Maintaining Sensitivity, Specificity and Efficiency During Mass Spectrometry Sample Prep

LM_Sample Prep_eBook_2024 Tips and tools for precise, consistent sample preparation COMPARING instruments and features IS AUTOMATION the right fit for your workflow? HOW TO REDUCE or eliminate solvent use Table of Contents 3Quality Sample Preparation, Meaningful Results5Slash Sample Preparation Time with MicrowaveDigestion8Selecting an Evaporator for Mass SpectrometrySample Prep10How to Choose the Right pH Meter12Automating Sample Preparation14Reducing Solvent Use During Sample Preparation 2 Lab Manager 16Tackling Sample Prep for Mass Spectrometry Introduction Quality Sample Preparation, Meaningful Results‌ Mass spectrometry demands consistent, precise sample preparation Mass spectrometry (MS) is a highly versatile, core analytical technique that many fields rely on, ranging from chemistry to pharmaceuticals to materials science. Frequently com- bined with other analytical techniques, such as liquid chromatography, gas chromatography, nuclear magnetic resonance, and more to create powerful hybrid methods, the adaptability, sensitivity, and specificity of MS is critical for numerous applications. Thhether applied to the identification and quantification of compounds, characterization of molecular structures, or other analyses, its high sensitivity and specificity demand meticulous sample preparation for successful outcomes. From benchtop instruments to automation solutions, there are many ways to optimize MS sample preparation to improve throughput, minimize costs, and achieve meaningful results. Sample preparation requires several benchtop tools including pH meters, microwave digest- ers, and evaporators, among others. There are many factors to consider when selecting these instruments, especially what the analysis requires and what types of samples the lab will be working with now and in the future. Introduction In addition to working with the right instruments, lab staff must possess the right knowledge and skills, and invest a substantial amount of time and attention to ensure consistency. This manual approach to sample preparation can be costly, and is subject to human error. Automat- ed solutions offer precision, consistency, and higher throughput, but require careful consider- ation-in the form of a feasibility study-to ensure a good return on investment. Adopting green chemistry practices, like reducing or eliminating solvent use, can also reduce the costs associated with sample preparation. If a solvent-free method isn't the right fit for your workflow, there are multiple tools available for solvent recycling. Meaningful mass spectrometry results depend on meticulous sample preparation. The right tools and automation can enhance throughput, reduce costs, and ensure precision and consistency. This resource guide includes detailed insights on instruments and techniques for mass spectrometry sample preparation. Learn what features to look for when selecting pH meters, microwave digesters, evaporators, and more for your sample preparation workflow. Not sure if automation is the right fit for your workflow? Learn how to conduct a feasibility study prior to implementing automation solutions. Also included is a comparison of solvent-free methods and solvent recovery/recycling options that can reduce the costs associated with mass spectrometry sample preparation. Slash Sample Preparation Time with Microwave Digestion‌ Microwave digestion offers increased speed and consistency over other sample prep methods by Colm O'Regan, PhD Sample preparation is the cornerstone of any lab operation, setting the stage for credible, accurate data, and subsequent analyses. This article outlines the basics of microwave digestion as a sample prep method. It also explains the ben- efits this technique brings to the lab, and why lab managers should consider it going forward. The need for microwave digestion Consider the classic process of hot plate digestion. Here, samples are painstakingly heated over time with the contin- ual addition of acids. The downside of this method is it can result in airborne contamination and poor volatile com- pound recovery. Such factors can be frustrating, as they add a level of unpredictability to the subsequent stages of analysis. Airborne contamination can also pose serious health risks. Microwave digestion addresses these challenges effectively, making it a compelling alternative for many labs. Understanding microwave digestion Microwave digestion offers numerous benefits over other sample preparation techniques. Even though it has existed for a number of years now, research groups are only just starting to realize its benefits for sample preparation in the lab. Traditional techniques such as hot plate digestion work via conductive heating, in which heat must pass through the walls of the vessel before reaching the sample. This process may be slow and inconsistent, as the speed at which the sample