These days everyone seems to be doing it–mass spectrometry, that is. As protein chemists, molecular biologists, and even crime scene investigators rush to adopt this once-esoteric technology, the basic science revolution that made mass spectrometry user-friendly is striving to make it even more powerful.

By Alan Dove

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.

The revolution in mass spectometry (MS) started in places like Carlos Lebrilla's laboratory at the University of California at Davis, California. About 15 years ago, when the young chemist was a new faculty member at the school, he hung a sign on his most expensive piece of equipment. The sign, still hanging today, boldly advertised "Mass Spectrometry for the Masses." It was not merely the expression of an inside joke—it was more like a mission statement.

"When I started my career, I saw mass spectrometry going the same way that NMR went," says Lebrilla, who is now a full professor. Initially used only by physical chemists to determine molecular structures, nuclear magnetic resonance (NMR), rebranded as MRI, is now nearly as ubiquitous in clinical settings as the X-ray. If it follows the same path, mass spectrometry, an astonishingly fast, sensitive method for identifying individual molecules in complex mixtures, could revolutionize everything from clinical diagnostic testing to crime scene investigation. Unfortunately, the technique has long been considered too expensive, too finicky, and too difficult for deployment outside of specialized spectroscopy laboratories.

Over the past few years, though, mass spectroscopists and equipment manufacturers have been chiseling away at those problems, and a new crop of relatively inexpensive, compact, user-friendly devices is finally bringing the method within reach of ordinary researchers. But a mass spectrometer is still a substantial purchase, so scientists considering bringing such a system into their labs should investigate all of their options first.

Getting Your Charge On

Since J.J. Thomson's invention of the technique at the turn of the 20th century, scientists have developed numerous variants of mass spectrometry, but all of them follow the same essential process. The device draws a sample into a component called the ionization source, which converts molecules in the sample into ions. The ions then enter a mass analyzer, which applies different electrostatic and magnetic forces to the ions in a vacuum, separating them according to their mass-to-charge (m/z) ratios. Finally, the mass analyzer reports these ratios to a connected computer, which graphs and analyzes the data. From the results, a spectroscopist can identify specific molecular structures. That is, assuming nothing malfunctions.

"The one thing that anybody who has performed mass spectrometry experiments is well aware of is mass spectrometers tended to break fairly often," says Gary Siuzdak, director of the Scripps Center for Mass Spectrometry in La Jolla, California. However, he adds that with newer devices, failures in the core components of the mass spectrometer are relatively rare. Instead, faults seem to now occur more often in the liquid chromatography (LC) system that is usually coupled to the spectrometer for sample preparation and sometimes fails due to the complexity and nature of the samples being injected. "You have to be aware of what the limitations are of the instrument, what you can put into it," says Siuzdak, who also wrote the standard introductory textbook for biological mass spectrometry (The Expanding Role of Mass Spectrometry in Biotechnology, 2006).

Even with careful technique, though, the machine could crash, highlighting one of the key features for which newcomers to mass spectrometry should shop. "It's not simply the technology or the instrument that's going to be important; it's going to be the support structure in getting the instrument installed, training the people on the instrument and the ongoing challenges," says Dave Hicks, senior director of the proteomics business group at Applied Biosystems in Foster City, California. Like other mass spectrometer makers, Applied Biosystems provides multiple levels of support with all of its devices, from field technicians who can fix common glitches to staff scientists who can answer high-level questions about experimental design and data analysis.

Besides a deep support network, researchers should look for the specific features they need, based on the types of samples they'll be analyzing. Though there are several kinds of ion sources and mass analyzers, workers in different fields tend to coalesce around specific arrangements.

In basic proteomics research, matrix assisted laser desorption/ionization (MALDI) has become the predominant ion source, and the mass analyzer is often an ion trap. MALDI is good for ionizing highly complex samples from protein-friendly buffers, and the ion trap can eject specific ion species from the sample seriatim, until all of the ions have been analyzed. It's a particularly good arrangement for researchers who aren't sure what they're looking for, and need to capture everything.

