by Jeffrey M. Perkel
For his 2007 study of insulin signaling targets in Caenorhabditis elegans1, Scripps Research Institute Professor John Yates III used two techniques to quantify changes in the abundance of several different proteins over time.
One was Western blotting, the gold standard for this type of problem, which relies on specific antibodies for each protein studied. The other was single reaction monitoring (SRM), a mass spectrometric approach that monitors a molecule's abundance by its degradation into specific fragments.
Western blotting may be tried-and-true, but SRM (or, as it is sometimes called, MRM, for multiple reaction monitoring) holds a significant advantage: it requires no antibodies and thus is amenable to studying proteins for which no antibodies exist.
"Increasingly I think these sorts of experiments will replace Western blots as a way to quantify proteins," Yates says, noting that though a mass spectrometer is a significant capital investment, it is balanced by the time and cost penalty inherent in the creation of custom antibodies.
Though Yates used an LTQ linear ion trap mass spectrometer from ThermoFisher Scientific for this particular study, triple quadrupoles are the more traditional choice.
"Triple-quads in general have better dynamic range and sensitivity than do ion traps for these sorts of experiments," says Yates, who has since purchased a Thermo Scientific TSQ Quantum Ultra triple quad.
Indeed, the triple-quad's design is uniquely suited to SRM. As their name implies, triple quadrupoles are built of three quadrupole mass spectrometers arranged in tandem. Quadrupoles are a bit like radios; by tuning the instrument, you can determine which ions are able to traverse the quadrupole. In this case, the first quad (Q1) is tuned to select parent ion(s) of interest. Those ions then pass into the second quadrupole, a collision chamber in which impacts with gas molecules cause the ion to shatter into characteristic pieces. These fragment ions then pass into the third quadrupole (Q3), which monitors for the presence of ions characteristic of the parent molecule.
Suppose, for instance, a researcher wanted to quantify the levels of an ion of mass-to-charge (m/z) ratio 523.9. Several molecules might have the same m/z, especially in complex biological fluids like blood and serum. But (in theory) only one will fragment to produce ions of, say, m/z 323.4 and 215.8. In practice, researchers can monitor anywhere from one to hundreds or thousands of such "transitions," and both small molecules (metabolites or drug compounds) and peptides may be so analyzed.
A key advantage of SRM/MRM is that, unlike many mass spec approaches, the technique is quantitative, not qualitative. A given ion's spectral signal can be converted into abundance by use of a standard curve and/or internal standards. Conversely, however, it is a targeted, hypothesis-driven technique, rather than a discovery-based fishing expedition. In other words, to use MRM, you need to know what you are looking for.
Thermo Scientific offers three triple-quad systems for life science applications, according to Lester Taylor, the company's director of marketing for life sciences mass spectrometry: the TSQ Quantum Access, TSQ Quantum Ultra, and TSQ Vantage. These systems provide entry, middle, and high-end performance, Taylor explains.
The TSQ Vantage, released earlier this year at the American Society for Mass Spectrometry (ASMS) annual meeting in Denver, enables high mass accuracy SRM, says Taylor. Typical triple-quadrupoles provide nominal mass resolution, meaning, for example, that they can distinguish parent ions of mass-to-charge (m/z) ratio 300 from ions of m/z 299 or 301. The TSQ Vantage, however, can resolve ions of 300.0 from 299.9 and 300.1, thereby providing greater confidence in the resulting data.
That increased resolution "has to do with the quadrupoles we use in our system," Taylor says. Those "hyperbolic cross-sectioned quadrupoles … more closely approximate the ideal field shape," he continues. "It's like looking through a lens with no chromatic aberrations."
Applied Biosystems' MIDAS workflow helps to develop better MRMs to provide more confident protein quantification, says Christie Hunter, senior staff scientist in the company's proteomics group.
Available only on Applied Biosystems' 3200 and 4000 QTRAP triple quad-ion trap hybrid, MIDAS, or MRM-initiated detection and sequencing, provides full MS/MS mass scans to ensure that the parent ions you count are in fact what you think they are, Hunter explains.
