Matrix-assisted Laser Desorption Electrospray Ionization Research Articles

Incorporation of Three Different Optical Trains into the IR-MALDESI Mass Spectrometry Imaging Platform to Characterize Artemisia annua.

Artemisinin is the leading medication for the treatment of malaria and is only produced naturally in Artemisia annua. The localization of artemisinin in both the glandular and non-glandular trichomes of the plant makes it an ideal candidate for mass spectrometry imaging (MSI) as a model system for method development. Infrared matrix-assisted laser desorption electrospray ionization MSI (IR-MALDESI-MSI) has the capability to detect hundreds to thousands of analytes simultaneously, providing abundance information in conjunction with species localization throughout a sample. The development of several new optical trains and their application to the IR-MALDESI-MSI platform has improved data quality in previous proof-of-concept experiments but has not yet been applied to analysis of native biological samples, especially the MSI analysis of plants. This study aimed to develop a workflow and optimize MSI parameters, specifically the laser optical train, for the analysis of Artemisia annua with the NextGen IR-MALDESI platform coupled to an Orbitrap Exploris 240 mass spectrometer. Two laser optics were compared to the conventional set up, of which include a Schwarzschild-like reflective objective and a diffractive optical element (DOE). These optics, respectively, enhance the spatial resolution of imaging experiments or create a square spot shape for top-hat imaging. Ultimately, we incorporated and characterized three different optical trains into our analysis of Artemisia annua to study metabolites in the artemisinin pathway. These improvements in our workflow, resulted in high spatial resolution and improved ion abundance from previous work, which will allow us to address many different questions in plant biology beyond this model system.

Imaging with mass spectrometry: Which ionization technique is best?

The use of mass spectrometry (MS) to acquire molecular images of biological tissues and other substrates has developed into an indispensable analytical tool over the past 25 years. Imaging mass spectrometry technologies are widely used today to study the in situ spatial distributions for a variety of analytes. Early MS images were acquired using secondary ion mass spectrometry and matrix-assisted laser desorption/ionization. Researchers have also designed and developed other ionization techniques in recent years to probe surfaces and generate MS images, including desorption electrospray ionization (DESI), nanoDESI, laser ablation electrospray ionization, and infrared matrix-assisted laser desorption electrospray ionization. Investigators now have a plethora of ionization techniques to select from when performing imaging mass spectrometry experiments. This brief perspective will highlight the utility and relative figures of merit of these techniques within the context of their use in imaging mass spectrometry.

A statistical approach to system suitability testing for mass spectrometry imaging.

Mass spectrometry imaging (MSI) elevates the power of conventional mass spectrometry (MS) to multidimensional space, elucidating both chemical composition and localization. However, the field lacks any robust quality control (QC) and/or system suitability testing (SST) protocols to monitor inconsistencies during data acquisition, both of which are integral to ensure the validity of experimental results. To satisfy this demand in the community, we propose an adaptable QC/SST approach with five analyte options amendable to various ionization MSI platforms (e.g., desorption electrospray ionization, matrix-assisted laser desorption/ionization [MALDI], MALDI-2, and infrared matrix-assisted laser desorption electrospray ionization [IR-MALDESI]). A novel QC mix was sprayed across glass slides to collect QC/SST regions-of-interest (ROIs). Data were collected under optimal conditions and on a compromised instrument to construct and refine the principal component analysis (PCA) model in R. Metrics, including mass measurement accuracy and spectral accuracy, were evaluated, yielding an individual suitability score for each compound. The average of these scores is utilized to inform if troubleshooting is necessary. The PCA-based SST model was applied to data collected when the instrument was compromised. The resultant SST scores were used to determine a statistically significant threshold, which was defined as 0.93 for IR-MALDESI-MSI analyses. This minimizes the type-I error rate, where the QC/SST would report the platform to be in working condition when cleaning is actually necessary. Further, data scored after a partial cleaning demonstrate the importance of QC and frequent full instrument cleaning. This study is the starting point for addressing an important issue and will undergo future development to improve the efficiency of the protocol. Ultimately, this work is the first of its kind and proposes this approach as a proof of concept to develop and implement universal QC/SST protocols for a variety of MSI platforms.

