DOI: 10.1126/science.opms.p0600007

Life Science Technologies
Mass Spectrometry in Drug Discovery and Development:
From Physics to Pharma

Long a quintessential tool of physicists, mass spectrometry has emerged as a critical technology for the pharmaceutical field. It gives researchers greater sensitivity, higher throughput, and extra information about molecules with druggable potential.

By Peter Gwynne and Gary Heebner

Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.

Drug discovery and development projects can generate hundreds of thousands of compounds that scientists must analyze to characterize structures and identify impurities. Once they have identified a drug target and have run bioassays to understand better how it functions, they can focus their efforts on finding small organic molecules that alter the target's function. To characterize these small entities, as well as larger biomolecules, life scientists increasingly rely on mass spectrometry (MS).

Originating in physics labs, MS has gradually gained a significant spot in the pharmaceutical world. "Its latest impact will be on the rational approach to drug design, via its capability to elucidate protein posttranslational modifications," says Emmanuel Raptakis, product manager of Shimadzu Biotech. "It gives an excellent view of molecules and their modifications." The technology's application has also begun to spread beyond small-molecule drugs. "With the more modern techniques, it also applies to biomolecules such as proteins, glycoconjugates, or oligonucleotides," explains Christoph Menzel, global product manager for mass spectrometry at Qiagen. Beyond that, adds Jasmine Gray, marketing director for protein sciences at GE Healthcare, "Biomarker discovery is the buzzword that everybody's using. MS is one of the critical parts of the identification and characterization of proteins."

'Ubiquitous and Indispensable'

Douglas Mautz, senior director of pharmacology (discovery absorption, distribution, metabolism, and excretion [ADME] and drugs and pharmacology metabolism) for contract research organization MDS Pharma Services, summarizes the situation. "I saw mass spectrometry was amazingly powerful in life science 15 years ago," he recalls. "Now I view it as nearly ubiquitous and indispensable." Lester Taylor, product manager for life science mass spectrometry at Thermo Electron, outlines the technology's clinical potential. "The development of drugs with higher potency that you can take once a day rather than more often has presented a challenge of analytical detection of low levels in plasma and urine," he says. "MS facilitates that."

The technology's value spans the entire drug pipeline. "In clinical trials, where you can generate thousands of samples, you need to be able to quantify drugs and metabolites quickly and accurately," points out Joe Anacleto, senior director of the small molecule business for Applied Biosystems. "MS is the method of choice for that." Indeed, says Robert Plumb, pharmaceutical application and development manager for Waters Corporation, "It has become almost a cornerstone of modern drug discovery and development, from target identification and validation up to combinatorial chemistry and parallel synthesis chemistry." Victor Fursey, assistant vice president for sales and marketing at Bruker Daltonics , echoes that thought. "You see mass spectrometry in everything from new chemical entities to formula determination to target profiling and preclinical work," he says. "Now, you even see it at the far end, in manufacturing. And many researchers are working to bring mass spectrometry into diagnostics."

Mass spectrometry doesn't stand alone. Pharmaceutical researchers routinely team it up with other techniques to obtain synergistic effects. "Liquid chromatography/mass spectrometry (LC/MS) is now the de facto technique for providing quantitative data for drug evaluation and submission for drug approval," Taylor explains.

Taylor makes another key point. "Instruments no longer have dedicated mass spectroscopists running them," he says. "Now, in many of the biological applications, biologists are using the tools. Vendors need to ensure the highest standards of performance in a way that's much more robust and easy to obtain on a routine basis."

Flavors and Configurations

Biological molecules produce several fragments when subjected to mass spectrometry. The technique does not yield the fragments' actual masses. Rather, it determines their mass-to-charge ratio; if scientists know an ion's charge, they can then calculate its mass. The fragments produce a mass spectrum that scientists can use to identify each molecule uniquely. Because biomolecules' mass spectra differ little from instrument to instrument, the information can be stored in a database and unfamiliar spectra searched against known ones.

