Accurate Mass Measurements with ESI-TOF Mass Spectrometers

A Powerful Combination: ESI-TOF Mass Spectrometers

MALDI-TOF. The two acronyms go together like peas and carrots, as Forrest Gump might say. But they don’t have to. It is possible, and in fact increasingly popular, to purchase a time-of-flight mass analyzer (TOF) with an electrospray ionization (ESI) source.

“You can’t do everything with MALDI-TOF,” explains Matthew Willetts, group leader for ESI applications at Bruker Daltonics. For one thing, not every molecule ionizes efficiently inside the laser-assisted MALDI (matrix-assisted laser desorption ionization) source. (To be fair, not every molecule ionizes efficiently with ESI either; the two sources are generally considered complementary.)

Another problem is that MALDI typically imparts a charge of +1 or +2 to the molecules it ionizes—that’s the z in m/z, the mass-to-charge ratio measured by a mass spectrometer. As a result, MALDI-ionized molecules typically have m/z values equivalent to their molecular weight (for z=1) or half their molecular weight (z=2). That isn’t necessarily an issue for small molecular weight compounds, such as drugs or peptides, but it can pose difficulties with large molecules such as proteins, says Sunia Trauger, director of the Small Molecule Mass Spectrometry Facility at the Harvard FAS Center for Systems Biology.

“For ions produced by MALDI that have a m/z of >20,000, isotopic resolution cannot be achieved by most MALDI-TOF mass spectrometers due to limitations of the ion optics [and] power supplies used in commercial instruments,” says Trauger. Furthermore, she says, large proteins tend to form “adducts with salts and other matrix molecules,” which can broaden otherwise sharp MALDI spectral peaks unless the sample is “cleaned up” prior to analysis.

Finally, there’s the matrix, the “M” in MALDI, which transfers the laser energy to the sample, vaporizing and ionizing it. But the matrix material itself is visible to the mass spectrometer, producing signal interference in the low molecular weight range.

ESI, on the other hand, requires no matrix. Instead, samples typically are injected into the instrument via either a syringe pump or liquid chromatography (LC) interface, such as HPLC (high performance liquid chromatography), UPLC (ultraperformance liquid chromatography) or nanoLC. That means samples can be cleaned and fractionated prior to analysis, which can simplify the spectra of complex mixtures such as serum.

ESI also produces more heavily charged ions than MALDI. Thus, proteins are charged not just to +1 and +2, but also to +3, +4 and so on (or, in negative ion mode, -1, -2, etc.), making it is possible to distinguish and measure even large molecular weight proteins at high resolution.

MALDI does offer two significant advantages, though. First, MALDI-TOFs typically have higher sensitivity than their ESI counterparts, says Willetts. The technology is also much faster, he adds, as samples often don’t need to be fractionated prior to analysis, and an entire MALDI plate’s worth of samples can be processed in just minutes.

The TOF mass analyzer

Single-stage time-of-flight analyzers can mass-analyze ions, but they cannot select a precursor to fragment for further analysis. Thus, they can profile a sample but can’t perform MS/MS experiments, for instance, to sequence a peptide.

But TOF analyzers do have two significant strengths, says Tim Schlabach, LC-MS product manager at Agilent Technologies: high resolution and high mass accuracy.

“Accurate mass is powerful,” Schlabach says. With high resolution, and the high mass accuracy that typically accompanies it, researchers can have greater confidence in their compound identifications. That’s because at high mass accuracy, only a very few compounds can share a given molecular formula.

These instruments can even resolve different isotopic forms of individual molecules, says Willetts. For instance, one peak may represent the molecule comprised entirely of carbon-12; another peak, one mass unit larger, might represent that same molecule containing a single carbon-13. The relative abundances of those two forms can help identify a molecule’s elemental composition, information that can narrow down the identities of unknown compounds such as metabolites, says Willetts, for instance using Bruker Daltonics’ SmartFormula software.

“It’s important that your instrument does not disturb this ratio,” Willetts says.

