Sample preparation is an important step for any experiment, whether in genomics, physics, chemistry, or in the clinic. Inadequate sample preparation yields variable and potentially inaccurate results. And while there is an incredible variety of sample prep kits and protocols, new applications are triggering novel method development, building on current approaches for additional uses.
This cannot be more true than for mass spectrometry. Mass spectrometry has long been a preferred method for identifying and quantitating compounds. Yet, sample analysis for MS provides unique challenges. “It’s important to ensure proper removal of interfering compounds such as salt, buffers, detergents, ion-pairing agents, drugs to name a few, that might impact sensitivity, specificity, instrument uptime, or the ability to increase ionization efficiency. Ineffective sample prep can impact the quality and reproducibility of results,” says Lisa Thomas, senior director, life science mass spectrometry, Thermo Fisher Scientific.
What’s your end goal?
When looking for the basic “clean-up” approach, there are a variety of sample prep methods one can choose from before sending a sample to mass spec. While some methods such as protein precipitation, dilution, and liquid extraction are the simplest and cheapest sample prep approaches, they also produce the dirtiest sample eluate. Ineffectively performed sample preparation ends in compromise, allowing more matrix components into the final sample and into LC-MS, contributing to ion suppression and reducing instrument and column performance. “Sometimes scientists looking for speed and low cost in sample preparation sacrifice sample cleanliness, reproducibility, and robustness of the whole system. This might become an issue for longer term quantitative studies and when low limits of detection are needed,” comments Kim Haynes, principal product marketing manager at Waters.
Availability of enhanced sample prep methods, including solid-phase extraction (SPE) or immunoaffinity purification techniques, have improved sample prep in recent years and target more matrix components to be removed, leading to a cleaner sample and more sensitive and specific results. Orthagonal methods can offer the highest specificity and sensitivity. Mixed-mode SPE combines functionality of reversed-phase and ion exchange into one sample clean up, useful for investigating therapeutic peptides or detecting low analyte levels. Though these methods provide a much higher degree of cleanliness, they are traditionally complex.
New technologies, such as the Oasis PRiME HLB, a reversed-phase SPE product from Waters that targets the removal of fats, phospholipids, salts, and other matrix components, offer researchers a simple, fast protocol to achieve a higher level of clean up. The product can be used to remove these matrix interferences from acids, bases, and neutral compounds to be flexible for any sample. “By obtaining a higher degree of sample cleanliness, researchers can see improvements in LOD and LOQ, cleaner LC columns, cleaner MS instruments and detection, and lessen the risk of matrix effects,” Haynes explains.
Tougher analytes such as polar, ionizable bases, which are common in most drugs, need special consideration when preparing for MS analysis. In order to combine the specificity of mixed-mode ion exchange with the cleanliness of Oasis PRiME HLB, Waters developed the Oasis PRiME MCX, designed to produce cleaner extracts faster than conventional ion-exchange protocols.
A new outlook
Can sample prep be further simplified or even eliminated? Thermo Fisher is researching new advancements in sample prep technologies that minimize sample prep before MS analysis. Paper spray ionization uses much smaller solvent volumes and produces very little solvent waste, potentially eliminating the need for liquid-liquid extraction and subsequently the need for additional processing steps, such as centrifugation and manual preparation.
Can sample prep be further simplified or even eliminated?
The technology is flexible and compatible with dried bloodspots, a sample collection method that is becoming routine in clinical laboratories. Paper spray ionization also shows promise in eliminating carryover, when high concentration samples are carried over from one sample to the next, by using a fresh disposable cartridge for each new sample. As a replacement for HPLC, the technology is significantly simpler, with no columns or high-pressure solvents, and enables less experienced operators to bypass typical problems seen with HPLC such as leaks in the fittings and deteriorations in the LC column.
Another new approach, field asymmetric waveform ion mobility spectrometry (FAIMS) technology, is beneficial for applications involving isobaric compounds, where multiple proteins result in numerous peptide fragments that are similar to the peptide of interest. “For proteomics and clinical proteomics, we see the potential for FAIMS to increase selectivity, specificity, and sensitivity, addressing the complexities often experienced with challenging analytes in complex matrices,” explains Thomas.
FAIMS restricts entry of ions into MS to those that are targeted, lessening the number of peptides in analysis and resulting in greater sensitivity and the ability to quantify more proteins faster. “In addition, coupling FAIMS with LC-MS could offer increased clarity needed to advance subsequent experiments faster with higher quality information,” Thomas adds.
