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.
Pop quiz: Which technique should you use to quantify a single protein in a complex mixture? Assuming an antibody to the protein is available, you likely answered either ELISA or Western blotting.
Your answer probably wouldn’t change for five proteins, or even 10—again, assuming antibodies are available. But as neither ELISA nor Westerns can be multiplexed much beyond a handful of proteins, workload and complexity largely increase with each new target. And of course, antibodies aren’t always available for every protein.
For higher levels of multiplexing, researchers in the proteomics community increasingly are keeping their antibodies in the freezer (PDF) and warming up their analytical hardware instead [1]. For these researchers, the key to multiplexing protein detection lies in a method long popular with small-molecule chemists and increasingly popular in the life sciences: SRM.
The ABCs of SRM
SRM, or single-reaction monitoring (also called selected reaction monitoring), is a triple-quadrupole mass spectrometry workflow in which the instrument is tuned to isolate a specific molecular ion—in this case, a peptide—in the first quadrupole, smash it into pieces in the collision cell and quantify particular fragment ions in the third quadrupole.
“It’s basically the mass spec equivalent of a Western blot,” says Michael MacCoss, professor of genome sciences at the University of Washington. Instead of antibodies providing specificity, he explains, only ions with specific target masses and fragment ions give a signal.
The technique offers several advantages. It is highly sensitive, says Jim Langridge, director of health sciences at Waters Corp., in part because the process is highly targeted and only looks at a single ion at a time. “So we remove a lot of the inherent chemical noise, giving very good limits of quantitation and limits of detection,” he says. The technique also has an “extremely high linear dynamic range” and “very good quantitative signal,” Langridge adds. “In the pharmaceutical industry, [quantification with the] triple quad is the gold standard.”
Perhaps most significantly, SRM can be highly multiplexed. Researchers can screen for anywhere from dozens to hundreds of targets by programming the instrument to scan for numerous so-called transitions—pairings of precursor and fragment ion pairs—cycling from one to the next like tuning a radio dial. (This is sometimes called MRM, or multiple-reaction monitoring.)
According to Langridge, Waters’ top-of-the-line Xevo TQ-S triple quadrupole can cycle through up to 16,000 MRM transitions. In one recent study, Steve Carr, director of the proteomics platform at the Broad Institute of MIT and Harvard, coupled the instrument with “heated, long, fused silica columns (>30 cm) packed with 1.9 μm of packing material” to quantify 2,400 transitions representing 800 proteins in a single run [2].
Parallel reaction monitoring
A related and emerging technique uses hybrid mass spectrometer configurations such as quadrupole-time-of-flights (qTOFs) and quadrupole-Orbitraps. The process is still targeted—only the products of a single parent ion are interrogated—but it produces richer data on the back end.
AB SCIEX’s MRM3 workflow, for instance, uses the company’s QTRAP quadrupole-linear ion trap configuration for more confident peptide identification by using two molecular transitions per parent ion rather than one.
An alternative approach, parallel reaction monitoring (PRM) collects a full spectral scan for selected parent ions in a qTOF or quadrupole-Orbitrap [3]. This offers some notable advantages over standard SRM. For one thing, an SRM assay is targeted—the instrument is programmed to look for specific transitions, and it ignores everything else. But it takes work to figure out which are the best SRM transitions for each parent peptide, and the instrument must skip from transition to transition, examining each one serially. With PRM, researchers can simply collect all the data up-front and then work out which peptides are most informative and quantitative after the fact, a faster and more comprehensive workflow.
Another advantage is that qTOFs (such as AB SCIEX’s TripleTOF® systems) and q-Orbitrap instruments (such as Thermo Scientific’s Q Exactive™) offer far higher resolution and mass accuracy than triple quads, which typically feature "unit-mass" resolution. That leads to more confident peptide identifications and characterization, says Reiko Kiyonami, senior marketing specialist for life sciences mass spectrometry at Thermo Fisher Scientific.
“The benefit of PRM on the Orbitrap is selectivity, accuracy, confidence, and it’s easy to set up an instrument method,” Kiyonami says. The approach also offers quantitative sensitivity on par with quadrupoles while sample complexity is high, she adds. Triple-quads, however, are most cost-effective, and Kiyonami recommends them, including Thermo’s new TSQ Quantiva™, for routine or high-throughput quantification tasks. “It’s a workhorse.”
Scheduling software
The TSQ Quantiva features relatively high mass resolution for a triple quad, 0.2 Da, and the ability to cycle through 500 transitions per second, Kiyonami says. But how does one actually select all those transitions and program the instrument to find them?
That’s the job of scheduling software. Combining LC elution data, SRM transition descriptions and collisional energy specifications, these tools simplify the task of instructing the instrument of precisely which transitions it should be looking for, and when. (It makes no sense to look for a particular transition at 5 minutes into the LC run, for instance, if you know the molecule won’t elute until minute 8.)
One popular option for peptide SRM method development is Skyline. An open-source package developed in MacCoss’ lab at the University of Washington, Skyline predicts precursor and fragment ion masses as well as the collision energies required to generate those fragments and outputs the method to the mass spectrometer. It can also build fragment libraries for future assay development, facilitate quantification and data analysis and “has an incredible amount of visualization tools,” says MacCoss.
Now at version 2.5, Skyline is compatible with instruments from AB SCIEX, Agilent Technologies, Bruker, Shimadzu, Thermo Scientific and Waters, and has been installed more than 28,000 times since 2009, MacCoss says. “We’re now at the point where Skyline is started up more than 6,000 times per week.”
Some companies, including AB SCIEX, offer Skyline with their own hardware. Others offer proprietary software, as well, such as Thermo Scientific’s Pinpoint™ 1.4, which was updated at the recent ASMS meeting to handle glycopeptide analysis, says Kiyonami.
Picking an instrument
As mass specs go, triple quads are relatively inexpensive. But they're hardly cheap, so when selecting one for SRM work Kiyonami encourages potential buyers to consider such variables as sensitivity, reproducibility, speed, selectivity and price.
For the most part, though, triple quads increasingly are becoming “commodity-type instruments,” says MacCoss, who describes his own lab as “very Thermo-centric.” Thus, he recommends users focus less on specifications and more on the big picture, such as confidence in the vendor and instrument ease of use.
In late 2012 Nature Methods named targeted proteomics its Method of the Year [4]. The technology, the editors noted, was not, per se, new. But it had reached a tipping point. "Mass spectrometry–based proteomics has long been viewed as far too complex for anyone but specialists to apply. A discovery-based proteomics experiment requires highly sophisticated bioinformatics aptitude to extract reliable results from the data. On the other hand, targeted mass spectrometry experiments are in principle simple to perform, once reliable protein assays are available, and data analysis is relatively straightforward."
Given those advantages, a mature toolset and the increasing desire among many researchers to look at ever more proteins at once, adoption of targeted proteomics, and SRM, should only increase.
References
[1] Aebersold, R, et al., “Western blots versus selected reaction monitoring assays: Time to turn the tables?” Mol Cell Proteomics, 12:2381–2, 2013. [PubMed ID: 23756428]
[2] Burgess, MW, et al., “Simplified and efficient quantification of low-abundance proteins at very high multiplex via targeted mass spectrometry,” Mol Cell Proteomics, 13:1137-49, 2014. [PubMed ID: 24522978]
[3] Peterson, AC, et al., “Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics,” Mol Cell Proteomics, 11:1475-88, 2012. [PubMed ID: 22865924]
[4] "Method of the Year 2012," Nat Methods, 10:1, 2013. [PubMed ID: 23547284]