Evaluating the optimal tissue thickness for mass spectrometry imaging using infrared matrix-assisted laser desorption electrospray ionization.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) utilizes a 2970 nm mid-IR laser to desorb samples with depth resolutions (Z) on the order of micrometers. Conventionally, 5-20 μm thick tissue sections are used to characterize different applications of the IR-MALDESI source, but an optimal thickness has not been systematically investigated. Mouse liver was sectioned to various thicknesses and analyzed using IR-MALDESI mass spectrometry imaging (MSI). Height profiles of tissue sections of various cryosectioned thicknesses were acquired to affirm tissue thickness. Tissue sections of each thickness were measured using a Keyence microscope. Paraffin wax was cryosectioned, mounted on microscope slides, and measured using a chromatic confocal sensor system to determine the cryostat sectioning accuracy. Analyzing sectioned tissues at higher thickness (>10μm) leads to lower ion abundance, a decrease in signal over long analysis times, and more frequent instrument cleaning. Additionally, increasing tissue thickness above the optimum (7μm) does not result in a significant increase in lipid annotations. This work defines an optimal sample thickness for IR-MALDESI-MSI and demonstrates the utility of optimizing tissue thickness for MSI platforms of comparable Z resolution.

Comparative analysis of sucrose-embedding for whole-body zebrafish MSI by IR-MALDESI.

Infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging (IR-MALDESI) conventionally utilizes fresh-frozen biological tissues with an ice matrix to improve the detection of analytes. Sucrose-embedding with paraformaldehyde fixation has demonstrated feasibility as an alternative matrix for analysis by IR-MALDESI by preserving tissue features and enhancing ionization of lipids. However, investigating multi-organ systems provides broader context for a biological study and can elucidate more information about a disease state as opposed to a single organ. Danio rerio, or zebrafish, are model organisms for various disease states and can be imaged as a multi-organ sample to analyze morphological and metabolomic preservation as a result of sample preparation. Herein, whole-body zebrafish were imaged to compare sucrose-embedding with paraformaldehyde fixation against conventional fresh-frozen sample preparation. Serial sections were analyzed with and without an ice matrix to evaluate if sucrose functions as an alternative energy-absorbing matrix for IR-MALDESI applications across whole-body tissues. The resulting four conditions were compared in terms of total putative lipid annotations and category diversity, coverage across the entire m/z range, and ion abundance. Ultimately, sucrose-embedded zebrafish had an increase in putative lipid annotations for the combination of putative annotations with and without the application of an ice matrix relative to fresh-frozen tissues which require the application of an ice matrix. Upon the use of an ice matrix, a greater number of high mass putative lipid annotations (e.g., glycerophospholipids, glycerolipids, and sphingolipids) were identified. Conversely, without an ice matrix, sucrose-embedded sections elucidated more putative annotations in lower molecular weight lipids, including fatty acyls and sterol lipids. Similar to the mouse brain model, sucrose-embedding increased putative lipid annotation and abundance for whole-body zebrafish.

Feasibility, Acceptability and Appropriateness of MedViewer: A Novel Hair-Based Antiretroviral Real-Time Clinical Monitoring Tool Providing Adherence Feedback to Patients and Their Providers.

Antiretroviral therapy (ART) adherence is key to achieving viral load suppression and ending the HIV epidemic but monitoring and supporting adherence using current interventions is challenging. We assessed the feasibility, acceptability and appropriateness of MedViewer(MV), a novel intervention that provides real-time adherence feedback for patients and providers using infra-red matrix-assisted laser desorption electrospray ionization (IR-MALDESI) for mass spectrometry imaging of daily ART concentrations in patients' hair. We used mixed methods to feasibility test MV at a busy Infectious Diseases (ID) clinic, enrolling 16 providers and 36 patients. Providers underwent standardized training; patients and providers watched an 8-min informational video about MV. We collected patient and provider data at baseline and within 24h of clinic visits and, with patients, approximately 1month after clinic visits. MedViewer was feasible, liked by patients and providers, and perceived to help facilitate adherence conversations and motivate patients to improve adherence. Trial Registration: NCT04232540.

Application of infrared matrix-assisted laser desorption electrospray ionization mass spectrometry for morphine imaging in brain tissue

Here, we present a method developed for the analysis of spatial distributions of morphine in mouse brain tissue using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) coupled to a Q Exactive Plus mass spectrometer. The method is also capable of evaluating spatial distributions of the antiretroviral drug abacavir. To maximize sensitivity to morphine, we analyze various Orbitrap mass spectrometry acquisition modes utilizing signal abundance and frequency of detection as evaluation criteria. We demonstrate detection of morphine in mouse brain and establish that the selected ion monitoring mode provides 2.5 times higher sensitivity than the full-scan mode. We find that distributions of morphine and abacavir are highly correlated with the Pearson correlation coefficient R = 0.87. Calibration showed that instrument response is linear up to 40 pg/mm2 (3.8 μg/g of tissue).Graphical abstract

Enhanced Detection of Charged N-Glycans in the Brain by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometric Imaging.