heats depends upon the thermal conductivity of the vessel. Essentially, microwave digestion is a process that uses microwave energy to heat samples in a closed-vessel sys- tem. The process is relatively simple: First, the user adds a known amount of sample material to a digestion vessel. They then pour in a concentrated acid, such as hydrogen perox- ide or hydrochloric acid, seal the vessel, and start applying microwave energy. The sealed vessels achieve very high "A full digestion can be completed in as few as 30 minutes, while conductive heating methods can take several hours or days." pressures as they heat up, similar to a pressure cooker. This pressure pushes the temperature of the vessel beyond the acid's boiling point, increasing acid's oxidative potential and breaking down the sample material faster. As a result, sam- ples are heated faster than in methods based on conductive heating-a full digestion can be completed in as few as 30 minutes, while conductive heating methods can take several hours or days. Along with faster digestion times, other ben- efits include increased sample throughput, more consistent digestion, and enhanced safety features. Increased sample throughput Sample throughput with microwave digestion is better than other methods by virtue of its quicker digestion times. Furthermore, microwave digestion systems are designed to handle multiple samples simultaneously. This is quite differ- ent from traditional methods, where usually one sample is processed at a time. The ability to process multiple samples at once substantially increases the lab's sample throughput. Simply put, more samples processed equals more data gener- ated in the same amount of time. Consistent and complete digestion A challenge with traditional methods is ensuring that each sample is treated identically. Thith microwave digestion, uniform heating in a closed vessel system provides consistent and complete digestion, ensuring reproducibility and reli- ability as well as protecting product quality. Thhether you're processing 10 samples or a hundred, each one undergoes the same exact treatment. Enhanced safety features Safety is paramount in any lab environment. Micro- wave digestion brings enhanced safety features due to its closed-vessel system. The containment minimizes the risk of contamination. It also minimizes the risk of exposure to hazardous chemicals, fostering a safer work environment. There's no open flame or hot surfaces, further reducing the risk of accidents. Justifying the cost of a microwave digester Undeniably, the upfront cost of a microwave digestion system can be higher than traditional apparatus like hot plates or reflux systems. It's akin to comparing the price of a high-end, multifunctional kitchen appliance to that of a simple stove. But let's look at it from a long-term perspective. Microwave digestion systems significantly reduce the con- sumption of expensive reagents and solvents. The savings from this reduction, though seemingly small on a per-sample basis, accumulate over time and can lead to a substantial decrease in operating costs. Moreover, microwave digestion enables you to process mul- tiple samples simultaneously, enhancing productivity. Sup- pose in your lab, you process hundreds of samples weekly. Thith microwave digestion, you could essentially double or triple your throughput without requiring additional person- nel or extending working hours. More productivity within the same timeframe equates to more data, more results, and ultimately, more value created. Why you should consider microwave digestion As lab managers or research scientists, embracing change and innovation in your methodologies can be the key to unlock- ing new levels of productivity. Microwave digestion presents an exciting leap in this direction. It's faster, more efficient, safer, and offers an environmentally friendly alternative to other methods. Thhile the upfront investment and learning curve may appear as initial challenges, the long-term bene- fits are too compelling to overlook. Consider the prospect of transforming your laboratory into a more efficient, safer, and greener space-that's the potential offered by microwave digestion. Selecting an Evaporator for Mass Spectrometry Sample Prep‌ Features to consider when comparing evaporators by Michelle Dotzert, PhD Evaporators are crucial for mass spectrometry sample prepa- ration. They can be used to concentrate samples, remove solvent, reduce the volume of complex matrices (biological fluids, for example), and much more. The choice of evaporator will depend on the sample type and application. The following are commonly used in mass spectrometry sample preparation to increase the concentra- tion of analytes in a sample: Rotary evaporators: The sample is placed in a round-bottom flask connected to the evaporator. It is heated using a water or oil bath and the flask rotates at a controlled speed while vacuum is applied. Nitrogen evaporators: The