Mass spectrometers typically include three components: an ionization device, a mass analyzer, and a detector. Since almost any ionization method can be coupled to any mass analyzer, commercially available mass spectrometers come in what Alan Millar, Waters's Synapt product manager, calls "several different flavors and configurations."

Life scientists usually rely on two ionization methods: matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI). MALDI is a solid phase technique that can analyze a digested protein sample from a 2-D polyacrylamide gel. ESI, a liquid methodology, is compatible with high pressure liquid chromatography (HPLC) and capillary electrophoresis (CE).

Mass analyzers commonly used for biochemical applications include ion trap, time-of-flight (TOF), triple-quadrupole, and quadrupole-TOF instruments. Ion trap systems capture ions in a small volume and then eject specific ranges of masses toward a detector. Their advantages include compact size and the ability to increase the signal-to-noise ratio of a measurement. In TOF systems, each ion has the same kinetic energy but moves at a speed that varies with its mass. Time-of-flight analyzers are highly compatible with very fast ionization methods such as MALDI. Quadrupole (or quad) analyzers permit only ions with specific mass-to-charge ratios to pass through an electrical field to the detector. Varying the field allows users to analyze different ions.

After they travel through any analyzer, ion species strike an ion detector. This ejects electrons that cause a voltage proportional to the number of ions that passed through the analyzer.

Researchers often couple quadrupole analyzers to ESI devices, and frequently use them in tandem configurations (referred to as MS/MS). Vendors now offer combinations of mass analyzers as solutions to specific problems in drug discovery and development. "We see a proliferation of hybrid instruments, ending up as one plus one equals three," Applied Biosystems' Anacleto notes.

Mother Nature's Cruel Way

The major barrier to productive MS analysis is not the instrumentation, but successful purification of biomolecules suitable for analysis. "The most important proteins in biomarker discovery are the low-abundant proteins; that's Mother Nature's cruel way of treating us," GE Healthcare's Gray explains. "That puts pressure on the front end — sample preparation — and the back end — mass spectrometry and data analysis. To look at low-abundant proteins, you need increasingly more sensitive techniques on the MS end and increasingly better fractionation on the front end. Sample preparation is the most critical phase of the whole process. Any sins you commit here cannot be recovered by even the most sophisticated mass spectrometer."

That's why sample preparation has become the most critical and challenging task in MS analysis. It involves purifying, storing, and recovering proteins, peptides, and other biomolecules and removing such contaminating species as buffers, salts, and detergents prior to MS analysis. "You're not looking to filter one protein away from the others; you're trying to reduce the complexity of a sample," Gray points out. "It's still a mixture that you want to enrich."

EMD Biosciences, Pierce, and Sigma-Aldrich, among other companies, offer a wide range of products and kits for purifying proteins and other biological molecules for MS analysis. GE Healthcare, which has long produced chromatography columns and systems, protein purification kits, and reagents, now offers specialized kits for use with MS studies. Those include its Ettan CAF MALDI Sequencing Kit for peptide-mass fingerprinting using MALDI- TOF mass spectrometry. "Chemically, it's a process that results in more reproducible fragmentation of the peptides in MALDI ionization," Gray explains.

Another preparatory method involves liquid chromatography. Termed Ultra Performance LC by Waters, the method takes advantage of the performance abilities of sub-two-micron particles. Used as front ends for mass spectrometry, UPLC systems can improve resolution by a factor of two, sensitivity by a factor of three, and separations by 10 times.

Chip-based Products

Qiagen also offers kits and reagents for sample preparation prior to MALDI- MS, including two chip-based products released this year. "Our Mass·Spec·Focus Chips are engineered for higher sensitivity and purification of samples from, for example, 2-D gel separation," Menzel says. "Their functionalities such as phosphopeptide enrichment derive from different zones on the chip's surface that retain particular peptides and simultaneously guide the droplet for a concentration in the analysis zone. This gives up to one thousand times the sensitivity compared with conventional targets."

