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UW-Madison Study Suggests High-Res Mass Spec Can Match Triple Quads for Protein Quantitation


New research by University of Wisconsin-Madison researchers suggests that high-resolution mass spectrometry now offers equivalent or better performance for protein quantitation as triple quadrupole-based selected-reaction monitoring methods.

In a study published last week as a "Paper in Press" at Molecular & Cellular Proteomics, scientists led by UW-Madison researcher Josh Coon used a targeted proteomic technique they termed parallel reaction monitoring, or PRM, to perform protein quantitation on a Thermo Fisher Scientific Q Exactive instrument. They then compared the Q Exactive results to quantitation done on a Thermo Scientific Quantum Discovery Max triple quadrupole, finding, Coon told ProteoMonitor, that the Q Exactive typically met or exceeded the performance of the triple quad.

"I think our data shows that for several synthetic peptides that we studied, the sensitivity and precision and linearity on the Q Exactive was almost identical," he said. "Sometimes [it was] better, sometimes [it was] a little worse, but I would say that if you look across all those figures of merit, it's performing at the same level."

Use of high-resolution machines like Q-TOFs and the Q Exactive has grown in recent years due to the relative ease of assay development and the ability to obtain both quantitative and qualitative measurements at the same time. However, despite this growing interest, many researchers and vendors have maintained that triple quad-based SRM remained the gold standard for targeted quantitation.

The MCP paper calls that assumption into question, Coon suggested.

In a triple quad-based SRM assay, the first quadrupole isolates a target precursor ion, which is then fragmented in the second quadrupole, after which a set of pre-selected product ions are detected in the third quadrupole. The PRM approach, by contrast, uses the upfront quadrupole of a Q TOF or Q Exactive machine to isolate a target precursor ion, but then monitors not just a few, but all of the resulting product ions using its time-of-flight or Orbitrap analyzer.

This offers several advantages compared to conventional SRM. For example, because the technique monitors all product ions instead of just a pre-selected few, researchers don't have to determine upfront what the best transitions to monitor will be, significantly reducing assay development time.

On a related note, the larger number of product ions monitored via PRM should improve the specificity of the analysis, since more transitions will be available to confirm a peptide ID. This might also reduce the effects of co-isolating background peptides.

Coon said that the technique could also help clarify some data analysis issues inherent in traditional SRM-MS.

"When you're dealing with low-resolution [triple-quadrupole] data, it's not trivial to say – particularly when you're looking at low-level targets in high background, when [a target] is there and when it isn't," he said. "There's not a clean statistical approach you can use like, for example, a decoy database."

"But we figured out that with our accurate mass MS2 data, it's pretty straightforward to confirm the presence or absence of a target in a spectrum, even in the presence of background because there's very little noise in these accurate mass channels," he added. "So I think one of the other things that came out of this is that processing accurate mass targeted data is a whole lot easier, and you can do it in a more defined way than with the SRM-type data."

Coon noted that while triple quadrupole detectors offer more raw sensitivity than a high-resolution analyzer like an Orbitrap, the higher specificity of the latter machine compensates for this.

"The Orbitrap isn't as sensitive, but it can [better] discriminate the ions of your target from the background, so what you lose in sensitivity you gain in selectivity, and it turns out you break even," he said.

Conceptually, the PRM technique is very similar to the Swath data acquisition technique released recently by AB Sciex for use on its TripleTOF 5600+ instrument, Institute for Systems Biology researcher Rob Moritz told ProteoMonitor. The main difference, he noted, is that while Swath uses wide fragmentation windows to analyze all the analytes in a sample, PRM uses very narrow windows in order to "hone in on discrete peptides."

In an interview with ProteoMonitor last year, Swiss Federal Institute of Technology Zurich researcher Ruedi Aebersold, one of the developers of the Swath method, said that that technique was also approaching parity with triple quad-based SRM, noting that an initial study using the technique showed it could identify proteins in a complex sample over four orders of magnitude of dynamic range with quantitation equivalent to that provided by SRM (PM 7/22/2011).

"In terms of sensitivity of detection, [Swath is] probably a factor of three or four off from SRM, but it's approaching the performance of SRM," he said.

Coon is not the only researcher investigating the Q Exactive for targeted quantitation. Even before the official launch of the instrument at the 2011 American Society for Mass Spectrometry meeting, Bruno Domon, director of the Luxembourg Clinical Proteomics Center, had begun comparing its performance to SRM-MS, using both the machine's SIM mode, which quantitates based on precursor ions, and an MS/MS workflow similar to the PRM approach.

At last year's annual meeting of the Human Proteome Organization, Domon presented results indicating that the instrument could compete with triple quads for quantitation (PM 9/16/2011).

In an e-mail to ProteoMonitor this week, he noted that his group has several papers under review presenting new findings comparing quantitation on the Q Exactive to SRM and that, while there are exceptions, they "usually obtain comparable or better performance" with the Q Exactive compared to a triple quad.

"High-resolution methods clearly add a new dimension in quantitative analysis in terms of increased selectivity," he said. "The workflows are different and easier in terms of experimental design, and allow more flexibility in the data analysis or re-analysis."

Domon noted that while assay development is simpler on the high-resolution machines, some setting and optimizing of instrument parameters is required. Coon said, however, that in the MCP paper his team intentionally spent little time tweaking the Q Exactive's parameters in order to demonstrate what could be done with a minimum of optimization.

"You could probably do a bit better here and there by tweaking things, but I think the point is you shouldn't have to," he said. "So we intentionally didn't do that ... Without much work we did pretty well."

Coon noted that because the Quantum Discovery Max instrument they used for their SRM analyses is several years old, the results didn't take into account the improvements in triple quad sensitivity made since then. However, he said, he expected that because the Q Exactive largely outperformed that instrument in the MCP paper, it would likely break even with new triple quads.

Although Coon used the Q Exactive for the work, the PRM technique should be applicable across a range of instrument types, he said. "A Q-TOF ought to be able to perform in the same way. You could also do the same thing on an ion trap-Orbitrap hybrid. Any device that can do an isolation, a fragmentation, and a high-resolution MS2 scan can in theory do PRM-type scanning."

Moving forward, Coon said, he hopes to combine the PRM technique with work his group published last year on using accurate mass instruments to make spectral matches in real time, allowing the machine to make peptide IDs as it goes.

"One of the benefits of doing that is that if you know in real time which peptides are eluting into the instrument you can then predict which of your targets will be eluting next and you can trigger a PRM scan on the target and it can all be automated," he said. "You can know in real time where you are [in your run] and so it can all be done in an automated way and tolerant to big changes in chromatography.

"So our goal is to merge the PRM approach with that real-time scheduling approach, and then you can just tell [the instrument] what targets you care about and it goes and finds them and you don't have to schedule them or pick transitions and you can do it all with the same sensitivity as a triple quad," he said.