sample is placed in a vial and positioned in the evaporator. A stream of nitrogen gas is directed just above the sample to help evapo- rate solvent by sweeping away evaporated molecules. Centrifugal evaporators: Sample tubes or plates are centrifuged under vacuum to improve evaporation at lower temperatures. Many units are equipped with tem- perature control for refrigerated or heated evaporation as required by the samples or application. Recent advances in evaporator technology can enhance safe- ty, efficiency, and precision. Thhen comparing evaporators, consider whether the following would benefit your workflow: Automated controls: in addition to precise tempera- ture, pressure, and speed control, settings can be programmed for complex procedures. Solvent recovery: integrated recovery systems can capture solvents to be reused, a greener practice that is also cost-effective. Safety features: automatic shut-off, over-temperature protection, and venting systems are moving from "nice to have" to standard features. Remote monitoring and control: connectivity features enable monitoring and control via phone or computer. MASS SPECTROMETRY Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a label-free technique that enables spatial analysis of thousands of molecules including metabolites, lipids, peptides, proteins, and glycans in a thin sample section. With continuous improvements to sample preparation techniques and instrumentation, as well as the development of methods for absolute quantitation of molecules, MSI is becoming more widely accepted in the pharmaceutical industry and clinical settings. WORKFLOW Sample collection Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a label-free technique that enables spatial analysis of thousands of molecules including metabolites, lipids, peptides, proteins, and glycans in a thin sample section. Download this free infographic, courtesy of Lab Manager to learn more about the MSI sample preparation. Advanced vacuum technology: improvements in pump technology mean quieter, more efficient pumps are being integrated into evaporators. How to Choose the Right pH Meter‌ Accuracy and resolution aren't the only factors to consider by Margaret Sivapragasam, PhD Thhen selecting the ideal pH meter for your research needs, you may feel a bit like a kid in a candy store-with an over- whelming array of choices, it is tough to know which option will truly hit the spot. Precise pH measurement is crucial for consistent results and product quality across laboratories and industrial settings. However, not all pH meters are the same. Even minor pH deviation levels can considerably impact outcomes and data interpretations. Here are four things to consider when select- ing a pH meter. Accuracy takes the cake The accuracy of a pH meter is determined by how closely its readings match the true pH of the tested material. Factors such as component quality, calibration techniques, and ambient conditions all influence a meter's accuracy level. For general laboratory usage, a pH meter with an accuracy of ±0.01 suffices. However, if your work requires even greater accuracy (down to 0.001 pH or lower), opt for a specialized research meter designed for advanced applications. Mind the resolution Thhile accuracy indicates how precise the reading is to its true value, resolution specifies the level of detail measured. It represents the smallest increment that a meter can display, revealing if minor fluctuations are detected. For many common lab applications, a resolution of 0.01 pH is sufficient. However, higher resolutions of 0.001 pH or even 0.0001 pH may be required to distinguish between very minute pH differences for sensitive processes. Optimize electrode compatibility Electrodes come in a variety of materials and designs, each customized to specific applications and sample properties. Glass bulb electrodes offer good all-around performance for aqueous samples ranging from zero to 14 pH at typical laboratory temperatures. Specialized electrodes, such as ones made of polymer or antimony, are better suited for applications such as low ionic strength solutions or at high temperatures. Future-proof your choice Maintenance Tip: Regularly replacing your pH meter electrode is important in maintaining its accuracy and reproducibility. Depending on how much abuse your unit takes and your specific application, you may need to replace your electrode in three to six months or 12 to 18 months. Apart from electrode material choice, prioritize pH meter models with compatible input circuitry and software for precise signal processing and data interpretation; this will minimize measurement errors. Factor in the recurring costs for replacement electrodes as they are relatively fragile and will likely need to be replaced over the lifetime of the pH meter. Finally, consider whether you will need to expand your measurement capabilities in the future. Automating Sample Preparation‌ Sample preparation can be