N-linked glycosylation represents a structurally diverse, complex, co- and posttranslational protein modification that bridges metabolism and cellular signaling. Consequently, aberrant protein glycosylation is a hallmark of most pathological scenarios. Due to their complex nature and non-template-driven synthesis, the analysis of glycans is faced with several challenges, underlining the need for new and improved analytical technologies. Spatial profiling of N-glycans through direct imaging on tissue sections reveals the regio-specific and/or disease pathology correlating tissue N-glycans that serve as a disease glycoprint for diagnosis. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a soft hybrid ionization technique that has been used for diverse mass spectrometry imaging (MSI) applications. Here, we report the first spatial analysis of the brain N-linked glycans by IR-MALDESI MSI, leading to a significant increase in the detection of the brain N-sialoglycans. A formalin-fixed paraffin-embedded mouse brain tissue was analyzed in negative ionization mode after tissue washing, antigen retrieval, and pneumatic application of PNGase F for enzymatic digestion of N-linked glycans. We report a comparative analysis of section thickness on the N-glycan detection using IR-MALDESI. One hundred thirty-six unique N-linked glycans were confidently identified in the brain tissue (with an additional 132 unique N-glycans, not reported in GlyConnect), where more than 50% contained sialic acid residues, which is approximately 3-fold higher than the previous reports. This work demonstrates the first application of IR-MALDESI in N-linked glycan imaging of the brain tissue, leading to a 2.5-fold increase in the in situ total brain N-glycan detection compared to the current gold standard of positive-mode matrix-assisted laser desorption/ionization mass spectrometry imaging. This is also the first report of the application of the MSI toward the identification of sulfoglycans in the rodent brain. Overall, IR-MALDESI-MSI presents a sensitive glycan detection platform to identify tissue-specific and/or disease-specific glycosignature in the brain while preserving the sialoglycans without any chemical derivatization.

Mass spectrometry imaging of hair identifies daily maraviroc adherence in HPTN 069/ACTG A5305.

Objective measures of adherence for antiretrovirals used as pre-exposure prophylaxis (PrEP) are critical for improving preventative efficacy in both clinical trials and real-world application. Current objective adherence measures either reflect only recent behavior (eg days for plasma or urine) or cumulative behavior (eg months for dried blood spots). Here, we measured the accumulation of the antiretroviral drug maraviroc (MVC) in hair strands by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) to evaluate adherence behavior longitudinally at high temporal resolution. An MSI threshold for classifying daily adherence was established using clinical samples from healthy volunteers following directly observed dosing of 1 to 7 doses MVC/week. We then used the benchmarked MSI assay to classify adherence to MVC-based PrEP regimens in hair samples collected throughout the 48-week HPTN069/ACTGA5305 study. We found that only ~32% of investigated hair samples collected during the study's active dosing period showed consistent daily PrEP adherence throughout a retrospective period of 30 days, and also found that profiles of daily individual adherence from MSI hair analysis could identify when patients were and were not taking study drug. The assessment of adherence from MSI hair strand analysis was 62% lower than adherence classified using paired plasma samples, the latter of which may be influenced by white-coat adherence. These findings demonstrate the ability of MSI hair analysis to examine daily variability of adherence behavior over a longer-term measurement and offer the potential for longitudinal comparison with risk behavior to target patient-specific adherence interventions and improve outcomes.

Automatic z-Axis Correction for IR-MALDESI Mass Spectrometry Imaging of Uneven Surfaces.

Two-dimensional mass spectrometry imaging (2D MSI) experiments mainly involve samples with a flat surface and constant thickness, but some samples are challenging to section due to the texture and topography. Herein, we present an MSI method that automatically corrects for discernible height differences across surfaces during imaging experiments. A chromatic confocal sensor was incorporated into the infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) system to measure the sample surface height at the location of each analytical scan. The height profile is subsequently used for adjusting the z-axis position of the sample during MSI data acquisition. We evaluated this method using a tilted mouse liver section and an unsectioned Prilosec tablet due to their exterior quasi-homogeneity and height differences of approximately ∼250 μm. MSI with automatic z-axis correction showed consistent ablated spot sizes and shapes, revealing the measured ion spatial distribution across a mouse liver section and a Prilosec tablet. Conversely, irregular spots and reduced signals with large variability were observed when no z-axis correction was applied.