The company's Mass·Spec·Turbo Chips, meanwhile, offer higher throughput and particular LC-MALDI applications. Each chip consists of a dense array of very homogenous matrix spots deposited by vacuum sublimation on an ultraphobic surface. "This allows simple, reproducible LC-MALDI applications with increased sensitivity without interfering with established protocols," Menzel says.

Further in preparation for mass spectrometry Qiagen's Qproteome series of protein fractionation kits is designed to "reduce the overwhelming complexity in biological systems," as Menzel explains. To supplement the pure fractionation kits, the company offers depletion kits that sort out high abundant proteins and total protein extraction kits that allow gentle but effective lysis of mammalian or bacterial cells.

GE Healthcare has recently released three new separation technologies. "Ettan nanoLC and Ettan MDLC give researchers flexibility in their peptides' separation methods upstream of MS. And nanoCOLLECT interfaces with the chromatography systems as a microfraction collector or spotting of fractions onto MALDI slides," Gray says. "We have created different chromatography solutions that interface to the different types of mass spectrometry. That's useful because you will tend to identify different proteins depending on what type of mass spectrometry technique you use — whether electrospray or MALDI; you have to choose your weapon." Since only a small proportion of identified proteins overlaps the different MS methods of upstream separation methods such as 2D electrophoresis and chromatography, she continues, "Researchers try to arm themselves with as many methods as possible to identify as many low-abundant proteins as possible."

No Standing Pat

Suppliers of mass analyzers refuse to stand pat on their technology. Several have recently introduced MS systems or enhancements that can provide additional data for researchers working in both drug discovery and life science research.

Waters recently introduced the first mass spectrometer to employ new ion- mobility technology and software, which it calls the Synapt High Definition MS System. "It's a new category of mass spectrometry," Millar explains. "It has a very innovative performance and functionality. It introduces an additional dimension of ion separation: Ions are separated according to size, shape, and charge prior to mass spectrometry. That results in more specificity, which means that we can extract more information."

A key feature of this new system is the patented Waters Triwave technology, a method for combining highly efficient measurement and separations based on ions' mobility with high performance quadrupole/TOF mass spectrometry. "This is the enabling technology that powers the Synapt system," Millar says. "It permits the high-efficiency ion mobility, which is very important. It also enables us to combine the ion mobility with the tandem MS. It's not just about performing the ion mobility but being able to combine it into a solution." Operational control and data acquisition and processing are performed through the company's MassLynx Software.

Shimadzu Biotech's AXIMA-TOF², launched in June, represents the next generation in MALDI collision-induced dissociation (CID) MS/MS. A TOF-TOF mass spectrometer with high energy MS/MS, it delivers information-rich spectra with greater sensitivity and higher confidence in identification. "It uses electron technology licensed from Johns Hopkins University that allows high- energy CID without the need to accelerate the ions; that gives better transmission," Raptakis says. "It's a very versatile tool — the highest energy collision-induced dissociations you can get. It has the ability to look at complex structures like peptides and lipids, which will break very effectively in a number of highly informative pathways. It is the most efficient way to discover, for example, double bond localization in lipid research and to generate cross-linked cleavage in oligosaccharides structural elucidation experiments."

Applied Biosystems recently launched another system aimed at drug discovery, that it calls the LightSight Software for Metabolic Identification. "It's a new piece of software especially for metabolite discoveries in the drug discovery phase," Anacleto explains. "We worked very closely with over a hundred scientists to understand their work flows in identifying metabolites quickly and easily. It's focused primarily on productivity for fairly routine work finding key metabolites." Scientists can use the system with the Applied Biosystems 4000 Q TRAP mass spectrometer, as well as other triple quadrupole and hybrid linear ion trap mass spectrometers from Applied Biosystems/MDS SCIEX.