labor-intensive and expensive. Is automation the answer? by Joe Liscouski Sample preparation demands precision and consistency. Achieving this manually is costly, as it requires skilled staff to invest a significant amount of time and attention. Manual sample preparation is also subject to human error, which can further increase costs when assays must be rerun, and valuable sample material is lost. A shift toward automating sample preparation workflows can drive improvements in efficiency, accuracy, and reproducibility, as well as reducing costs. Cost vs return The goals for improvements in sample prep include: Overall cost reduction, including labor and materials More consistent preparation Higher productivity-more samples processed, which may be coupled with automated instruments The ability to work with hazardous materials More extensive analysis-work that might be too expensive for manual efforts such as statistical experi- mental design and high-throughput screening The potential for 24/7 operations For any ROI equation, there are two sides to consider. The first is what you want out of it, which includes some or all of the points above plus metrics-what level of performance are you looking for, what are you willing to spend, and what is the schedule requirement for implementation? You also have to evaluate the alternatives to automated systems, which include increasing head count or outsourcing work for comparison. Those last points would have to include an understanding of whether the need is a temporary spike in testing throughput or a long-term requirement; it is going to take time to implement a solution, and you don't want it coming online as the need evaporates. The other side of the equation covers the costs. They include the development of the user and system requirements, a feasi- bility study, and prototype work, followed by the actual design, implementation, documentation, testing, validation, and user education. Given a set of requirements, the next major step is the feasibility study-this is going to provide the basis for the go/no-go decision on the project and guide the design effort. The first step in that study is an evaluation of the sample prepa- ration procedure, the underlying process of the system. Feasibility Unless the process is specifically designed for automated implementation, the process is going to have to be analyzed to see what it will take to make it suitable for semi-automated or fully automated work. If there is a make-or-break step in the project, this is it. Sample preparation consists of a series of steps lab personnel need to take to ensure the sample is ready for mass spectrometry analysis, but often these protocols rely on an individual's knowledge and experience to fill in any gaps or solve problems. The first item that has to be determined is whether you are using the current, up-to-date description of the process, including any undocumented knowledge. Next, the feasibility analysis has to evaluate whether there is anything about the process that precludes automation. This would include working with objects or materials that depend on human dexterity and might not be usable with robotic systems. Another consideration is whether the process can be optimized to meet or exceed performance goals. Mass spectrometry is both a powerful technique and a tricky one to master. Proper sample preparation is crucial for success, and there are a few tips that can improve your chances of obtaining meaningful results. Download this free infographic courtesy of Lab Manager. Mass spectrometry is a powerful technique that can detect, identify, specify, and quantify molecules separated by their mass-to-charge (m/z) ratio. Ditterent mass spectrometry techniques function in very ditterent ways. Before introducing your sample into a mass spectrometer, proper sample preparation must be performed. Here are a few sample preparation tips to improve your results. For the optimal analysis conditions, use HPLC- or mass spectrometry-grade reagents throughout your protocol. Use an extraction method that will ettectively concentrate the analyte of interest. It is important to ensure that the chemical properties of your solution are what they're supposed to be. Prepare the samples properly for GC, LC, or direct sampling. Autoclaved tips, tubes, and plastics may leach, potentially contaminating your experiment. The right internal standard at the right concentrations will improve the accuracy of the results SAMPLE PREP TIPS FOR MASS SPECTROMETRY Use the right extraction method. Reagent quality matters. Check the pH of your solutions. Know the introduction method. Use proper internal standards. Do not use autoclaved plastics. Automating sample preparation may be an effective way to improve efficiency, accuracy, and consistency while reducing overall costs. The decision to invest in automation requires careful consideration of several factors, and an assessment of current processes to determine