of more than one sample per second. Such speed is important for compound library, assay and future clinical screening of millions of samples within a reasonable time frame. Herein, we present a liquid atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) setup for high-throughput large-scale sample analysis (>5 samples per second) for three substance classes (peptides, antibiotics, and lipids). Liquid support matrices (LSM) were used for the analysis of standard substances as well as complex biological fluids (milk). Throughput and analytical robustness were mainly dependent on the complexity of the sample composition and the current limitations of the commercial hardware. However, the ultimate limits of liquid AP-MALDI in sample throughput can be conservatively estimated to be beyond 10–20 samples per second. This level of analytical speed is highly competitive compared with other label-free MS methods, including electrospray ionization and solid state MALDI, as well as MS methods using multiplexing by labeling, which in principle can also be used in combination with liquid AP-MALDI MS.

Bacterial identification by lipid profiling using liquid atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry.

Background In recent years, mass spectrometry (MS) has been applied to clinical microbial biotyping, exploiting the speed of matrix-assisted laser desorption/ionization (MALDI) in recording microbe-specific MS profiles. More recently, liquid atmospheric pressure (AP) MALDI has been shown to produce extremely stable ion flux from homogenous samples and 'electrospray ionization (ESI)-like' multiply charged ions for larger biomolecules, whilst maintaining the benefits of traditional MALDI including high tolerance to contaminants, low analyte consumption and rapid analysis. These and other advantages of liquid AP-MALDI MS have been explored in this study to investigate its potential in microbial biotyping. Methods Genetically diverse bacterial strains were analyzed using liquid AP-MALDI MS, including clinically relevant species such as Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae. Bacterial cultures were subjected to a simple and fast extraction protocol using ethanol and formic acid. Extracts were spotted with a liquid support matrix (LSM) and analyzed using a Synapt G2-Si mass spectrometer with an in-house built AP-MALDI source. Results Each species produces a unique lipid profile in the m/z range of 400-1100, allowing species discrimination. Traditional (solid) MALDI MS produced spectra containing a high abundance of matrix-related clusters and an absence of lipid peaks. The MS profiles of the bacterial species tested form distinct clusters using principle component analysis (PCA) with a classification accuracy of 98.63% using a PCA-based prediction model. Conclusions Liquid AP-MALDI MS profiles can be sufficient to distinguish clinically relevant bacterial pathogens and other bacteria, based on their unique lipid profiles. The analysis of the lipid MS profiles is typically excluded from commercial instruments approved for clinical diagnostics.

High Spatial Resolution Imaging Mass Spectrometry Reveals Chemical Heterogeneity Across Bacterial Microcolonies.

Microbes interact with the world around them at the chemical level. However, directly examining the chemical exchange between microbes and microbes and their environment, at ecological scales, i.e., the scale of a single bacterial cell or small groups of cells, remains a key challenge. Here we address this obstacle by presenting a methodology that enables matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) of bacterial microcolonies. By combining optimized sample preparation with subatmospheric pressure MALDI, we demonstrate that chemical output from groups of as few as ∼50 cells can be visualized with MALDI-IMS. Application of this methodology to Bacillus subtilis and Streptomyces coelicolor revealed heterogeneity in chemical output across microcolonies and asymmetrical metabolite production when cells grew within physiological gradients produced by Medicago sativa roots. Taken together, these results indicate that MALDI-IMS can readily visualize metabolites made by very small assemblages of bacterial cells and that even these small groups of cells can differentially produce metabolites in response to local chemical gradients.

Phospholipid imaging of zebrafish exposed to fipronil using atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry

Mass spectrometry imaging can effectively detect and reflect the information of molecular spatial distribution, and has been widely used for in situ analysis of endogenous or exogenous molecules in organisms. The present work applied the atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry (AP-MALDI-MS) in the imaging of whole zebrafish slices exposed to fipronil. The chemical fingerprints in the range of m/z 600–950 showed significant differences in phospholipids between the fipronil exposed and untreated zebrafish groups. The major perturbed phospholipids were identified as PC(34:2), PC(34:1), PC(34:2)+Na, PC(36:4), PC(38:6), and PS(18:0/22:6), PI(18:0/20:4), PI(18:1/20:4) etc. Our results indicated that the exposure of fipronil obviously affected the phospholipid metabolism of zebrafish, especially of the fish eye region. Our work provides a new method or possibility for toxicological study and related metabolic analysis of pesticides in animals.