Going universal
Introducing new methods can advance sample prep in novel ways, particularly in consolidating various sample prep protocols for different sample types into one assay. Chromotek and PreOmics have developed a new in-stage tip (iST)-GFP-trap method for sample processing that caters to labs running multiple sample types. “Peptides can be eluted in several fractions or in one step for single-run proteome analysis. The three-step protocol can be used with any starting material, taking only three hours instead of the standard two days,” describes Garwin Pichler, managing director of PreOmics.
The method was developed with clinical research labs in mind, specifically those that process thousands of different samples and optimize protocols for each sample type. Creating a robust protocol that can run multiple samples by any user and adapt to high-throughput instruments makes proteomics studies instantly more accessible. By supplementing an additional method that introduces multiplexing to MS through isobaric labeling, called tandem mass tagging (TMT), even more samples can be processed in fewer runs. Different samples are labeled, each with a specific tag, then mixed to measure all samples in one run, increasing throughput and sensitivity per sample.
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ChromoTek has a significant portion of its customers doing co-immunoprecipitation of GFP-fusion proteins of interest using GFP-Trap for analysis of interacting partners by MS. A partnership with PreOmics, a company seeded from the Mann Lab within MPI Munich, has led to a universal sample prep kit capable of such clean sample prep that core facilities have reportedly come to recommend it for consistently reproducible results independent of user skill set. Using the combined iST GFP-Trap Kit users can both effectively pull-down GFP fusion proteins from their cells and process that sample for MS analysis within less than four hours’ time.
The new kit simplifies proteome-sample preparation by eliminating or combining multiple steps and performing all processing in a single, enclosed volume. To validate the iST method, the group used the kit to obtain the largest proteome coverage to date for budding and fission yeast in one day. They also applied the in-stage tip method to quadruplicate measurements of a human cell line and obtained copy-number estimates for 9,667 human proteins, observing robust quantitative reproducibility between replicates.
Integration and automation
A new trend that Brian Feild, life science product manager at Shimadzu Scientific Instruments, has noticed is the desire to load a sample and walk away, leaving a system to perform sample prep, LC, and MS independently. Not only does this enable faster processing of samples but also allows any user, whether proficient in MS or not, to perform an experiment as well as seamlessly transition workflows from one lab to another.
“Automation is no longer enough,” Field explains. “You also need integration of the automated platform in the LC-MS system to help improve reproducibility and achieve higher lab efficiency. Integrated workflows can facilitate ease of use, minimize error, and enable quick turnaround of samples.” The CLAM-2000 is the first of its kind fully integrated system with sample prep automation for LC-MS geared toward small molecule analysis. The instrument processes samples in parallel through sample prep to injection for common applications including research of immunosuppressants, vitamin D, therapeutic drug monitoring, and drug development and detection.
For more complex workflows, Shimadzu is investing in chemistry protocols specifically for sample prep before mass spec. “Recently there has been increased interest in large molecule workflows that can work for monoclonal antibodies and DMPK studies, where sample prep can take more time and planning. One method that can be used against any molecule simplifies the process for sample prep and LC-MS,” explains Gurmil Gendeh, Shimadzu’s pharmaceuticals marketing manager. Using a new technology called nSMOL, Shimadzu has brought nanotechnology to MS sample prep. Nanoparticles are enlisted to capture antibodies against any molecule for use with all available therapeutic antibodies. nSMOL comprises one universal kit for any clinical sample in one workflow, taking far less time than traditional methods like ligand binding assays and conventional LC-MS.
To take advances one step further, the Perfinity Workstation and integrated digestion platform automates affinity capture, buffer exchange, rapid trypsin digestion, desalting, and reverse-phase chromatography. The process shortens digestion to 1–8 minutes instead of overnight by streaming proteins through a trypsin tunnel for easy access to lysines and arginines on the proteins. This system accelerates the movement of MS from discovery and development to the more regulated space of QA/QC in manufacturing, merging the entire process from sample prep to MS. For example, bringing Perfinity inline with other systems such as a bioreactor and C2MAP enables constant and automated cell culture monitoring. These technologies are enabling a move away from batch manufacturing to continuous manufacturing, turning a 24-hour workflow into a 30-minute process saving the most time in sample prep.
Speed, high throughput, and versatility encompass new technologies for sample prep in mass spec. Automation and standardization both offer the ability to transfer methods from lab to lab and user to user without compromising quality and reproducibility. Given current and upcoming advancements, mass spec appears to be on track to become a primary quantitation and detection method in any field and industry.