Matrix-Assisted Laser Desorption/Ionization Imaging - Mass Spectrometry (MALDI-MS): revolutionizing scientific research by automating sample preparation.

Mass spectrometry (MS) is a powerful analytical technique that has revolutionized the way scientists study and understand organic compounds. It has the remarkable ability to perform label-free detection of analytes (as opposed to classic microscopy imaging), and more importantly, have the capability to monitor thousands of molecules in a single experiment. MS enables researchers to determine the molecular mass and chemical structure of various analytes within a sample, ranging from proteins in proteomic research to genetic targets in genomics. Mass spectrometry remarkable power and diverse applicability provide unique opportunities for supporting drug discovery and development efforts throughout the drug development life cycle, including drug discovery, development, quality monitoring and manufacturing.

As part of the Hamilton family, BioFluidix provides the most precise and accurate liquid dispenser technology. With unique solutions tailored to your application, you can automate your workflows. Liquid-independent, contactless, production-optimized, and limitless. Precise sample preparation or reproducible matrix application are just a few example applications of our flexible non-contact dispensing technology.

Mass spectrometry begins with the process of ionization, in which the molecules composing the sample are converted into ions. The specific method used for ionization can vary, depending on the analytes and chosen MS analysis technique.


In this exploration of MS and automation, we will focus on Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS). This soft ionization method is suitable for analyzing large biomolecules like proteins, peptides, nucleic acids, and other high-molecular-weight compounds, and is widely used in proteomics and biomolecular research. MALDI-MS has become increasingly important in the last decades due to its significant impact on scientific research (eg, tissue imaging) and clinical diagnostics.

UNDERSTANDING MALDI

Efficient sample preparation is a crucial step in performing MALDI-MS projects. Samples are prepared by co-crystallizing them with a matrix, typically composed of small acidic molecules. The matrix solution mixed with the sample is spotted or sprayed onto a MALDI plate, which facilitates the crystallization of the sample-matrix mixture. MALDI plates come in various formats, including stainless steel plates with sample spots or wells, and are specifically designed to accommodate different types of samples. The matrix absorbs ultraviolet light (emitted by a laser) and transforms it into heat energy, resulting in the desorption and ionization of the sample. Therefore, when a laser beam is directed at these matrix-embedded samples, the energy absorbed by the matrix causes the analyte to vaporize and become ionized.
Once the sample is ionized, the generated ions are accelerated using an electric field. This ensures that all ions, irrespective of their size, have the same kinetic energy as they enter the TOF (Time-of-Flight) mass analyzer.
In the TOF mass analyzer, ions travel down a "flight tube." The time taken for ions to traverse this tube is measured. Lighter ions (or ions with higher charge) will reach the detector faster than heavier ions. Because all ions started with the same kinetic energy, the time taken (or "time of flight") to reach the detector will be directly proportional to the square root of their mass-to-charge ratio (m/z).
At the end of the flight tube, there is a detector that measures the time each ion takes to arrive. This information is used to determine the m/z ratio for each ion.
A mass spectrum is a plot with m/z values on the x-axis and ion abundance (intensity) on the y-axis. Peaks on a mass spectrum represent ions of different m/z ratios, with the height of each peak indicating the relative abundance of that ion. This technique can provide vital information about the molecular weight of compounds in a sample. In some cases, when a molecule is fragmented into several ions, the spectrum can also offer clues about the molecule's structure.

Other Common Ionization Methods

Electron Ionization (EI): a vaporized sample is exposed to a high-energy electron beam (typically around 70 eV). This beam strips electrons from the sample molecules, resulting in the creation of positively charged radical species. The molecular ion formed is typically unstable and can fragment into smaller ions.

Suitable for small, volatile, and nonpolar compounds.

Chemical Ionization (CI): the sample is introduced into a chamber filled with an excess of reagent gas, like methane. Electrons ionize the reagent gas, forming a plasma containing species such as CH5+, which then reacts with the sample to form the pseudomolecular ion [M+H]+. CI can also operate in negative mode to generate anions by using different reagent gases.

