How useful are 6600 QTofs in a Proteomics Core Facility?

Posted on 13th May, 2020

 

Ten years ago, it would have been hard to imagine a Core Facility MS lab without access to a QTOF. Proteomics had become well established and researchers wanted to know what?, how much?, how modified? and what with?, amongst other biological questions. A combination of a Triple Quad and QTOF enabled most of these questions to be answered or at least attempted with some degree of success. Indeed, back in 2004, our lab opted for a combination of QSTAR and QTRAP to fulfil the group’s proteomic needs.

 

But what of today? How far away from this situation are we now?

 

Well, whilst the unique configuration of the QTRAP (triple quad with linear ion trap) is

still going strong – albeit several generations later – the QSTAR is sadly no more.

 

So, what does a core lab do to fill this gap?

 

The (relatively) new kid on the block, is of course the QExactive with orbitrap detection. The mass of marketing hype could make the less experienced mass spectrometrist feel that QTOFs have had their day and are no longer fit for purpose. That, perhaps, technology had overtaken them and other configurations were the state-of-the-art way to go. However, this assumption does not stand up to close scrutiny. Nanoflow columns become overloaded at around 1 µg of digest, with 500 ng being a sweet spot for most complex digests. With this amount of material, a super-fast QTOF, such as the TripleTOF 6600, can acquire data at 50 Hz, ie 100 ms Full Scan plus 50 x 20 ms MS/MS scans.

 

How good is this in the context of other option?

 

Orbitrap (OT) analysers rely on the generation of an image current for ion detection. Without the critical number of ions (ion flux), no image current is generated and therefore no signal detected. The detection effectively falls off a cliff when sufficient ion numbers are not present. In recent years, OT detectors with higher fields have facilitated a faster scanning capability. However, the utility of these faster scanning routines is severely limited because their efficient employment necessitates shorter ion fill times that often render an inadequate image current with the resulting spectra devoid of information (as mentioned above). There exists therefore an apparent trade-off between speed of acquisition and MS/MS signal. The headline rates of scanning at 40 Hz seem attractive until you interrogate the MS2 spectra to find you have acquired large numbers of spectra, empty of analytical value.

 

What about the unrivalled resolution?

In addition to the increased number of “empty” spectra, shorter ion fill times result in a significant fall in resolution. At 40 Hz, a resolution at 200 m/z (the best marketing figure to quote) is 7.5k i.e. not dissimilar to our old QSTAR from 2004, and unlike a QTOF, this resolution falls with increasing m/z values. A TripleTOF can maintain its 40,000 resolution across the entire spectral mass range with no drop-off. And that’s in both MS and MS/MS modes.

 

Let’s return to the original premise that all Core Facility Labs should have access to a fast QTOF. How versatile are they?

 

In short, they are remarkably versatile. Data-Dependent Acquisition (DDA) of complex mixtures at 50 Hz speaks for itself, but that’s only a small part of the picture. SWATH-enabled Data-Independent Acquisition (DIA) is rapidly becoming a challenger to DDA for the in-depth analysis complex samples. The fast-scanning QTOF provides the perfect platform for such analysis strategies with a large number of SWATH windows being possible on the chromatographic timeframe.

 

Anything else?

 

Well, whilst SRMs on a triple quadrupole instrument provide a highly sensitive method of detection/quantification of lower abundance peptides, method set-up, especially for large numbers of transition is very time-intensive. Running a TripleTOF in MRMHR mode offers much easier method set-up along with tremendous selectivity due to the 40,000 resolution of product ions across the mass range.

 

This begs the question “Can a Core Facility Lab really do without a fast QTOF”?

 

Having the fast-scanning QTOF along with SWATH-enabled DIA fulfils two major requirements of a core facility lab – flexibility and robustness. Analysis of complex samples (microflow LC with DIA or DDA), gel bands such as pull-downs (nanoflow LC with DIA, DDA or MRMHR), or enriched phospho samples are all covered by the same analyser. This well-established platform has evolved over many generations to become THE essential mass spectrometer for proteomics analysis. Although the TripleTOF is a distant relative of the QSTAR the major advancements in hardware, software and methods of operation mean that it is now capable of things only dreamed of only a decade ago.

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