Scientists working in a more applied setting, such as a clinical lab, usually need to process samples more quickly than their basic research colleagues, but may only need to detect a comparatively small, defined set of analytes. For these users, electrospray ionization may be a good choice, combined with a triple-quadrupole mass analyzer. The triple-quadrupole design isn't quite as versatile as the ion trap, but it is efficient and inexpensive.

Less Than a Lamborghini

Of course, the term "inexpensive" is relative. For an entry-level triple-quadrupole mass spectrometer, complete with a liquid chromatography system capable of handling a particular class of samples, expect to spend around $100,000. Adding more features, more speed, or more versatility can quickly double that figure.

A six-digit purchase will certainly give most researchers pause, especially in grant-starved academic labs, but the prices on the latest entry-level mass spectrometers are still a historical bargain. "No matter what instrument you buy, you're most likely not going to lose your job because you bought that instrument," says Siuzdak. He adds that a decade ago, when even a cheap mass spectrometer could cost $300,000 and the technology was less reliable, a poor choice could indeed shorten one's career.

Nonetheless, scientists will still want to shop around before settling on a particular machine; there are many similar products to choose from, and each has its own strengths and weaknesses. "I would say that we're all competing head to head, but then the practical result is that we tend to have our respective areas of strength," says Ken Miller, director of marketing for the LC/MS group at Agilent Technologies in Santa Clara, California.

Agilent is a relatively recent entrant in the mass spectrometry field, but the company has quickly developed a large base of users. "I think we've been seeing dramatic growth across whole markets," says Miller, adding that the customers seem to be a mix of those migrating from other brands and those entering mass spectrometry for the first time. In response, Agilent has introduced several entry-level models for different types of users, as well as a series of high-end systems for experienced spectroscopists.

The same demands have driven longtime mass spectrometer maker Thermo Fisher Scientific in Waltham, Massachusetts, to expand its product lines. "Historically one of our key focuses has been on the high-end systems and the high-end users. But we're seeing this transfer of utilization of mass spectrometry to the non-mass-spec experts," says Lester Taylor, director of product marketing for mass spectrometry at the company. Accordingly, Thermo Fisher has introduced a series of new devices designed for these users. "There was a strong need to ensure that the operation and use of these systems is simplified, and that required [revising] the interface and the software that's used to run the system," says Taylor. Indeed, user-friendly software has become a major selling point for most entry-level mass spectrometers (see "Mac, PC, or MS?").

While the streamlined interfaces certainly simplify training—Siuzdak recently taught a high school student to do mass spectrometry experiments during a rotation in his lab—advances in the fundamental technology of mass spectrometry have made the initial purchase more complex. "Even though many people can use them, you need quite a bit of expertise in helping you pick out the one that would best suit your needs," says UC Davis's Lebrilla. He adds that while salespeople are a good source for technical information, prospective buyers should also talk to other researchers in their field who have bought mass spectrometers. The American Society for Mass Spectrometry (www.asms.org) also provides extensive resources for newcomers.

Pocket-sized Power

The growing population of entry-level mass spectroscopists is a diverse crowd, as manufacturers have already discovered. "I represent what we call the proteomics mass spec side of the business," says Applied Biosystems' Hicks. In academic and pharmaceutical proteomics, many researchers are thinking of buying their first mass spectrometer, but it won't be their first exposure to mass spectrometry. "Many of these sites already have some sort of a core lab or a core mass spectrometry facility," Hicks explains, so the question is not whether to use the technology, but whether to bring it into one's own laboratory. Users in this category often need a fairly advanced system, and are probably less intimidated by complex hardware and software.

When advocates like Siuzdak and Lebrilla talk about bringing the technology to the "masses," though, they are usually referring to users who have a specific problem in mind, but little or no background in mass spectrometry. "We are definitely seeing good growth in these markets and increasing adoption of mass spectrometry," says Joe Anacletto, senior director of applied markets and clinical research at Applied Biosystems. Unlike Hicks's customers, Anacletto's often have mass spectrometry thrust upon them by regulatory changes.