Effective for less volatile and polar compounds, especially for compounds that are difficult to ionize with EI.

Atmospheric Pressure Chemical Ionization (APCI): APCI involves delivering the sample as a neutral spray, which is then ionized by corona discharge, producing ions.

Well-suited for analyzing low molecular weight, nonpolar species that may be challenging to analyze by other methods. It is also used for environmental analysis and drug metabolite studies.

Field Ionization/Desorption: in Field Ionization (FI) molecules undergo ionization when their electrons are removed by tunneling in a strong electric field. It's typically used for nonpolar or slightly polar organic compounds. In Field Desorption (FD), a high electric field is applied to an emitter's sharp surface, leading to ionization of gaseous analyte molecules. Low-vapor pressure materials are desorbed and ionized by alkali metal cation attachment upon emitter heating.

Primarily used for studying organic compounds, especially aromatic and high-vapor-pressure analytes.

Fast Atom Bombardment (FAB): operates by directing a beam of accelerated atoms, typically Ar or Xe, with kilovolt energies, onto a sample placed in a vacuum. The sample is typically mixed with a matrix, and the ions generated by FAB become adducts to the target molecules.

Historically used for polar and nonvolatile compounds, but it has been largely replaced by more modern techniques due to their greater sensitivity.

Electrospray Ionization (ESI): is a method for generating ions from liquid samples, typically in solution. In ESI, a high voltage is applied to a fine capillary tube through which the liquid sample is pumped. This strong electrical field at the tip of the capillary causes the liquid to break up into tiny, charged droplets. As these droplets evaporate, they leave behind charged ions of the sample molecules, which can then be analyzed using a mass spectrometer.

Ideal for large and complex molecules, such as proteins, peptides, and biomolecules, making it commonly used in proteomics and pharmaceutical research.

Matrix Assisted Laser Desorption Ionization (MALDI): Involves uniformly mixing the sample with a substantial quantity of matrix material. The matrix absorbs ultraviolet light and transforms it into heat energy, resulting in the desorption and ionization of the sample.

Suitable for analyzing large biomolecules like proteins, peptides, nucleic acids, and other high-molecular-weight compounds, and is widely used in proteomics and biomolecular research.

COMMON APPLICATIONS OF MALDI-TOF MS

Pathogen Diagnosis

Accurate pathogen diagnosis for effective treatment.

Accurate pathogen diagnosis is critical for effective treatment. Traditional methods like gene sequencing are time-consuming. MALDI-TOF MS is an efficient technique for identifying pathogens using protein and peptide profiles, enabling the differential identification of various microorganisms. This method is particularly useful for yeast, Mycobacterium tuberculosis, fungi, and even virus detection. However, its accuracy relies on comprehensive databases. Despite some limitations, MALDI-TOF MS proves invaluable in clinical diagnostics and research, providing rapid, accurate identification across microbial and viral species, including Hepatitis B and C, and monitoring influenza A mutations.

Dereplication

A powerful tool for identifying and categorizing microorganisms.

MALDI-TOF MS can differentiate closely related strains, species, and subspecies based on protein fingerprints. This technology has been effective in various fields, including clinical microbiology, environmental studies, and food science, enabling precise strain identification and insights into microbial diversity and taxonomy.

Biomarker Detection for Disease Diagnosis

Overcoming False Positives/Negatives: Refining Diagnostic Accuracy

By diluting the fluid and mixing it with a matrix, mass spectrometry characterizes biomolecules, providing valuable insights into physiological and pathological changes. Proteomic tools like MALDI-TOF/TOF and 2D electrophoresis have uncovered disease-specific protein expression profiles, enabling the identification of predictive biomarkers. MALDI-TOF MS Imaging (MSI) has also been instrumental in diagnosing diseases, such as prostate cancer, by revealing tissue-specific lipid metabolism differences. In summary, mass spectrometry-based approaches, particularly MALDI-TOF/MS, offer greater accuracy, transforming the detection of disease biomarkers and improving diagnostics and personalized treatment strategies for various diseases, including cancer.

