With all the marketing hype surrounding the introduction of the MALDI-TOF/TOF mass spectrometer, it may be difficult for some proteomics researchers to attain any insight into what the instruments can actually accomplish. Perhaps the easiest way to find out is to run one’s samples on the instrument while visiting a vendor’s exhibition lab, but as an alternative, ProteoMonitor spoke with a sampling of the researchers who have already acquired a TOF/TOF to assay their initial feedback on the instrument.
By far the most notable observation concerning the TOF/TOFs — be they Bruker Daltonics or Applied Biosystems — was that researchers have yet to take advantage of the highly vaunted improvements in throughput that the instrument was meant to bring. Currently, the researchers interviewed simply do not have enough samples to require running the TOF/TOF at its most productive capacity. This may change, as more researchers turn to a facility’s TOF/TOF to analyze their samples, or as protein mass spectrometrists devise new experiments to use the instrument’s capacity in other ways, but for the time being the benefit of owning a TOF/TOF is rendered in other ways.
The most obvious of these is in the instrument’s increased ability over other mass spectrometers to perform protein identifications rapidly and accurately, researchers said. For both Bruker and ABI’s instruments, the ability to select precursor ions for MS/MS analysis has led to significant improvements in the number of peptide fragments that the database search software can match with fragments generated from protein databases. Said Hanno Langen, Roche’s chief of proteomics of his Bruker Ultraflex TOF/TOF: “I just recently looked at 800 spots [from a 2D gel], and I expected to get 60 to 70 percent identification [in regular MS mode], and with the TOF/TOF an additional 20 percent. Instead, I got 90 percent in the first round, and a total of 95 percent [by adding the TOF/TOF analysis].”
ABI’s 4700 Proteomics Analyzer
Early users of ABI’s version of the TOF/TOF, which the company refers to as the 4700 Proteomics Analyzer, had both praise and suggestions for improvements. The ability of the ABI instrument to perform high-energy collision-induced dissociation (CID) of peptide fragments earned praise, because the resulting increase in peptide sequence coverage brings more accurate protein identifications. The improved sequence coverage also could bring additional insights into protein composition, such as picking out posttranslational modifications, users said.
Phil Andrews, a protein mass spectrometrist at the University of Michigan and director of the Michigan proteomics consortium, said that prior to the TOF/TOF a researcher could never account for 100 percent of the protein, even in an MS/MS experiment. With his ABI TOF/TOF, however, “there’s the potential for getting better sequence coverage because you can analyze the larger fragments,” he said. “You’re more likely to get posttranslational modifications. It’s logical.”
Other researchers expressed confidence in the high-energy CID capabilities of the ABI instrument to produce reliable data for de novo peptide sequencing. Al Yergey, a protein mass spectrometrist at the NIH, explained that he performs pairs of experiments on the same sample with the mass spectrometer — first without CID, and then comparing the resulting spectra with CID data. “In general, my argument is that this is only possible with this instrument, because the fragments are more complete,” he said.
Predictably, several of the researchers with ABI TOF/TOFs lamented the lack of sophisticated software for interpreting the kinds of complex spectra produced when the instrument does such a thorough job of fragmenting peptides. Although the software packages installed in the ABI instruments in the field vary according to their update status, some researchers said they hoped to soon be able to automatically export spectral data from the mass spectrometer directly into database search software.
Others, while wishing for software to take away some of the manual data interpretation, expressed satisfaction with the Oracle database installed on the instrument for archiving data.
Bruker Ultraflex TOF/TOF
As for Bruker’s version of the MALDI-TOF/TOF, known as the Ultraflex, the users interviewed also expressed content with the instrument’s improved sequence coverage. While the Bruker technology differs from ABI’s, primarily in its ability to re-accelerate selected parent and daughter ions by “lifting” their voltage, it also allows for the operator to run the instrument in CID mode, users said, giving researchers the ability to collect spectra containing fragments generated by ion decay or by collision with a noble gas.
From the perspective of the users interviewed by ProteoMonitor, who all hailed from industry, the capabilities of the Bruker TOF/TOF — in terms of resolution, sensitivity, and throughput exceeded the requirements posed by the researchers’ sample demand. Thomas Krogh, a protein mass spectrometrist at Novo Nordisk in Bagsvaerd, Denmark, said that he hasn’t found it necessary to resort to CID mode to collect highly fragmented peptide ions in order to make accurate protein identifications. “It’s a nice instrument,” he said. “As a confirmation tool for peptide mapping, database searching, and to confirm a couple [peptide ion identifications] with MS/MS, it’s working quite well.”
Where Bruker users see room for improvement — as with ABI TOF/TOF users — is in the realm of software. Krogh complained that he found it frustrating to jump between three software programs to control the acquisition, analysis, and database searching when running his instrument, while others placed additional automation software on the top of their wish list. Langen, at Roche, said his instrument can automatically load sample plates into the spectrometer, but currently cannot automatically select particular ions for MS/MS analysis. “At the moment the instrument runs in batch mode, but in the future there should be a feedback loop for automating the protein identification runs,” he said.
How To Compare?
Given that only a few of the TOF/TOF users interviewed had experience working with both the ABI and Bruker versions of the instrument, there was no clear consensus on which instrument most researchers preferred. In general, ABI users claimed their instrument is superior based on its higher energy CID mode, which generates a more diverse array of peptide fragments, but Bruker users countered that not only was this additional data unnecessary, but made interpreting spectra unnecessarily complicated.
“It depends on the application,” said Martin Kussman, a researcher at GeneProt who works with a Bruker TOF/TOF but has also run experiments on the ABI instrument. “Using the TOF/TOF in CID mode yields a lot of additional diagnostic ions, but CID mode does not create a more complete b and y ion series.”
Countering a comment from one ABI user, who claimed that the Bruker instrument was “inferior,” Kussman said his company chose to acquire a Bruker TOF/TOF because of its superior reliability. Bruker’s Ultraflex sells for about $575,000, compared with $650,000 for an ABI 4700.