Dealing with More Data

As a consequence of their increased sensitivity and higher throughput, MS technologies generate significantly more data than in the past. "You might acquire a mass spec in 20 to 30 seconds and find yourself looking at 20-30 megabytes of data," says Ian Brookhouse, development manager for MALDI mass spectrometers at Shimadzu Biotech. To manage this increase, companies such as Agilent, Ciphergen, PerkinElmer, and Waters offer software for data management and analysis, often integrated into the MS system. "We've been working on application managers — dedicated software tools that permit you to put a particular focus on specific tasks such as proteomics or metabolic identification, or food safety," Waters's Plumb reports. "We also have the ability to store an archive and retrieve it, and to combine data from different projects, such as MS and X-ray crystallography."

Thermo Electron has introduced what it calls QuickQuan for automating high- throughput LC-MS/MS assays in early drug discovery. "This is a software and hardware solution that allows users to analyze multiple sets of compounds in an automated fashion without having to optimize and tune for a particular compound," Taylor says. "You can automatically set up to run for each compound you're checking on. The system tunes itself automatically and analyzes assay samples overnight without needing to have people on hand. It works in combination with our triple quadrupole system."

Last year, GE Healthcare launched its DeCyder MS differential analysis software. "It creates a three-dimensional plot of the retention time and m/z ratio," Gray says. "Instead of traditional peaks, you have a signal intensity spot map like a 2-D electrophoresis map. It basically helps you to compare the different peptides between different runs and tease apart all the nuances and find that needle in the haystack."

Fursey of Bruker Daltonics highlights an emerging field facilitated by MS. "Metabolomics is gaining a lot of momentum in the effort to understand the pharmaceutical world," he says. "Much of what's going on in metabolomics stems from nuclear magnetic resonance [NMR] technology. We're finding that mass spectrometric data can be very complementary to NMR." His company and Bruker BioSpin offer the Metabolic Profiler, an integrated platform for metabolic studies and analyzing complex mixtures. It features an HPLC-microTOF ESI-TOF system and an optional Avance NMR spectrometer and integrated software for data acquisition, evaluation, and statistical analysis in such applications as assessing the metabolic profile of a living organism to distinguish between normal and altered states and studying the mechanism of drug-induced toxicity. Fursey notes that the Metabolic Profiler is fully integrated into the Bruker Compass OA software environment.

The CRO Connection

Although vendors have made their MS systems increasingly user-friendly, some drug discovery and development groups — particularly in small and medium- sized pharmas — prefer to keep the technology at a distance. They turn to contract research organizations (CROs) such as Covance, MDS Pharma Services, PPD, and Quintitles for MS and related services.

MDS, for example, provides a full spectrum of resources, including mass spectrometry, to meet the pharmaceutical and biotechnology industries' drug discovery and development needs. "While access to capacity is certainly one advantage," Mautz says, "we find that our sponsors value access to expertise and our experience in handling compounds with less than ideal biophysical properties."

While multiple lines of business in MDS use mass spectrometry, Mautz's team has a particular focus on discovery ADME. "We offer standard quantitative work," he says. "And because we are an ADME group, we move from quantitative into qualitative aspects of mass spectrometry. When we ask what are the metabolites, fragmentation analysis by mass spectrometry is the most powerful tool to provide an answer."

In addition, Mautz's team is working on MALDI imaging/profiling for analysis of tissues. "MALDI imaging represents a new and exciting technique for improving our understanding of distribution while still in discovery," Mautz explains. "If the ADME sciences can provide a better distribution picture earlier on, discovery teams may choose compounds that are more likely to get to the site of actions or to avoid those going somewhere they don't want them to go. This may in turn improve the clinical failure rates."

Mass spectrometry has rapidly found a new home in drug discovery and development. Manufacturers who have already developed a wide range of capable systems will continue to focus on integrating different components and streamlining upstream sample preparation steps.

Peter Gwynne (pgwynne767@aol.com) is a freelance science writer based on Cape Cod, Massachusetts, U.S.A. Gary Heebner (gheebner@cell-associates.com) is a marketing consultant with Cell Associates in St. Louis, Missouri, U.S.A.

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