Lipid Profiling

MALDI-TOF MS enables the identification and characterization of lipids in diverse organisms.

This technique aids in microorganism differentiation, structural analysis of lipids, and profiling specific lipid classes. It has been instrumental in exploring lipid-related markers for diseases and environmental studies. MALDI-TOF MS, with its sensitivity and high throughput, is a valuable asset in microbiology, proteomics, and lipidomics research, providing key insights into complex biological systems.

Proteomics and Metabolomics

MALDI-TOF MS: unraveling the intricate biochemical processes within organisms.

In proteomics, MALDI-TOF MS expedites protein identification, facilitates high-throughput screening, aids stress response studies, and delves into metabolic pathways. For metabolomics, MALDI-TOF MS enables the screening and profiling of metabolites, particularly in bioremediation and pigment analysis, while mapping metabolic pathways. This technology significantly contributes to our understanding of complex biological phenomena, fostering advancements in environmental and biological research.

Understanding MALDI-TOF MS challenges

MALDI-TOF MS, is a cutting-edge technology that has significantly advanced the field of analytical chemistry.
This technique is used for the analysis of large biomolecules, such as proteins, peptides, nucleic acids, and other high-molecular-weight compounds. Sample preparation is a crucial step to ensure accurate and reproducible results. The precise dosing technologies of BioFluidix support scientific researchers in optimally leveraging the advantages of MALDI-TOF MS. With flexible applicability, precise and reliable dosing of samples and matrices, and intuitive programming of devices, substrate independent processes can be developed quickly and effectively.

Important Steps in MALDI-TOF Sample Preparation

Sample selection: Choose a suitable sample that can be ionized by MALDI. Common samples include peptides, proteins, DNA, and synthetic polymers.

Matrix Selection: Select an appropriate matrix material. Common matrices are small organic molecules, such as sinapinic acid or α-cyano-4-hydroxycinnamic acid. The matrix helps desorb and ionize the analyte molecules when struck by a laser.

Matrix Preparation: Prepare a concentrated solution of the matrix in a suitable solvent. The matrix solution should be applied to the sample in a way that forms a thin, uniform layer. This can be done by mixing the matrix solution with the sample, spotting it onto a target plate, and allowing it to dry.

Sample Matrix Co-crystallization: The mixture of the sample and matrix is ​​allowed to co-crystallize on the target plate. This step is critical for successful ionization. Proper crystallization enhances ion formation and improves mass spectral quality.

Drying: The sample-matrix crystals are dried thoroughly to remove solvent. This can be done using a vacuum or by air-drying.

MASS SPECTOMETRY IMAGING: MSI

When the analysis requires the spatial resolution on a tissue of interest, the matrix can be applied directly onto biological specimen (generally a tissue section of a biological sample). Similarly, the matrix helps desorb and ionize the analyte molecules when struck by the laser. Two methods exist to prepare a sample for imaging. One method spreads the matrix all over the sample, which is flexible for capturing detailed images but might cause molecules to spread sideways more than desired. The other method uses automated spotters to coat the sample with evenly spaced droplets, and the distance between these droplets determines the image resolution. The efficiency of the spotter determines how accurately molecules can be traced back to their specific originating cells, akin to identifying specific locations on a map. This process involves converting mass spectrometry data into visual images, allowing for the detailed mapping of individual molecules within the tissue.

The precision of sample preparation is a cornerstone of successful MALDI analysis. Enhancing sample preparation demands specialized technologies and systems capable of precise and contamination-free dispensing of small liquid volumes onto various substrates, achieved by pre-storing specific reagents. This precision and reproducibility are essential for improving the sample preparation process.

To fully harness the potential of mass spectrometry imaging, automation of sample preparation becomes essential. This is where BioFluidix and the BIOSPOT® automation platform steps in, offering numerous advantages that streamline the process and improve the quality of results.

Discover the MALDI-TOF Benefits

Enhanced Analytical Capabilities: MALDI-TOF MS enables scientists to identify and differentiate microorganisms, proteins, carbohydrates, lipids, and other biomolecules with remarkable accuracy and ease. It has largely replaced traditional phenotypic and biochemical methods for microbial identification.

