MALDI-TOF Instruments

In MALDI, a sample (such as a mixture of peptides, oligonucleotides, or intact proteins) is combined with a matrix material, such as 2,5-dihydroxybenzoic acid or alpha-cyano-4-hydroxycinnamic acid, spotted onto a metal target plate, and dried. When the spot is excited with laser light, the matrix heats up, causing the sample to ionize, generally with a charge of +1. These ions are then resolved by mass, most typically in a time-of-flight (TOF) analyzer that determines the mass of a particle based on how long it takes to reach the detector.

Though frequently the tool of choice for protein identification from one- and two-dimensional polyacrylamide gels, MALDI can also complement traditionally ESI-based liquid chromatography workflows, where it can be used to analyze chromatographic fractions off-line, according to Dominic Gostick, director of product marketing for the biomarker and proteomics business unit at Applied Biosystems.

"There's some real advantages [to LC-MALDI mass spectrometry]," he said. "You've decoupled the LC from the analyzer. You can use trifluoracetic acid in the solvents, which gives sharper peaks in chromatography but is not compatible with electrospray ionization. And you can decouple the [single-stage] MS from tandem MS/MS, and that has a real advantage when you want to dig deeper, to see the maximum number of peptides per sample."

That's because decoupling the mass analysis from the liquid chromatography means you are no longer limited to the information you can extract before the next peak comes off the column.

MALDI-TOF mass spectrometry has value beyond protein biochemistry labs, too, said Ronan O'Malley, mass spectrometry product manager at Waters Corporation in Manchester, UK. "MALDI-TOF is important in proteomics, there's no two ways about that. But it's also important for oligonucleotide work, for instance in quality control, and also for the identification and structure of glycans."

And there are other applications, including genotyping and MALDI imaging, an approach in which the protein or metabolite components of a tissue sample are identified by rasterizing across the sample with a laser, much as a dot-matrix printer operates: left to right and top to bottom.

In tandem mass spectrometry, an ion of a particular mass is selected (that's the first stage of the analysis) and fragmented. Its constituent fragment ions are then mass-analyzed a second time (that's the tandem stage) to reveal data about the molecule's structure or sequence; single-stage TOF instruments lack this capability (though some fragmentation does occur via "post-source decay" as the ions traverse the flight tube).

Though single-stage MALDI is often sufficient for peptide mass fingerprinting, sequencing and structure-determination cannot be accomplished without tandem capabilities of some form or another. Gostick said he has seen a corresponding shift in sales trends over the last few years as customer needs have become more sophisticated.

"When single-stage MALDI-TOF mass spectrometry first came onto the market in proteomics, a lot of people were looking to identify proteins using only the mass of the peptides coming from a tryptic digest, and we saw a real sharp increase in the product, it really grew, along with the two-dimensional gel world," Gostick said. "But I think over the last few years people are less inclined to just buy a single-stage MALDI. Customers are looking for more performance than just accurate molecular weight."

Applied Biosystems offers both single-stage and tandem instruments, including the single-stage Voyager mass spectrometers (DE-PRO and DE-STR) and the 4800 MALDI TOF/TOF Analyzer. Bruker Daltonics' Ultraflex III TOF/TOF also employs a tandem TOF configuration.

Some companies offer tandem MALDI instruments based on hybrid mass analyzer configurations. Applied Biosystems' QSTAR, for instance, couples an optional MALDI source with a quadrupole-time-of-flight mass analyzer, as does Waters Corporation's MALDI Q-Tof Premier.

According to Victor Fursey, assistant vice president for sales and marketing at Bruker Daltonics, the reflectron provides "a double whammy" in terms of ion separation, providing both energy compensation and a longer overall flight path relative to linear mode. Yet linear instruments also have a place in the lab, he added. They work well for things like basic quality control work, and are indispensable for visualizing very large ions, such as large intact proteins, he said. "The minute you try to bend ions or extract them orthogonally, it becomes more difficult for the ion optics to handle those extremely high mass-to-charge particles."

Though most MALDI-TOF spectrometers now contain both linear and reflectron detectors, not all do. Bruker Daltonics' microflex LT, for instance, is available only in a linear configuration.

In axial instruments, the laser-activated ions travel in a straight line (that is, axially) down the flight tube; in orthogonal systems, the flight tube is rotated 90 degrees relative to the initial ion flight path.

The advantage of orthogonal geometry, says Gostick, is that it decouples the detector from the ion source—or more precisely, from variations in the target position. "However, on the 4800, we now have a 96-well plate size, and that's a big piece of real estate to maintain a constant mass accuracy across," he said. "Aberrations in that can cause shifts in mass accuracy." According to O'Malley, MALDI imaging also presents target-position challenges, owing to differences in tissue thickness.

"With an orthogonal system, you're measuring the time of flight from pusher to detector," said O'Malley. "You don't have to take into account time of flight from the source, so any energy spread at the source has no detrimental effect on resolution and therefore, mass measurement." As a result, O'Malley said, orthogonal systems (such as Waters' MALDI Q-Tof Premier and Perkin Elmer’s single-stage PrOTOF 2000) provide "constant resolution over the entire mass range, better mass accuracy, and better calibration stability over time," than do axial systems.

Applied Biosystems recently introduced new software for protein quantification and identification called ProteinPilot, said Gostick. "We've added some algorithms that make the software very easy to use," he said. "We now ask biologically relevant questions, let the software make the hard decisions, rather than the user."

Salim Merali, director of the proteomics core facility at Temple University's Fels Institute, said the software on his new Bruker AutoFlex II TOF/TOF was one of the points he liked about the system. "It’s just overall more friendly," Merali said. "You have direct interaction with the instrument, and the way you maneuver it on the screen, it’s quite attractive, easy, foolproof, whereas other instruments [in his facility] are more clumsy."

According to O'Malley, other variables to consider in making a purchasing decision include the usual mass spec parameters—mass accuracy, resolution, and specificity—as well as precursor ion resolution (that is, how selective you can be in selecting a precursor ion for tandem analysis) and fragmentation approach (does the instrument include a collision cell for ion fragmentation, or does it rely on post-source decay to produce limited, random fragmentation).

Finally, said Gostick, think long-term. Just because the instrument is intended as a workhorse for proteomics workflows does not mean other users will not have other applications, such as analyzing oligonucleotides or carbohydrates. "Typically people buy this as a workhorse instrument, but sometimes they want some versatility too, so they can look at other things."

"For us, the key thing was sensitivity across a very wide mass range, up to 100,000 molecular weight or so," said Cornett, who does MALDI imaging of diseased tissue samples. He took sections from "basically the same tissue" to both companies, and compared those against the results he obtained on the instruments he already had.

Before deciding to commit to any mass spectrometer, if at all possible, you should take advantage of vendors’ demonstration facilities and take their instruments out for a spin.