feasibility. Reducing Solvent Use During Sample Preparation‌ Solventless sample preparation methods may be applied to a variety of sample types by Mike May, PhD and Michelle Dotzert, PhD The move toward greener chemistry extends to sample preparation with the reduction or elimination of solvents. Going solvent-free can reduce background contamination from other materials such as glassware used in sample prepa- ration, and allows one to move directly from sample collec- tion to analysis. Advances in solventless techniques continue to increase the possibilities for labs to eliminate, or at least reduce, the use of solvents. Use more efficient extraction methods Solid-phase microextraction (SPME) can combine sam- pling, extraction, concentration, and sample introduction in one solvent-free step. A standard form of SPME uses a fiber coated with material that will extract the desired components from a sample, which can be solid, liquid, or gas. A polymer-coated fiber goes in the headspace over a sample, and heating the sample releases components that are absorbed by the fiber and later extracted from it. SPME may have different formats, such as a fiber, membrane, or various other shapes made of different materials, all of which can be modified to accommodate an application. Direct analysis in real time (DART) is a solvent-free technique based on ambient ionization of the sample prior to introduction into the mass spectrometer. Desorption electrospray ionization (DESI) is also an ambient ionization technique. It relies on a fine spray of charged droplets to de- sorb analytes from a surface directly into the spectrometer. Matrix-assisted laser desorption/ionization (MALDI) is primarily used for large biomolecules. A matrix compound is mixed with the sample then irradiated to produce ions. Consider automation Automated sample preparation systems offer a high degree of control and precision. Compared to manual processes, they can reduce the amount of solvent wasted during sample prepa- ration. These systems can also be programmed to use optimal amounts of solvent, avoiding the need for large volumes. Their higher-throughput capabilities also reduce the need for large volumes of solvent required for manual processes. Recover and recycle A variety of solvent recovery systems are available. Distilla- tion units are ideal for recovering large volumes of solvent. The unit heats the solvent until it begins to evaporate, then 15 Lab Manager condenses it back to a liquid and collects it. Vacuum dis- tillation is suitable for solvents with high-boiling points. The unit reduces pressure in the system, and the solvents boil at lower temperatures to preserve and recover sensitive compounds. A rotary evaporator can also be equipped with a solvent recovery system. Often used in high-throughput labs, closed-loop systems combine recovery, purification, and reuse processes to ensure efficient recovery. Some automated sample preparation systems also include integrated sol- vent-recovery mechanisms. Thhen solvents are reduced or eliminated whenever possible, science grows more sustainable and, in many cases, simpler. The results can be good for everyone. "Automated sample preparation systems offer a high degree of control and precision. Compared to manual processes, they can reduce the amount of solvent wasted during sample preparation." Sample Preparation Resource Guide Product Spotlight Streamline Your Sample Preparation with PRO Homogenizer Kits PRO Homogenizer Kits simplify sample preparation by providing all the necessary equipment for your specific homogenizing needs. Each kit is tailored to the process and volume range, ensuring you have the right tools for efficient sample preparation. The Micro-Sample Homogenizer is perfect for small-scale work, using 0.5ml and 1.5ml/2ml micro-tubes. For higher throughput, the Multi-Sample Homogenizer processes multiple samples in tubes ranging from 1.5ml to 50ml, minimizing cross-contamination concerns. The Universal Homogenizing Package handles larger samples within the same tube range of 1.5ml to 50ml. For even larger volumes, the Max-Homogenizing Package is ideal, accommodating samples up to 5 liters. With over 25 years of experience, PRO Scientific offers high-quality homogenizer kits that streamline sample preparation, providing everything you need to start homogenizing quickly and effectively. CLICK HERE TO LEARN MORE 16 Lab Manager Sample Preparation Resource Guide Tackling Sample Prep for Mass Spectrometry‌ Expert insights on analyzing small molecules and proteins from diverse samples using mass spectrometry by Tanuja Koppal, PhD Thomas Neubert, PhD, professor of cell biology and director of the New York University Protein Mass Spectrometry Core for Neuroscience, talks to contributing editor Tanuja Koppal, PhD, about analyzing small molecules and proteins from diverse samples using mass spectrometry (MS). He discusses some of the common issues that researchers often overlook