Broad Application Range: This technique has found applications in various fields, including medical diagnostics, ecological studies, disease detection, and biomarker profiling. It can identify microbes at the genus and species levels, making it invaluable for research and clinical microbiology.

High Throughput Profiling: MALDI-TOF MS allows for high-throughput profiling of microbial biomolecules, shedding light on their roles in ecosystems. For example, ribosomal protein spectra provide microbe-specific information, making it a potential tool for dereplication.

Versatility in Sample Sources: Scientists have successfully used MALDI-TOF MS to identify organisms in diverse environments, from biofilm habitats to spacecraft surfaces and nosocomial settings. It has also been applied to the accurate identification of mammalian cell types, contributing to more effective and affordable diagnostic procedures.

Biomolecule Profiling: MALDI-TOF MS has facilitated the discovery of numerous biomarkers for diseases, including cancer. It can analyze biological fluids, cells, and tissues under normal or altered conditions, aiding in disease diagnosis and research.

BIOFLUIDIX: PIONEERING PRECISION SOLUTIONS FOR MALDI SAMPLE PREPARATION:

BioFluidix stands at the forefront of precision solutions, offering groundbreaking technologies like the PIPEJET® nanoDispenser and the BIOSPOT® workstation. These innovations are not just technological marvels; they are instrumental in overcoming significant challenges in sample preparation.

Process Standardization for Reliable Results

With the BIOSPOT® automation platform ensures the creation of a uniform layer of small matrix crystals, thus optimizing the resolution of mass spectra.

Enhance your precision dispensing with our SMARTDROP® System, a state-of-the-art droplet analysis solution that ensures unparalleled accuracy and consistency. Using high-speed optical imaging, this advanced system captures and analyzes every droplet in flight at up to 40 Hz, providing real-time, non-contact volume calibration and verification. The system's high-performance camera stands as a sentinel for quality control, validating each dispensed droplet and storing images for comprehensive QC documentation.

Learn more about the SMARTDROP® System

High-Precision, Versatile Dispensing

Our high-precision, non-contact nanoliter dispensing system is designed to accommodate a wide range of liquids, from aqueous solutions to organic solvents, beads, and more. It's also compatible with a variety of substrates, including MALDI plates, but also chips, cartridges, slides, and electrode arrays, offering exceptional versatility and precision for your applications.

Learn more about our non-contact nanoliter dispensing

Liquid Independence and Contactless Operation

Experience the advantages of liquid independence and contactless operation with BioFluidix. This approach helps limit contamination and opens up a world of limitless possibilities for your research and processes, ensuring data integrity and exceptional performance.

BioFluidix's PIPEJET® technology utilizes robust polyimide pipes, offering a minimal dead volume of 0.4-6.4 µl and compatibility with various applications through customizable lengths, diameters, and anti-wetting coatings. These pipes connect seamlessly to disposable 1 ml reservoirs, ensuring precise, contamination-free dispensing and exceptional durability in every process

Explore our dispensing consumables

Increased Throughput

Enhance your workflow efficiency with BioFluidix's automated solutions, expertly designed to handle precise liquid transfers, removing the need for manual pipetting. Our versatile equipment serves a wide range of applications, offering everything from standard workstations to customized platforms. Discover how our innovative liquid handling technologies can transform your ideas into reality through seamless integration and user-friendly interfaces.

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Dispense on target

The vision guided dispense on target function deposits a droplet wherever you want it to be. If you wish to target a region or cells of interest on a sample, or simply compare the same regions of different specimens, navigate the crossbar to your desired position on the spotting area and click the “dispense” button in the software. A droplet will be delivered - exactly to your needs! Alternatively, the print and move function will allow the BIOSPOT® user to design a regular pattern of droplets onto the surface to print, easily designing grids, where the spot size and pitch can be fully controlled.