Predicting Sialic Acid Content of N-Linked Glycans Using the Isotopic Pattern of Chlorine.

Sialic acids play several roles in both physiological and pathological processes; however, due to their labile nature, they are difficult to analyze using mass spectrometry. Previous work has shown that infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is able to detect intact sialylated N-linked glycans without the use of chemical derivatization. In this work, we describe a new rule that can predict the number of sialic acids on a glycan. Formalin-fixed paraffin-embedded human kidney tissue was prepared using previously established methods and analyzed using IR-MALDESI in negative-ion mode mass spectrometry. Using the experimental isotopic distribution of a detected glycan, we can predict the number of sialic acids on the glycan; #sialic acids is equal to the charge state minus the number of chlorine adducts, or z - #Cl-. This new rule grants confident glycan annotations and compositions beyond accurate mass measurements, thereby further improving the capability of IR-MALDESI to study sialylated N-linked glycans within biological tissues.

New Platform for Label-Free, Proximal Cellular Pharmacodynamic Assays: Identification of Glutaminase Inhibitors Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry.

Cellular pharmacodynamic assays are crucial aspects of lead optimization programs in drug discovery. These assays are sometimes difficult to develop, oftentimes distal from the target and frequently low throughput, which necessitates their incorporation in the drug discovery funnel later than desired. The earlier direct pharmacodynamic modulation of a target can be established, the fewer resources are wasted on compounds that are acting via an off-target mechanism. Mass spectrometry is a versatile tool that is often used for direct, proximal cellular pharmacodynamic assay analysis, but liquid chromatography-mass spectrometry methods are low throughput and are unable to fully support structure-activity relationship efforts in early medicinal chemistry programs. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is an ambient ionization method amenable to high-throughput cellular assays, capable of diverse analyte detection, ambient and rapid laser sampling processes, and low cross-contamination. Here, we demonstrate the capability of IR-MALDESI for the detection of diverse analytes directly from cells and report the development of a high-throughput, label-free, proximal cellular pharmacodynamic assay using IR-MALDESI for the discovery of glutaminase inhibitors and a biochemical assay for hit confirmation. We demonstrate the throughput with a ∼100,000-compound cellular screen. Hits from the screening were confirmed by retesting in dose-response with mass spectrometry-based cellular and biochemical assays. A similar workflow can be applied to other targets with minimal modifications, which will speed up the discovery of cell active lead series and minimize wasted chemistry resources on off-target mechanisms.

Lipidomic Analysis of Mouse Brain to Evaluate the Efficacy and Preservation of Different Tissue Preparatory Techniques by IR-MALDESI-MSI

Numerous preparatory methods have been developed to preserve the cellular and structural integrity of various biological tissues for different -omics studies. Herein, two preparatory methods for mass spectrometry imaging (MSI) were evaluated, fresh-frozen and sucrose-embedded, paraformaldehyde (PFA) fixed, in terms of ion abundance, putative lipid identifications, and preservation of analyte spatial distributions. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI)-MSI was utilized to compare the preparatory methods of interest with and without the use of the conventional ice matrix. There were 2.5-fold and 1.6-fold more lipid species putatively identified in positive- and negative-ion modes, respectively, for sucrose-embedded, PFA-fixed tissues without an ice matrix relative to the current IR-MALDESI-MSI gold-standard, fresh-frozen tissue preparation with an exogenous ice matrix. Furthermore, sucrose-embedded tissues demonstrated improved spatial distribution of ions resulting from the cryo-protective property of sucrose and paraformaldehyde fixation. Evidence from these investigations supports sucrose-embedding without ice matrix as an alternative preparatory technique for IR-MALDESI-MSI.

3D printer platform and conductance feedback loop for automated imaging of uneven surfaces by liquid microjunction-surface sampling probe mass spectrometry.

