Featured Article
Tuesday September 23, 2008
by Jeffrey M. Perkel
Try this thought exercise: Imagine you're blindfolded, and asked to identify the object someone puts into your hand.
The object is a bottle filled with jellybeans. Given its texture, weight, and shape, you probably can surmise you've been handed a bottle. But what are its contents? To figure that out—we'll assume you don't have a bottle opener—you break the bottle against a wall, and determine its contents from the pieces on the floor.
What you've just done is a sort of macroscale version of a technique commonly used by biologists and chemists alike: tandem mass spectrometry. In standard mass spectrometry, the instrument measures the mass (or more precisely, the mass-to-charge ratios) of the ions that are injected into the system. But as useful as that data is, it often is difficult, if not impossible, to unambiguously identify a molecule from its mass alone.
That's where tandem approaches come into play. In tandem mass spectrometry, you select one or more of those ions for further study, fragment them (generally by collision with a gas, by laser excitation, or by chemical reaction), and analyze them again. The result is a richer dataset, which can help confirm a tentative molecular identification, sequence a peptide, or even, when combined with isobaric reagents like Applied Biosystems' iTRAQ system, quantify changes in the abundance of certain ions of interest.
Several techniques can be used to smash the metaphorical bottle. Two involve laser excitation: laser-induced dissociation on MALDI-based instruments, and infrared multiphoton dissociation (IRMPD), in which high-energy laser photons induce fragmentation within a FT-ICR mass spectrometer.
One of the most common techniques is collision-induced dissociation, or CID (also called collisional-activated decomposition, CAD). According to Keith Waddell, LC-MS applications solution manager for Agilent Technologies, CID is the "classic technology" for protein sequencing by tandem mass spectrometry, and it is one that is supported on both the company's triple-quadrupole and quadrupole-time-of-flight instruments.
Both systems employ identical hexapole-based collision cells, says Waddell, in which fragmentation occurs by accelerating the parent ion and smashing it into a gas molecule.
"You fill the collision cell with gas, usually helium," he says. "What happens in practice is the molecular ion that passes through the first quad goes into the collision cell, accelerates, and hits a gas particle, and the molecule will fragment."
It is the molecular equivalent of smashing a bottle against a wall, and it is really the only game in town for small molecule fragmentation work. But for peptides, several options exist in addition to CID.
According to Waddell, CID fragmentation of peptides predominantly produces so-called B and Y ions; these ions result from cleaving the backbone between the C and N of a peptide bond (B refers to the N-terminal fragment, while Y refers to the C-terminal one). But, he notes, the process "tend[s] to be fairly random," and not every peptide bond will break by this method.
That means a peptide fragmentation series may contain gaps, hindering identification of its sequence. For more reliable peptide sequencing, he says, try electron-transfer dissociation, or ETD.
ETD is a chemical process in which reaction with fluoranthene radical anions disrupts the peptide backbone at regular intervals. The reaction "gives you a different ionization pattern, which is good for labile-group-containing peptides like phosphopeptides, and you get a more even distribution of characteristic C and Z ions from the peptide backbone," Waddell says. (C and Z ions are those formed by cleavage immediately N-terminal to the alpha-carbon of the peptide chain.) "It is easier to read off the sequence," he concludes.
Agilent supports ETD fragmentation on its ion traps. ThermoFisher supports this method, as well as CID, on its LTQ Orbitrap systems, while Bruker Daltonics incorporates it in its HCTultra ETD II system.
According to Lester Taylor, director of marketing for life sciences mass spectrometry at ThermoFisher Scientific, ETD "preserves the intact information about labile modifications, which are not observed directly [using CID]. For instance, phosphate groups are good leaving groups, so they are easily lost in the excitation process, and therefore mass spectrometry is used to infer their location. However, by using ETD one can directly observe fragments that contain the intact phosphopeptides."
Waddell cautions, however, that ETD is less sensitive than CID, because of lower ionization efficiency. As a result, he says, "I would use CID to start with, and if I couldn't determine where the phosphate was, I would switch to ETD."
ThermoFisher supports a related form of fragmentation on its FTICR-based instruments, electron-capture dissociation, or ECD. "ECD and ETD generate very similar types of fragmentation," Taylor says. "ECD uses an electron beam to induce dissociation rather than a reagent ion [as in ETD]."
ThermoFisher's Orbitrap systems also support a relatively new fragmentation method, called high-energy collisional dissociation, or HCD, which occurs in a dedicated collision cell in the Orbitrap. According to Lester, the process is similar to CID in the LTQ Orbitrap's ion trap component, but because it occurs at a higher energy, more (and different) fragmentation occurs.
"The fragment ion observed depends on how much energy is used to induce fragmentation," he explains. "The more energy used tends to generate more and different types of fragments."
As a result, HCD fragmentation spectra tend to complement CID spectra, he says. "It extends the range of their analytical capability."
Applied Biosystems' mass spectrometers support neither ECD nor ETD, says Aaron Hudson, senior marketing manager in the company's proteomics group. Instead, its ESI-based QSTAR Elite and 4000 QTRAP support CID. So does the company's MALDI-based 4800 TOF/TOF, which, Hudson says, is "the real differentiator for us."
"Most TOF/TOFs use post-source decay as a fragmentation method," he says. "We are unique in the fact that we have a collision cell between the first and second TOFs."
Hudson says this feature is most useful in areas like carbohydrate analysis, where the high energy of the CID cell can shatter sugar rings to reveal the linkages and branching patterns that make them unique. For peptide analysis, however, CID on the TOF/TOF is basically overkill, he says, though it can be useful in differentiating the isobaric amino acids, leucine and isoleucine.
Waters Corp. combines the CID cell in its Synapt High-Definition MS system with ion mobility spectroscopy (IMS). IMS resolves nominally identical molecules based on their conformation, size, shape, and charge, providing another dimension of fractionation prior to mass analysis. (ThermoFisher also offers IMS capabilities, with a "high-field asymmetric waveform" IMS interface to its ion trap mass spectrometers.)
The ion mobility separation occurs in the second of three modules—a quadrupole, Triwave device, and time-of-flight mass spectrometer—in the Synapt instrument. The Triwave module is comprised of three traveling wave (T-Wave) ion guides; the first traps ions, the second separates them by their ion mobility, and the third transfers the separated ions to the TOF detector that lies downstream. Ions emerging from the front-end quadrupole are thus trapped and accumulated in the first module, separated, and then accelerated downstream.
Ions may be fragmented in either the first and third modules of the Triwave, or in both—a process called Time Aligned Parallel (TAP) Fragmentation—so that fragments can be tied back to their parent ions.
According to Michael Balogh, principal scientist at Waters, it's all about simplifying the data.
"You can do fragmentation, but if you have a complex environment, you can get all kinds of bits and pieces that are very hard to interpret," says Balogh. "That's something you have to deal with in the biological world, and that's where ion mobility comes in—reducing the complexity of the problem."