Suppose you're studying histone modifications, and you want to know whether a specific amino acid is modified with an acetyl (CH
3CO), or with a trimethyl (C
3H
9) group. Both modifications produce nominal mass increases of 42. How then can you distinguish the two?
If you're using a run-of-the-mill mass spectrometer, you're out of luck. But if your instrument features high mass-accuracy and resolution, you’re in business. Such instruments are capable of distinguishing ions that differ by just a fraction of a mass unit, and so can tell the difference between an acetyl group (mass 42.0372) and a trimethyl group (42.0804), or between ethylene (28.0536) and carbon monoxide (28.0104).
For small molecule experts, that accuracy reduces uncertainty in metabolite identification. Protein chemists like John Yates, a mass spectrometry expert and professor of cell biology at the Scripps Research Institute in La Jolla, Calif., gain confidence in their peptide sequencing efforts, as better mass accuracy means fewer possible amino acid compositions—sequences—that can fit the mass. Common post-translational modifications, such as phosphorylation events, can be assigned with higher confidence, while novel or unexpected modifications can be analyzed to identify their elemental composition.
"The basic value of mass accuracy is that as the mass gets more and more accurate, you're limiting the potential elemental compositions that can fit that mass," said Yates.
High mass-accuracy instruments implement such configurations as magnetic sector and Fourier transform ion-cyclotron resonance (FTICR) analyzers, quadrupole-time-of-flight hybrids, and a relatively new design from Thermo Fisher Scientific, the Orbitrap, which is sometimes described as FT-ICR lite. Yates's lab sports a pair of LTQ Orbitraps, a hybrid linear ion trap-Orbitrap instrument. FTICR instruments provide the highest accuracy and resolving power, but also the highest price—Bruker's apex-Qe FTMS can cost up to $2.5 million.
Mass accuracy describes how closely an observed mass matches the expected (or "true") mass. It is typically given in parts-per-million (ppm), and is defined as the true mass minus the observed mass, divided by the true mass, times one million. There is no hard and fast rule, but high mass accuracy instruments are generally described as having accuracies of 5 ppm or below. Such an instrument can thus distinguish ions of mass 1000.002 from those of mass 999.998; the Orbitrap has an accuracy of less than 2 ppm (with an internal standard).
Accuracy is related to, yet distinct from, another parameter: resolution. Resolution (sometimes defined as the peak mass divided by its width at full width-half maximum) describes the sharpness of spectral peaks, and according to Anthony Ziberna, North American manager for advanced mass spectrometry at Thermo Fisher Scientific, varies with both mass and acquisition time. The narrower the peaks, the more ions that can be distinguished over a given mass range. As mass increases, resolution decreases proportionately; similarly, if you double the scan time, you double the resolution. The Orbitrap's resolution is 60,000 at mass 400 with a one second scan speed.
"Think about a Gaussian curve," said Yates. "Try to measure the absolute center of that curve. The wider it is, the harder it is to measure. As resolution improves, it gets easier to measure the center of the peak and mass accuracy gets better. So there's a relationship between resolution and mass accuracy."
Philip Gafken, director of the Fred Hutchinson Cancer Research Center Proteomics Shared Resource in Seattle, has among his fleet of five mass spectrometers Thermo Fisher Scientific's other high mass accuracy instrument, an LTQ FT, a hybrid linear ion trap-FT ICR instrument.
FTICR instruments use a cryogenically cooled magnet and pulses of radio frequency energy to cause ions to orbit around the inside of the cylindrical mass analyzer at a rate that is proportional to their mass; a fast Fourier transform algorithm is used to resolve those orbital frequencies into mass spectra.
Thermo's FT Ultra features mass accuracy below 1 ppm (internal standard) and a resolution of 100,000 at mass 400 every second, according to Ziberna. (The Orbitrap employs the same basic idea, but it uses an electrode to induce the rotation, rather than a supercooled magnet.)
Gafken uses his FT both for mapping histone modifications and quantitative mass spectrometry—using heavy atom (e.g., O18) labeling or SILAC to quantify the differences between two biological samples. "In very complex mixtures, it can be a challenge determining quantitative ion pairs in the mass spectrometry data. The combination of very high resolution and mass accuracy greatly enhances our ability to match quantitative ion pairs and to ultimately obtain peptide quantitation ratios," Gafken said.
Bruker Daltonics offers its hybrid quadrupole-FTICR spectrometer, the apex-Qe, at a range of magnetic field strengths up to 15 Tesla; the base unit includes a 7-Tesla magnet and costs $700,000.
Though all magnetic field configurations benefit from the high accuracy and resolving power of the FTICR, at higher magnetic field strengths it becomes possible to resolve higher molecular weight ions, including large proteins, said Paul Speir, vice president of Bruker Daltonics' FTMS business. That, added Speir, has made the higher field systems popular choices for "top-down" proteomics—a strategy that analyzes intact proteins rather than protein digests.
Not all high resolution and high accuracy mass spectrometers rely on cycling ions. Applied Biosystems' entries in this market include the 4800 TOF/TOF and the QSTAR Elite, a quadrupole-time-of-flight. According to Julie Wingate, the company's senior product manager for mass spectrometers, one of the advantages of the QSTAR Elite is that unlike cyclotron-based mass spectrometers, the resolution and mass accuracy of the QSTAR Elite are independent of scan speed. "It doesn't matter if it's 10 or 20 spectra per second, you still have the same resolution and mass accuracy."
Bruker Daltonics' high mass accuracy QTof, the micrOTOF-Q, lists for around $320,000, according to Ian Sanders, the company's director of sales and applications. Sanders says that in addition to accurate mass (3 ppm with internal standard), the micrOTOF-Q provides rich isotopic detail—information that can help pin down a chemical formula by measuring the proportions of naturally occurring isotopes in a sample (for instance, the relative ratio of carbon-12 to carbon-13 in a sample).
Other companies, including Varian, also sell high mass accuracy mass spectrometers, and more are surely coming. Although better accuracy and resolution are always advantageous, especially when looking for the unexpected, there is a point of diminishing returns.
"It's clear that when you get below 10 ppm some benefits of high mass accuracy kick in, in terms of limiting amino acid compositions," said Yates. "But in terms of database searching, the value of increased mass accuracy diminishes below 10 ppm. Better and better mass accuracy tells nothing more about composition below that level. Going to 1 ppm doesn't change that."
For more information on high mass-accuracy and high-resolution mass spectrometers, be sure to visit the links below.