But the hardest part was actually the control software. “In this day and age, the hardware work always ends up taking the least amount of time,” he says.
“Everything is all about timing, because you're doing different things with the ions at different times. If you can't break into our analysis scheme as we're controlling the ions, it's nearly impossible to synchronize what you want to do with what we're gonna try to do,” explains Mike Senko, director of research at Thermo Fisher Scientific.
Ion trap-based instruments from Thermo (including its Orbitraps) are controlled by scripts written in a proprietary language known as ion trap control language (ITCL); these scripts are what tell the machine how to run a given protocol. Most users will never interact with the instrument at this level, says Senko, since access to the language is highly restricted (perhaps six laboratories worldwide have access, including Coons’).
Learning the language isn't like picking up Perl from online tutorials or printed how-to guides. “You can't go to the bookstore and buy an ITCL manual,” says Senko. If the company agrees to partner with a lab — for instance, because the lab proposes a project that is scientifically or commercially intriguing — Thermo brings in a graduate student or postdoc to either its San Jose or Bremen (Germany) location “for anywhere between four weeks and an entire summer [to] teach them how to use the software.”
Westphall notes that the Coons lab generally sends a student every one to two years. He has never gone himself — he learned the language from a student — but this year a Ph.D. student who is graduating in December will take a job at Thermo's Bremen facility, the lab's first to accept such a position.Fire up the ‘Bruce-o-pole’
Neil Kelleher's lab at Northwestern University also has ITCL access from Thermo.
Kelleher focuses on top-down proteomics, which involves resolving intact protein isoforms in the mass spectrometer. Naturally, these experiments require exceptional mass accuracy and resolution, so Kelleher uses Thermo Orbitrap and FT-ICR mass spectrometers.
But that doesn't mean the instruments can't use some tweaking to push their capabilities. For example, Kelleher notes that off-the-shelf instruments like his 7T FT-ICR have ion traps that are too small — or more precisely, have too small a “space-charge capacity” for top-down work.
“I'm the first guy to say that size doesn't matter,” quips the 5-foot-tall Kelleher, “but in this case …”
Space-charge capacity refers to how many ions (charges) a given mass spectrometer can hold. Kelleher's ICR is relatively spacious, but the ion trap in front of it, used to accumulate ions prior to analysis, is not — it has a capacity of perhaps 500,000 charges. In top-down, where each protein molecule could contain 50-odd charges, this means the trap is limited to perhaps 10,000 molecules. To have the sensitivity required to identify and, especially, characterize intact proteins in complex mixtures, Kelleher would like orders of magnitude more than that — 10 million or 100 million charges.
For a possible fix to this dilemma, Kelleher turned to his head of instrumentation, Phil Compton. “I would like to give him some street cred, too,” says Kelleher. “He's fantastic.”
Compton's interest in gadgetry goes way back. When he took “interest inventories” in high school — those surveys given to students that supposedly match a person with his or her ideal career — they always said: “I should do something with my hands,” such as auto mechanic, plumber, or electrician.
Instead, Compton pursued a degree in chemistry at the University of Illinois, Urbana-Champaign, eventually working for Kelleher. He was drawn to the analytical power of mass spectrometry. “The type of resolution that you could get with these instruments compared to other techniques, was just amazing to me,” he says. “And the fact that you could move ions around with electric and magnetic fields so precisely, it hooked me.”
Compton left Illinois for graduate studies at the University of Virginia, working in mass spec pioneer Don Hunt's lab where he designed a novel front-end ETD interface called f-ETD (the original ETD ion source moved the reactive anions from the back of the instrument forward; the new, simpler design had the anions enter from the front along with the ions under study).