when it comes to sample preparation. These issues, although seemingly trivial, have a significant impact on the separation of samples and analysis of data and could lead to false discovery and misinterpretations. Q: Can you describe your work and the types of analyses you do? A: I have been running a mass spectrometry core lab at the New York University School of Medicine since 1998. The collaborate with many researchers to use MS for analysis, and at any given time we have many different projects going on. Our main interest is in neuroscience, although we do work on other projects as well. Because we work on many projects we have to process a variety of samples and use different types of MS instruments. Some samples are tissues, others are cell culture, plasma, or serum. The analyze both proteins and small molecules in these samples and do most of the sample preparations ourselves. Q: How important is sample preparation for your analysis? A: Sample preparation is very important. For analyzing pro- teins, we usually get the protein in the form of a pellet or in a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. The main processing steps include digest- ing the protein into peptides using trypsin and fractionating or cleaning the peptides before putting them in the MS instrument. The exact processing steps depend on whether we are studying a protein or a small molecule, as the two workflows are very different. For studying post-translational modifications, such as phosphorylation, ubiquitination, and glycosylation, on proteins, we have to enrich the modified peptides so they can be seen using MS. So that's an import- ant step. However, if we just have to identify the proteins and not measure their quantities, then we do only fractionation of the complex mixture and no enrichment. After the sam- ples are processed, we inject them into a nanoflow high-per- formance liquid chromatography (HPLC) column, which is coupled to MS. Thhile some labs study intact proteins, we typically study only peptides, which makes it easier for MS analysis. The small molecules that we study are typically metabolites found in the cell. Q: With some journals requesting researchers submit raw data with the manuscript, has there been a concomitant improvement in sample preparation and data analysis? A: The field is improving but there continue to be challenges as new technologies are introduced and data sets get larger. Experiments have to be done carefully, and controls have to Thomas Neubert, PhD, professor of cell biology and director of the New York University Protein Mass Spectrometry Core for Neuroscience be used appropriately, so there are no mistakes in data in- terpretation. Experimental conditions have to be monitored, and there can be no bias when selecting samples, especially for clinical research. Even in cell biology experiments, con- ditions have to remain identical, with the exception of a few variables, when it comes to making accurate comparisons. As instruments like MS become more sensitive, research- ers can analyze very small amounts of samples. However, sample processing also has to improve to accommodate these small amounts of material. Technologies have now moved to single-cell analysis with RNA sequencing, but analyzing proteins and small molecules in single cells is still quite dif- ficult. To do that, you have to be able to process the cell and extract the analyte in a reliable way, which is very difficult and often involves microfluidics. At the same time, there are innovations taking place all the time to help with sample preparation. Q: How do you overcome some of the challenges with sample preparation? A: Thorking with different types of samples can be challeng- ing, so it's important to hire people who are skilled and col- laborative, so they are willing to share their knowledge and techniques with others. I rely heavily on senior lab members to teach the junior members, so our protocols can be passed down to the next generation. Everyone who joins my lab learns the basics of sample preparation, which is extracting the proteins, digesting them, purifying, and fractionating them. However, some people develop expertise working with a particular sample type. For instance, some have more experience working with small tissue samples, while others work better with cell culture. Every individual has a niche, and sometimes they have to develop new methods for a spe- cific type of sample based on their expertise. PRO Scientific is a global leader in the production of mechanical homogenizing equipment and systems, designed to handle everything from micro volumes to larger multi-liter samples. Our product range includes high-quality handheld devices, benchtop equipment, and advanced automated systems, all proudly made in the USA. Our innovative DPS-20 and Multi-Prep Homogenizing Systems offer cutting-edge high-throughput homogenization solutions. 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