Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
A mass spectrometer is basically a very high-tech balance: Put in an ion, and the system will tell you what it weighs. But just as the scale in your grocery store’s vegetable aisle lacks the precision of your laboratory Mettler, so it is with mass spectrometers.
Any mass spectrometer is capable of what’s called “nominal” mass measurement. Given an isolated molecular species, a triple quadrupole or ion trap can report its mass to within 1 m/z (m/z is the ratio of an ion’s mass to its charge; a molecule of mass 1,000 containing two positive charges will yield a peak of m/z 500).
But many samples—blood sera, for instance, or food extracts—aren’t so simple. Maciej Bromirski, global product marketing manager for Q Exactive products, supports the “applied markets” side of the life science mass spectrometry business at Thermo Fisher Scientific, including such applications as food-safety analysis, environmental analysis, forensic toxicology, clinical research and small-molecule pharma. In all these areas, Bromirski says, sample “matrix”—the chemical background of the sample itself—can complicate analysis. Researchers interested in identifying pesticides in tea, for instance, must be able to efficiently find and identify that one peak, often at relatively low concentrations, in an overwhelming background of hundreds of compounds. The problem is comparably complex in the research market, he adds, especially in the fields of metabolomics and proteomics.
Another complication: Many samples contain isobaric species, or molecular entities of more or less equivalent mass, which cannot be resolved on a nominal mass analyzer.
Acetyl and trimethyl modifications, for instance, add 42.0106 and 42.0470 Da, respectively, to a peptide’s mass. Similarly, many pesticides and pharmaceuticals can be processed in the body into a series of discrete, but highly isobaric, species, only some of which may be toxic.
To overcome both issues, instrument vendors offer high-performance mass spectrometers. These systems are more commonly (though not exclusively) used for research work. They tend to cost considerably more than their “nominal mass” cousins and historically were intended for sophisticated users who are well versed in mass spec operation. But for those needing to eke every ounce of insight from their precious samples, there’s often no substitute.
High-performance configurations
High-performance mass spectrometry typically encompasses a handful of geometries, including quadrupole-time-of-flight (qTOF) hybrids, Orbitraps and Fourier transform-ion cyclotron resonance (FTICR) mass spectrometers. The former is like a triple-quadrupole system in which the final mass analyzer has been replaced with a high-resolution TOF; Orbitraps and FTICRs are basically exceptionally high-resolution electrostatic traps, capable of multiple rounds of ion isolation, analysis and fragmentation.
Whatever the configuration, these systems all excel in two parameters, mass accuracy and resolution. The former is expressed in “parts per million” (ppm), a reflection of how close the measured mass is to the “actual” mass, so smaller values are better; the latter is typically measured in terms of “full-width at half-maximum” (FWHM), a measure of the ability to resolve the smallest mass difference between peaks of equal magnitude, with larger numbers equating to higher resolution. These parameters are somewhat interconnected, as the ability to accurately measure an ion can depend on your ability to resolve it from interferences.
“The more you’re able to pull interfering peaks apart, the more accurate your measurements are,” says Jim Langridge, health sciences director at Waters Corp.
According to Langridge, researchers traditionally have considered resolution greater than about 5,000 FWHM to be high resolution compared to quadrupole technology. The specification sheet for the Waters SYNAPT G2-Si qTOF, for instance, lists a positive ion-mode resolution of 60,000 FWHM “measured on the (M + 6H)6+ isotope cluster from bovine insulin (m/z 956),” and a mass accuracy of 1 ppm “based on 10 consecutive repeat measurements of the [M + Na]+ ion of raffinose (m/z 527.1588).”
One should note the caveats in these definitions, says Langridge.
Resolution and accuracy are not necessarily fixed quantities, as they depend on, among other things, the mass of the ion being measured and the time dedicated to measuring it.
That time latter factor can become particularly important when analyzing compounds as they elute off chromatography columns, as today’s fast chromatography systems can produce peaks that may be only a few seconds wide. But time-of-flight analyzers, at least, can cope with that speed, he says. “You’re pushing your instruments to acquire data faster and faster, and the beauty of TOF is that resolution is constant, irrespective of scan rate.”
On-board separation and fragmentation
Though most mass spectrometers can be coupled to online chromatography systems, some, including the SYNAPT G2-Si and Vion IMS QTof systems from Waters and the SCIEX TripleTOF® 6600, also offer additional on-board separation via a process called ion mobility separation.
Water's ion mobility separation (IMS) system resolves molecules based not on mass-to-charge but rather their three-dimensional shape. In the SYNAPT G2-Si, the IMS chamber is located between the quadrupole and TOF, while the VION places the IMS in front of the quad, meaning it can be used to provide an orthogonal separation prior to the first mass filtration step.
SCIEX's SelexION® technology is a "differential mobility" separation technique that occurs at atomospheric pressure and sits between the ion source and the entrance to the mass spec itself. Among other things, says Christie Hunter, director of omics applications at SCIEX, that makes differential mobility spectrometry useful in teasing apart similar lipids. “Differential mobility spectrometry allows lipid classes to be separated by the dipole moment of their head groups in the gas phase, allowing the effects of isobaric overlap to be reduced,” she explains.
Another key distinguishing factor between the various mass spec instruments is the kind of fragmentation they allow. All of the high-performance systems discussed here enable tandem mass spec (MS/MS) workflows, in which an ion is isolated, fragmented and measured. But the kinds of fragmentation researchers can use vary from system to system.
Thermo Fisher Scientific’s top-of-the-line, research-grade Orbitrap, the Orbitrap Fusion Lumos “tribrid,” for instance, offers collision-induced dissociation (CID) and high-energy collisional dissociation (HCD), as well as a popular proteomics strategy called electron transfer dissociation (ETD) and a hybrid of the latter two called EThcD. “It’s really a flagship for proteomics,” Bromirski shares, , “as well as biopharma applications where detailed protein structural characterization is required.” The company’s more affordable Q Exactive Focus Orbitrap, on the other hand, which is intended for more routine applications, has an HCD cell for fragmentation and screening applications plus high-performance features for quantitation.
The Synapt provides both CID and ETD fragmentation, and has also been coupled with surface-induced dissociation(SID) for protein complex analysis, while the benchtop VION provides CID fragmentation.
Look to the future
As with all instrument purchases, the one question researchers must answer as they shop is whether the instrument will empower them to run their planned experiments, both now and in the future. Therefore, says Bromirski, researchers should consider such variables as sensitivity, resolution, robustness and flexibility. An instrument that can, for instance, perform both quantitative and qualitative analyses simultaneously enables researchers to squeeze more information from less sample, as it eliminates the need for multiple runs on multiple instruments.
But, says Langridge, a mass spec is also more than just the sum of its specifications, so consider the instrument holistically. For instance, will you require sophisticated upstream separation? Do you have time to linger over samples, or will you be linking the analyzer to a high-speed chromatography separation? “Don’t be led down the path by impressive-sounding specifications,” he says. “Look instead at the instrument in terms of what you’re trying to do and the questions you are trying to answer.”