Learn more about these features here

MALDI sample preparation

SCIENTIFIC RESEARCH ENABLED BY BIOFLUIDIX TECHNOLOGY

Scientists across both academia and the biopharmaceutical industry trust BioFluidix technologies to propel their research forward. Below, we've highlighted selected examples showcasing how our customers leverage the versatility and power of BioFluidix technologies, particularly in the realm of mass spectrometry imaging (MSI).
In the subsequent section, we delve into specific research studies and publications that have benefited from BioFluidix technology to enhance their investigations.

Dannhorn et al. 2021

This study presents a decontamination method using ultraviolet-C (UV-C) light to safely eliminate infectious pathogens in clinical tissue samples for mass spectrometry imaging while preserving analyte distributions. The authors found that lower UV-C doses maintain pathogen inactivation with less damage to the tissue's metabolome and enable the analysis of endogenous metabolite distributions and even the abundance of a specific inhibitor in decontaminated biopsies.

https://doi.org/10.1021/acs.analchem.0c03430

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In this study, the BIOSPOT® workstation was employed to deposit precise 50 nL droplets of several drug solutions at a precise concentration onto tissue sections. These droplets were used to perform kinetic experiments for photodegradation of the dosed drugs. The BIOSPOT® workstation ensures accurate and reproducible liquid dispensing, enabling researchers to spot the drug solution onto the tissue sections in triplicate.

Luptáková et al. 2021

The aim of the study is to quantitatively and visually assess drug transport across the region-specific blood-brain barrier (BBB) ​​using in vivo and in vitro neuropharmacokinetic investigations with mass spectrometry imaging. This approach enables the differentiation of regional and subregional BBB transport characteristics in small brain regions, providing valuable insights for molecular psychiatrists in understanding regional target-site exposure and decision-making.

https://doi.org/10.1038/s41380-021-01267-y

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In the study, BIOSPOT® was used for sample preparation in MALDI-qMSI by spotting calibration standard solutions of the drug and quality-control (QC) solutions directly onto the tissue, ensuring precise calibration and QC application during the analysis.

Hamm et al. 2022

The study aimed to assess the in vivo effects of pharmacological inhibition of the receptor tyrosine kinase MERTK using a multi-modal imaging platform. The study employed mass spectrometry imaging (MSI) to characterize the spatial distribution of the MERTK inhibitor AZ14145845 in the eye. It provides a strategy to investigate the preclinical toxicity of MERTK inhibitors and potentially other chemotypes.

https://doi.org/10.1007/s00204-021-03197-8

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The BIOSPOT® was used to generate a calibration curve for quantifying AZ14145845 in the samples. The results revealed high localized compound concentrations in the retinal pigment epithelium (RPE) and retinal lesions, suggesting a MERTK-induced mechanism of photoreceptor cell death.

BIOSPOT® AUTOMATION PLATFORM: ELEVATING MALDI-TOF MS

BioFluidix's BIOSPOT® automation platform is a game-changer when combined with MALDI-TOF MS, enabling scientists to streamline workflows, enhance precision, reduce costs, and achieve consistently reliable results. This dynamic synergy offers unprecedented possibilities across various scientific fields, from microbiology to drug discovery and beyond. With BIOSPOT® at the forefront of sample preparation, precision becomes paramount.

 

Our liquid-independent, contactless, and production-optimized dispensing technology delivers a level of accuracy that forms the bedrock of scientific research and diagnostics. It ensures that even the tiniest liquid volumes are dosed with impeccable reliability.

Beyond simply enhancing data quality, our solutions also offer versatility, from straightforward dosing tasks to highly complex, automated systems.

As part of the Hamilton family, BioFluidix is ​​dedicated to driving the benefits of MALDI-TOF MS, resulting in better care for patients and a brighter future for researchers.

Next-generation dispensing systems are pivotal in advancing mass spectrometry, enabling precise, efficient, and error-free sample preparation while optimizing medical processes, ultimately saving time and costs.

Our customized solutions, developed in close collaboration with customers, empower them to achieve progress more swiftly and efficiently. In the world of liquid dosing, BioFluidix is ​​synonymous with precision and limitless potential.