The data sheet for Agilent Technologies’ 6230 ESI-TOF specifies a resolution of better than 20,000 FWHM (full width, half maximum) at m/z 1,522 and a mass accuracy of less than 2 ppm. Bruker Daltonics’ micrOTOF II ESI-TOF specs indicate resolution of at least 16,500 FWHM and less than 2 ppm mass accuracy, as well.

ESI-TOF applications

According to Willetts, ESI-TOFs are popular in mass spectrometry core labs, “where they are routinely [used] to get accurate mass measurements of small molecules, peptides or even intact proteins.”

Schlabach identifies three primary applications for the Agilent 6230: characterization of therapeutic biomolecules, for instance, to ensure a therapeutic protein is properly modified; toxicity analysis (e.g., identifying unknown poisons in biological samples); and quantifying drugs of abuse.

Trauger’s facility has both ESI- and MALDI-TOF systems available, but the ESI-based instrument is used about three to four times more frequently, she estimates. (The latter is a Waters Micro MX MALDI-TOF.) Researchers in her facility routinely use their ESI-based Agilent 6220 to measure small molecules, peptides and proteins of 75,000 to 100,000 Da molecular weight with high mass accuracy—high enough to detect post-translational modification differences such as glycosylation, for instance.

The mass spectrometry facility in the Department of Chemistry and Biochemistry at the University of Maryland also has both ESI- and MALDI-TOF instruments available. But the ESI-TOF (a JEOL AccuTOF-CS) is used about 10 times more frequently than the MALDI-TOF (a Shimadzu Axima-CFR), estimates director Yue Li.
In part, that’s because the Axima-CFR is older than the AccuTOF-CS, Li says, and has lower resolution. In addition, the MALDI instrument cannot be used for small molecular weight compounds because of matrix interference.

Li says his users fall into two categories. Organic chemists use the ESI-TOF to characterize and confirm small organic compounds synthesized in their labs. Biochemists, on the other hand, tend to study peptides, proteins and nucleic acids.

Buying advice: key questions when looking for your ESI-TOF instrument

According to Schlabach, the single most important question to consider when shopping for an ESI-TOF is: Do you need MS/MS capabilities? If so, consider a triple-quadrupole or quadrupole-time-of-flight hybrid (qTOF), both of which can be interfaced to an ESI source.

“If you are doing a lot of fragmentation studies, forget it—it [ESI-TOF] is not the instrument for you,” says Willetts.

Next, consider flexibility. Will you be running the same types of experiments over and over? If so, and if an ESI-TOF can handle that experiment, then you’re good. But if you are in an academic lab that runs many different types of experiments, it might make sense to consider a qTOF.

In terms of specifications, ESI-TOFs offer excellent resolution, mass accuracy and sensitivity. Willetts advises looking for resolution of at least 10,000 FWHM (and preferably higher) and mass accuracy of 2 ppm or lower. “If it’s not 1 [to] 2 ppm, you don’t have accurate mass and you miss out on the point of TOF,” he says. Also, does the system come with the appropriate software to make the most of the data?

Consider if you’ll need additional or alternate LC-based ionization sources, such as APCI (Atmospheric Pressure Chemical Ionization) or APPI (Atmospheric Pressure Photoionization). These are generally customer-swappable options. Some TOFs also can be coupled to gas chromatographs. Agilent offers its customers an ESI variant called JetStream, which uses a heated stream of flowing gas to concentrate and focus the sample, producing five-fold or higher sensitivity, says Schlabach.

Other variables, says Trauger, include the instrument’s dynamic range (she recommends four orders of magnitude), scan speed (it needs to keep up with your LC system) and mass stability, so there’s no “mass drift” or “jitter” in the mass measurements.

Finally, Trauger recommends checking out the system software. Agilent’s software, for instance, is easy for newcomers to learn and can handle calibration, as well as both the LC and MS, from one interface, she says. “It’s definitely one of the easiest instruments to use.”

As for Bruker Daltonics, Trauger says, “Their instruments are great, too, and the software is fantastic.”

Ultimately, of course, there’s no denying that, for those with the financial wherewithal, a qTOF is a far more flexible mass spec. But an ESI-TOF is no slouch, says Trauger. “You get very high accuracy, very high resolution, and you can get elemental formulas.”

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