Environmental testing, food science, and police forensic laboratories exemplify this trend. As the public, legislatures, and courts have learned more about environmental pollutants and trace evidence, they have raised their standards for detecting and analyzing specific compounds. "This drives these testing labs to look for newer technologies that can handle the broader array of things they're going to be looking for," says Anacletto.

Indeed, the advance of mass spectrometry technology in basic research labs will likely increase the number of important biological and environmental markers even further. With the current crop of robust, sensitive mass spectrometers, biomarker researchers have embarked on ambitious efforts to find new indicators of disease, often by comparing blood or serum samples from patients with and without a particular condition.

Separation Anxiety

Though the mass spectrometry has gotten easier in these studies, the sample preparation problem has been harder to address. Serum, for example, consists mainly of a handful of highly abundant proteins, which tend to swamp sensitive mass spectrometry assays and conceal the much scarcer proteins that might change during pathogenesis.

In response, several companies have introduced immunoaffinity columns specifically designed to remove these abundant proteins. The columns, first introduced around 2003 and now available from companies such as Sigma-Aldrich in St. Louis, Missouri, and Agilent, have also been refined for greater efficiency and selectivity.

Prefiltering serum samples can certainly simplify a biomarker study, but the columns also have some disadvantages. Because they add a step to the sample preparation, they inevitably deplete some molecules by nonspecific binding. If an important biomarker is extremely scarce, it might disappear entirely after serum filtration. Also, scientists hoping to develop assays in animal systems before moving to human trials may find their protocols hard to translate; relatively few immunodepletion columns are available for animal systems, as companies have focused on developing the more popular human serum columns.

The Matrix and Beyond

Serum is not the only place biomarker investigators have been searching, of course. For those more interested in characterizing the markers in solid tissues, a major challenge has been to determine which cells in a complex sample are actually producing particular proteins. One elegant option, recently introduced by mass spectrometry giant Bruker Daltonics of Billerica, Massachusetts, is an integrated tissue imaging, sample preparation, and spectrometry platform called ImagePrep. After fixing a layer of tissue to the system's stage, researchers can overlay a precisely calibrated matrix, dry the sample, and perform MALDI spectrometry in situ. The result is a complete picture of the tissue structure, overlaid with spectra of the proteins expressed in each area.

As the new gear helps generate waves of additional data on everything from biomarkers to environmental chemicals, experts expect demand to increase for even simpler mass spectrometry platforms, especially for use in the field or at a patient's bedside. "The next stage of that development is to make more portable mass spectrometers. There are a lot of other groups looking at miniaturization of mass spectrometers, so you can actually just carry it around with you," says Lebrilla. Spectrometry for the masses, indeed.

Alan Dove is a science writer and editor based in Massachusetts.

DOI: 10.1126/science.opms.p0800022

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Metabolite Identification Software

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Proteomics Mass Spectrometry

With the addition of Compass 1.3 ion trap software, the high-capacity trap HCTultra PTM Discovery System is the first commercial mass spectrometer equipped with electron transfer dissociation (ETD) and proton transfer reaction (PTR) capabilities. The instrument features a unique and innovative ion optics design and chemistry setup for ETD/PTR that allows rapid, routine top-down characterization of large peptides and mid-size proteins in the high-capacity ion trap with superior sensitivity. The usefulness of ETD/PTR is demonstrated by the unambiguous characterization of posttranslational modifica-tions. Compass 1.3 also introduces a novel collision-induced dissociation (CID) fragmentation mode that eliminates the low mass cut-off of ion traps in MS/MS. It enables multiplexed quantitative proteome analyses on Bruker’s high-capacity ion-trap systems. A new AutoMSn mode in Compass 1.3 automatically eliminates the most abundant ions in order to further enhance dynamic range.

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