Characterizing and alleviating ion suppression effects in atmospheric pressure matrix-assisted laser desorption/ionization.

As a powerful ambient ion source, atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) enables direct analysis at atmospheric pressure/temperature and minimal sample preparation. With the increasing usage of AP-MALDI sources with Orbitrap instruments, systematic characterization of the extent of ion suppression effect (ISE) in AP-MALDI-Orbitrap mass spectrometry imaging (MSI) is desirable. Recently, a new low-pressure MALDI platform has been introduced that reportedly provided better sensitivity. While extensive research efforts have been devoted to improving spatial resolution, fewer studies focused on the characterization and sensitivity improvement of these MALDI platforms that, coupled with high-resolution Orbitraps, provide powerful strategy for MSI. We compared the analytical performance of AP and low-pressure (subatmospheric) MALDI sources to study the effect of pressure control in the ion source. Using a model peptide/protein mixture, we systematically evaluated the factors influencing ISE. Furthermore, the effect of laser spot size was evaluated through tissue imaging analysis of lipids and neuropeptides. The effects of ion suppression and laser spot size have also been examined by comparing the number of identified molecular species during MSI analysis. Several key operating parameters including source pressure, laser energy, laser repetition rate, and microscopic slide coating materials were optimized to minimize the ISE. Under the optimal conditions, the subatmospheric AP-MALDI-Orbitrap platform with high spatial and mass spectral resolution enabled significantly improved coverage of several lipid and neuropeptide families in the MS analysis of mouse brain tissue sections. The new SubAP-MALDI source coupled with an Orbitrap mass spectrometer was established as a viable platform for in situ endogenous biomolecular analysis with increased sensitivity compared with conventional AP-MALDI sources as evidenced by the confident identification of neuropeptides from mouse brain imaging analyses. The alleviated ISE was key to substantial performance improvement due to optimized intermediate pressure conditions and better ion collection by the ion funnel.

Comparison of Vacuum MALDI and AP-MALDI Platforms for the Mass Spectrometry Imaging of Metabolites Involved in Salt Stress in Medicago truncatula.

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is routinely used to determine the spatial distributions of various biomolecules in tissues. Recently, there has been an increased interest in creating higher resolution images using sources with more focused beams. One such source, an atmospheric pressure (AP) MALDI source from MassTech, has a laser capable of reaching spatial resolutions of 10 μm. Here, the AP-MALDI source coupled with a Q Exactive HF Orbitrap platform is compared to the commercial MALDI LTQ Orbitrap XL system using Medicago truncatula root nodules. AP-MALDI parameters, such as the S-lens value, capillary temperature, and spray voltage, were optimized on the Q Exactive-HF platform for optimal detection of plant metabolites. The performance of the two systems was evaluated for sensitivity, spatial resolution, and overall ability to detect plant metabolites. The commercial MALDI LTQ Orbitrap XL was superior regarding the number of compounds detected, as at least two times more m/z were detected compared to the AP-MALDI system. However, although the AP-MALDI source requires a spatial resolution higher than 10 μm to get the best signal, the spatial resolution at 30 μm is still superior compared to the 75 μm spatial resolution achieved on the MALDI platform. The AP-MALDI system was also used to investigate the metabolites present in M. truncatula roots and root nodules under high salt and low salt conditions. A discriminative analysis with SCiLS software revealed m/z ions specific to the control and salt conditions. This analysis revealed 44 m/z ions present at relatively higher abundances in the control samples, and 77 m/z enriched in the salt samples. Liquid chromatography-tandem MS was performed to determine the putative molecular identities of some of the mass ions enriched in each sample, including, asparagine, adenosine, and nicotianamine in the control samples, and arginine and soyasaponin I in the salt treated samples.