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Thermo Fisher pSMART Mass Spec Method Aims to Combine Benefits of DIA, DDA

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NEW YORK (GenomeWeb News) – Thermo Fisher Scientific has developed a new mass spec workflow that, according to company researchers, combines the advantages of data-independent and data-dependent analyses.

Called, pSMART, the method collects quantitative data at the MS1 level and then uses DIA-style analysis at the MS2 level for confirmation of the peptide sequence. The approach allows for use of narrowed isolation windows which leads to less complicated spectra, improving peptide identification rates, Mary Lopez, director of Thermo Fisher's Biomarker Research Initiatives in Mass Spectrometry (BRIMS) Center, told ProteoMonitor.

In DDA mass spec, the instrument performs an initial scan of precursor ions entering the instrument and selects a sampling of those ions for fragmentation and generation of MS/MS spectra. Because instruments can't scan quickly enough to acquire all the precursors entering at a given moment, many ions – particularly low-abundance ions – are never selected for MS/MS fragmentation and so are not detected.

In DIA, on the other hand, the mass spec selects broad mass windows and fragments all precursors in that window, allowing the machine to collect MS/MS spectra on all ions in a sample. This means researchers can look at the same sets of peptides across different experiments, allowing for high levels of reproducibility, which is essential to quantitative analyses.

The complexity of the spectra generated by a DIA approache, however, can limit the number of peptides it can identify. Therefore, while DIA has become a popular approach for gathering reproducible, quantitative data on large numbers of peptides, DDA generally remains the optimal approach to go as deep as possible into a particular sample.

Perhaps the most popular DIA approach currently is AB Sciex's Swath method, which is based on methods developed in the lab of Federal Institute of Technology Zurich researcher Ruedi Aebersold. That method cycles through 25-Da isolation windows (AB Sciex's recently released Swath 2.0 allows for variable isolation windows) and uses MS2-level data for identification and quantitation of the peptides.

By contrast, the pSMART approach, which Thermo Fisher introduced at the 2014 American Society for Mass Spectrometry annual meeting and which company researchers including Lopez detailed in a paper published last month in the Journal of Proteome Research, collects quantitative data at the MS1 level and uses MS2 data collected in isolation windows ranging from 6 Da to 21 Da to confirm peptide sequences. Using a Q Exactive, they performed the MS scan at 140,000 resolution and the DIA MS2 scans at 35,000 resolution, acquiring an MS spectrum every 5 seconds along with 20 DIA events.

The notion underlying the method is that the high-resolution, accurate-mass capabilities of the Orbitrap allow for good quantitation at the MS1 level. This allows for MS2 data – acquired independently, as opposed to triggered by the MS1 analysis as in DDA – to be used to confirm the identify of the peptide. That only one MS2 spectra is required for this confirmation allows for generation of high-quality spectra, offering a potential improvement in sensitivity and, consequently, the number of peptide IDs.

"In a nutshell, the Orbitrap allows us to obtain very comprehensive quantitative information in the full scan at at least seven separate points across a peak and then also get confirmatory MS2 information," Lopez said.

The idea for the approach stemmed from the fact that, in looking through the literature and considering their own experiments, the Thermo Fisher researchers noticed that "the complaint the community seems to have with [DDA] is just the stochastic nature of triggering an MS2," Scott Peterman, a senior scientist at BRIMS, told ProteoMonitor.

"I really haven't come across any criticism of doing the quantitation at the MS1 [level] with high resolution and very tight mass tolerance," he said. "It's always the fact that you don't trigger the same peptides for MS2 every time. So if the community agrees that you can quantitate efficiently in MS1... then you just need to add MS2 onto that to [confirm], and then by going to the [pSMART] method you could really maximize those five-dalton [DIA] windows to get high-quality data."

Waters' MSE technique, which was the first commercially available DIA approach when it launched in 2006, also uses MS1 data for quantitation. Instead of using m/z-based fragmentation windows, the Waters method uses co-elution times to match precursor ions to the fragment ions generated, allowing them to be searched against databases to make peptide IDs.

In a recent interview with ProteoMonitor, Stefan Tenzer, a researcher at Johannes-Gutenberg University Mainz who has done significant work developing DIA methods for Waters instruments, noted that – somewhat in contrast to the direction taken by the Thermo Fisher pSMART work – he and his colleagues were working on DIA methods for Waters instruments that used MS2 instead of MS1 for quantitation.

Such an approach could expand the method's dynamic range, he said, due to the fact that it might "have an advantage with lower-[abundance] signals because they are looking at multiple fragments at one time and not just one ion. So we could be able to identify more [proteins] and more reliably in the lower orders of magnitude."

Peterman said, though, that in their work, he and his BRIMS colleagues had found that on the Q Exactive, the pSMART method's combination of MS1 quantitation and confirmation in MS2 allowed them to consistently identify more peptides than either Swath-style DIA or standard DDA methods on the same instrument while also measuring a higher percentage of peptides reproducibly across different runs than those techniques.

The pSMART approach "seems to have the advantages of DIA and DDA combined, and we haven't run into a downside yet," Lopez said.

It's worth noting, though, that according to some recent studies, DIA methods have already equaled DDA in terms of breadth of coverage, arguably representing a similar combination of the two approaches' capabilities.

For instance, in a recent analysis, researchers from targeted proteomics firm Biognosys found that, using a Swath-style analysis of HEK-293 cell lysate on the Q Exactive, they were able in a single run to identify more peptides in DIA mode than in DDA mode – around 29,000 for DIA compared to around 17,000 for DDA.

Then, running additional replicates, they found – as would be expected – that the number of peptides identified consistently by DDA dropped off significantly – down to around 6,000 by run 24 – while with DIA they were able to consistently identify 90 percent of the proteins they detected in their first run.

In a paper published last year in Nature Methods, meanwhile, Tenzer's team found that using their UDMSE DIA method to analyze HeLa cell digest on a Waters Synapt G2-S they were able to identify 36 percent more proteins than they were using an equivalent DDA workflow on the Q Exactive.

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