"When you get your [MRM] signal, it triggers the QTRAP to switch into linear ion trap mode, which collects a full-scan MS/MS, and because it is in ion-trap mode, it has orders of magnitude higher sensitivity than if you did this on a quadrupole, typically 100-to-500 times better sensitivity," she says.
At this year's ASMS meeting, Applied Biosystems launched two software packages specifically to simplify development of MRM assays, Hunter says: the "scheduled MRM algorithm" and MRM Pilot.
Suppose you want to monitor multiple MRM transitions. Traditionally, users program the mass spectrometer to switch rapidly from one m/z to another, as it looks for ions for the ions corresponding to each compound analyzed. As a result, the more ions you want to measure, the less time the instrument can "dwell" on any one ion, and the lower the sensitivity.
Scheduled MRM divides that task into smaller batches, by programming the instrument to look for each ion only when it is expected to enter the instrument from an upstream liquid chromatography system. For instance, if you wanted to monitor 20 ions, which emerge over the course of a 10-minute LC run, you might instruct the instrument to scan for each ion during twenty different two-minute or longer time slices; each time slice is optimized for the retention time of each compound.
The resulting increase in sensitivity can be significant. According to Keith Waddell, LC/MS applications solution manager at Agilent Technologies, reducing the number of MRMs per time-slice by a factor of four improves sensitivity two-fold.
The "scheduled MRM algorithm," Hunter says, uses Q1 and Q3 m/z values, plus chromatographic retention times for each desired ion, to build a map of the experiment and optimize dwell times. The MRM Pilot, in turn, uses the MIDAS workflow to optimize the best MRM transition, which it then passes to the scheduled MRM algorithm.
Users of Waters' new Xevo TQ (tandem quad) mass spectrometer can use MRM to trigger collection of full fragment ion spectra, a mode called PICS ("product ion confirmation spectral" mode). "These spectra could be compared to a previously acquired reference spectrum to provide extra confirmation of the identity of the compound being quantified using the MRM data," Little explains.
Released at ASMS 2008, the Xevo TQ MS replaces the company's earlier Quattro Premier XE and represents the high end of a three-tier triple-quad product line, according to Little. At the bottom end is the Quattro micro, which is optimized for use with standard HPLC. The newer, mid-range Acuity TQD has the collision cell and detection speed necessary to work with Waters' faster Acuity UPLC systems, as does the Xevo TQ, though the latter also boasts higher sensitivity.
Agilent Technologies has two instruments in its 6400 series line of triple-quads. The 6410B is "a very capable, robust, workhorse instrument," says Waddell. For those who need greater sensitivity, however, he recommends the 6460, introduced at ASMS. Both mass spectrometers can be hooked up either to a conventional LC system, or to a microfluidic peripheral called an HPLC Chip, which enables nanoflow applications.
"The primary performance difference [between the two instruments] is that the 6460 has some new technology, which we call Agilent Jet Stream Technology," he explains. The Agilent Jet Stream "allows collimation of the ion stream so you can take more ions into the analyzer," he continues. That "significantly enhances sensitivity," particularly for conventional (as opposed to nano) flow applications of up to 1 ml/min.
Like its competitors, Agilent's triple-quads can perform scheduled MRM. According to Waddell, the 6460 supports 500 MRMs per time segment, which can be as short as 0.1 minutes each. Surprisingly, adds Ken Miller, senior global marketing director of Agilent’s LC/MS Division, some researchers actually need that kind of performance.
"People come up with lists of proteins they want to monitor in samples," Miller says. "You can go in and do targeted MRMs on those peptides, and typically you want many proteins per sample and many peptides per protein and many transitions per peptide, so the number of MRMs adds up surprisingly quickly."
At the moment, scheduling those MRMs can be a chore—but not for long; later this summer, Agilent will release Optimizer, a software module for its MassHunter package that simplifies the development and optimization of MRM methods.
References:
1MQ Dong et al., “Quantitative mass spectrometry identifies insulin signaling targets in C. elegans,” Science, 3;317(5838):603-4, 2007.