Molecular imaging of samples using mass spectrometric techniques, such as matrix-assisted laser desorption ionization or desorption electrospray ionization, requires the sample surface to be even/flat and sliced into thin sections (c. 10μm). Furthermore, sample preparation steps can alter the analyte composition of the sample. The liquid microjunction-surface sampling probe (LMJ-SSP) is a robust sampling interface that enables surface profiling with minimal sample preparation. In conjunction with a conductance feedback system, the LMJ-SSP can be used to automatically sample uneven specimens. A sampling stage was built with a modified 3D printer where the LMJ-SSP is attached to the printing head. This setup can scan across flat and even surfaces in a predefined pattern ("static sampling mode"). Uneven samples are automatically probed in "conductance sampling mode" where an electric potential is applied and measured at the probe. When the probe contacts the electrically grounded sample, the potential at the probe drops, which is used as a feedback signal to determine the optimal position of the probe for sampling each location. The applicability of the probe/sensing system was demonstrated by first examining the strawberry tissue using the "static sampling mode." Second, porcine tissue samples were profiled using the "conductance sampling mode." With minimal sample preparation, an area of 11 × 15 mm was profiled in less than 2h. From the obtained results, adipose areas could be distinguished from non-adipose parts. The versatility of the approach was further demonstrated by directly sampling the bacteria colonies on agar and resected human kidney (intratumoral hemorrhage) specimens with thicknesses ranging from 1 to 4mm. The LMJ-SSP in conjunction with a conductive feedback system is a powerful tool that allows for fast, reproducible, and automated assessment of uneven surfaces with minimal sample preparation. This setup could be used for perioperative assessment of tissue samples, food screening, and natural product discovery, among others.

Quasi-continuous infrared matrix-assisted laser desorption electrospray ionization source coupled to a quadrupole time-of-flight mass spectrometer for direct analysis from well plates.

High-throughput screening (HTS) is a technique mostly used by pharmaceutical companies to rapidly screen multiple libraries of compounds to find drug hits with biological or pharmaceutical activity. Mass spectrometry (MS) has become a popular option for HTS given that it can simultaneously resolve hundreds to thousands of compounds without additional chemical derivatization. For this application, it is convenient to do direct analysis from well plates. Herein, we present the development of an infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) source coupled directly to an Agilent 6545 for direct analysis from well plates. The source is coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometer to take advantage of the high acquisition rates without sacrificing resolving power as required with Orbitrap or Fourier-transform ion cyclotron resonance (FTICR) instruments. The laser used for this source operates at 100 Hz, firing 1 pulse-per-burst, and delivers around 0.7 mJ per pulse. Continuously firing this laser for an extended duration makes it a quasi-continuous ionization source. Additionally, a metal capillary was constructed to extend the inlet of the mass spectrometer, increase desolvation of electrospray charged droplets, improve ion transmission, and increase sensitivity. Its efficiency was compared with the conventional dielectric glass capillary by measured signal and demonstrated that the metal capillary increased ionization efficiency due to its more uniformly distributed temperature gradient. Finally, we present the functionality of the source by analyzing tune mix directly from well plates. This source is a proof of concept for HTS applications using IR-MALDESI coupled to a different MS platform.

High-Throughput Deconvolution of Intact Protein Mass Spectra for the Screening of Covalent Inhibitors.

Deconvolution from intact protein mass-to-charge spectra to mass spectra is essential to generate interpretable data for mass spectrometry (MS) platforms coupled to ionization sources that produce multiply charged species. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) can be used to analyze intact proteins in multiwell microtiter plates with speed matching small molecule analyses (at least 1 Hz). However, the lack of compatible deconvolution software has limited its use in high-throughput screening applications. Most existing automated deconvolution software packages work best for data generated from LC-MS, and to the best of our knowledge, there is no software capable of performing fast plate-based mass spectral deconvolution. Herein we present the use of a new workflow in ProSight Native for the deconvolution of protein spectra from entire well plates that can be completed within 3 s. First, we successfully demonstrated the potential increased throughput benefits produced by the combined IR-MALDESI-MS - ProSight Native platform using protein standards. We then conducted a screen for Bruton's tyrosine kinase (BTK) covalent binders against a well-annotated compound collection consisting of 2232 compounds and applied ProSight Native to deconvolute the protein spectra. Seventeen hits including five known BTK covalent inhibitors in the compound set were identified. By alleviating the data processing bottleneck using ProSight Native, it may be feasible to analyze and report covalent screening results for >200,000 samples in a single day.

Interrogating the Metabolomic Profile of Amyotrophic Lateral Sclerosis in the Post-Mortem Human Brain by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) Mass Spectrometry Imaging (MSI).

Amyotrophic lateral sclerosis (ALS) is an idiopathic, fatal neurodegenerative disease characterized by progressive loss of motor function with an average survival time of 2-5 years after diagnosis. Due to the lack of signature biomarkers and heterogenous disease phenotypes, a definitive diagnosis of ALS can be challenging. Comprehensive investigation of this disease is imperative to discovering unique features to expedite the diagnostic process and improve diagnostic accuracy. Here, we present untargeted metabolomics by mass spectrometry imaging (MSI) for comparing sporadic ALS (sALS) and C9orf72 positive (C9Pos) post-mortem frontal cortex human brain tissues against a control cohort. The spatial distribution and relative abundance of metabolites were measured by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MSI for association to biological pathways. Proteomic studies on the same patients were completed via LC-MS/MS in a previous study, and results were integrated with imaging metabolomics results to enhance the breadth of molecular coverage. Utilizing METASPACE annotation platform and MSiPeakfinder, nearly 300 metabolites were identified across the sixteen samples, where 25 were identified as dysregulated between disease cohorts. The dysregulated metabolites were further examined for their relevance to alanine, aspartate, and glutamate metabolism, glutathione metabolism, and arginine and proline metabolism. The dysregulated pathways discussed are consistent with reports from other ALS studies. To our knowledge, this work is the first of its kind, reporting on the investigation of ALS post-mortem human brain tissue analyzed by multiomic MSI.

Next-Generation Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Source for Mass Spectrometry Imaging and High-Throughput Screening.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a hybrid, ambient ionization source that combines the advantages of electrospray ionization and matrix-assisted laser desorption/ionization, making it a versatile tool for both high-throughput screening (HTS) and mass spectrometry imaging (MSI) studies. To expand the capabilities of the IR-MALDESI source, an entirely new architecture was designed to overcome the key limitations of the previous source. This next-generation (NextGen) IR-MALDESI source features a vertically mounted IR-laser, a planar translation stage with computerized sample height control, an aluminum enclosure, and a novel mass spectrometer interface plate. The NextGen IR-MALDESI source has improved user-friendliness, improved overall versatility, and can be coupled to numerous Orbitrap mass spectrometers to accommodate more research laboratories. In this work, we highlight the benefits of the NextGen IR-MALDESI source as an improved platform for MSI and direct analysis. We also optimize the NextGen MALDESI source component geometries to increase target ion abundances over a wide m/z range. Finally, documentation is provided for each NextGen IR-MALDESI part so that it can be replicated and incorporated into any lab space.

High-Throughput Intact Protein Analysis for Drug Discovery Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry.

Mass spectrometry (MS) is the primary analytical tool used to characterize proteins within the biopharmaceutical industry. Electrospray ionization (ESI) coupled to liquid chromatography (LC) is the current gold standard for intact protein analysis. However, inherent speed limitations of LC/MS prevent analysis of large sample numbers (>1000) in a day. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI-MS), an ambient ionization MS technology, has recently been established as a platform for high-throughput small molecule analysis. Here, we report the applications of such a system for the analysis of intact proteins commonly performed within the drug discovery process. A wide molecular weight range of proteins 10-150 kDa was detected on the system with improved tolerance to salts and buffers compared to ESI. With high concentrations and model proteins, a sample rate of up to 22 Hz was obtained. For proteins at low concentrations and in buffers used in commonly employed assays, robust data at a sample rate of 1.5 Hz were achieved, which is ∼22× faster than current technologies used for high-throughput ESI-MS-based protein assays. In addition, two multiplexed plate-based high-throughput sample cleanup methods were coupled to IR-MALDESI-MS to enable analysis of samples containing excessive amounts of salts and buffers without fully compromising productivity. Example experiments, which leverage the speed of the IR-MALDESI-MS system to monitor NISTmAb reduction, protein autophosphorylation, and compound binding kinetics in near real time, are demonstrated.

Developing transmission mode for infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging.

RationaleThe development and characterization of the novel NextGen infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI) source catalyzed new advancements in IR‐MALDESI instrumentation, including the development of a new analysis geometry.MethodsA vertically oriented transmission mode (tm)‐IR‐MALDESI setup was developed and optimized on thawed mouse tissue. In addition, glycerol was introduced as an alternative energy‐absorbing matrix for tm‐IR‐MALDESI because the new geometry does not currently allow for the formation of an ice matrix. The tm geom was evaluated against the optimized standard geometry for the NextGen source in reflection mode (rm).ResultsIt was found that tm‐IR‐MALDESI produces comparable results to rm‐IR‐MALDESI after optimization. The attempt to incorporate glycerol as an alternative matrix provided little improvement to tm‐IR‐MALDESI ion abundances.ConclusionsThis work has successfully demonstrated the adaptation of the NextGen IR‐MALDESI source through the feasibility of tm‐IR‐MALDESI mass spectrometry imaging on mammalian tissue, expanding future biological applications of the method.