Mass spectrometry (MS) measures the mass (m) to charge (z) ratio—m/z—of the ions in a sample. Those ratios provide an atomic signature for what makes up a sample. In many situations, z = 1, so the ratio provides the mass of the ions. In original instruments, the mass could only be measured to single-digit units, or integers. With high-resolution mass spectrometry (HRMS), the mass can be measured to several decimal places. Often, ordinary MS is said to measure nominal mass and HRMS measures exact mass. By obtaining data that provide a more accurate representation of the mass of ions in a sample, this technique becomes useful in a wider range of applications. Likewise, HRMS can answer questions that previous generations of the technology couldn’t. To get the most from HRMS, it often requires upstream sample preparation plus downstream data analysis.

“Advances in high-resolution mass spectrometry have created renewed interest for studying global lipid biochemistry in disease and biological systems, explains Susanne Breitkopf, instructor of medicine, and her colleagues at Beth Israel Deaconess Medical Center.1 In fact, HRMS can be applied to a wide range of biological and medical questions.

In fact, HRMS can be applied to a wide range of biological and medical questions.

This article will compare three kinds of HRMS: time-of-flight MS (TOFMS), Orbitrap MS, and Fourier transform ion cyclotron resonance (FT-IRC) mass spectrometry. An MS platform is often characterized by mass accuracy and resolution. For HRMS, in general, the mass accuracy is below 5 parts per million. The mass resolution indicates a platform’s ability to distinguish peaks, and a higher number provides more separation. A team of scientists from the University of North Texas (UNT) in Denton reported that TOFMS, Orbitrap and FT-ICR-MS deliver mass resolutions of 60,000, 240,000 and more than 1,ooo,ooo, respectively.2

Take your time

In TOFMS, a time measurement is used to calculate an ion’s m/z. When asked about the benefits of TOFMS, Jennifer Gushue, marketing manager for the mass spectrometry division at Agilent Technologies, notes: “Time-of-Flight instruments are used for both qualitative and quantitative analyses. They provide high-resolution, full-spectral data with excellent mass accuracy and isotopic fidelity.” She adds that “advances in Agilent TOF acquisition systems have significantly improved dynamic range, enabling our TOF and QTOF instruments to detect low-level compounds in challenging matrices, all at fast acquisition speeds.”

TOFMS can be put together in various ways. “When coupled in a hybrid instrument with one or more quadrupoles in an orthogonal configuration, the resulting QTOF instrument is an excellent choice for MS/MS analysis,” says Dominic Gostick, senior director of product management and informatics at SCIEX. “QTOF’s were initially used extensively for qualitative analysis, where they were the work horse of instruments in many core laboratories.”

These platforms can analyze large molecules. “This makes them ideal for intact protein analysis, as they can provide high-resolution, accurate-mass data on proteins with molecular weights of hundreds of kilodaltons,” Gostick explains. “Today’s biopharma industry has adopted QTOF technology in the characterization of their biotherapeutic drug molecules.”

With some versions of QTOF, the data can be qualitative or quantitative. These features can lead to new analytical techniques, such as SCIEX’s SWATH Acquisition. “SWATH is a data-independent acquisition strategy that provides the most comprehensive quantitative analysis for many analytical challenges,” Gostick says. “In fact, for a typical SWATH Acquisition on the SCIEX 6600 TripleTOF, our high-end QTOF, it is possible to quantify more than 3,000 proteins or 15,000 peptides with coefficients of variation less than 20% with greater than 4 orders of biological dynamic range.”

Focusing on frequency

Although TOFMS uses time as a key metric, Orbitrap and FT-ICR-MS use frequency. The two later techniques use accurate measurements of frequency, and then a Fourier transform provides m/z.

FT-IRC-MS, for instance, involves a series of steps. First, ions are formed and then cooled, focused, and accumulated. Next, the ions go in a Penning trap, which stores the ions and excites them to their cyclotron frequencies (the frequency of switching an electric field required to accelerate a particle in a cyclotron). This frequency is related to an ion’s m/z, and further processing is followed by a Fourier transform of the data, which reveals the mass spectrum of a sample.

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The Orbitrap technology employs an ion trap to analyze mass. This platform traps ions between an outer electrode, built like a barrel, and an inner electrode, where the ions orbit around the inner electrode. A Fourier transform of the resulting current reveals a mass spectrum.

The team of UNT scientists examined the application of FT-IRC and Orbitrap MS to study metabolomics and lipidomics.2 They wrote that the FT-ICR-MS platforms “are the most advanced mass analyzers in terms of high accuracy and resolving power with sub-parts-per-million mass accuracy.” Nonetheless, they point out that speed is not one of FT-ICR-MS’s strong points. They explain: “At a scan rate of 1 Hz with mass resolution of 100,000 at m /z 4000, the number of points over the chromatographic peak, especially if additional MS/MS scans are required, is low when FTMS is combined with modern fast chromatography systems.

Comparing the components

As noted above, mass resolution generally increases from TOFMS to Orbitrap to FT-ICR-MS, but there are pros and cons to each technique. For the highest level of performance, scientists typically pick FT-ICR-MS, but that technology comes with its own limitations. For one thing, this method uses large superconducting magnets, which are expensive. Orbitrap and TOFMS don’t require magnets, which makes them more affordable.

Vendors also provide upstream separation methods that can easily be added to HRMS platforms. For example, adding ultra-high performance liquid chromatography (UHPLC) provides fast separation of sample components. This requires high speed acquisition to ensure sufficient data points across narrow chromatographic peaks, for which TOFMS instruments are best suited. A QTOF with a data-independent workflow, such as SWATH Acquisition, can provide comprehensive quantitative analysis.

Among the diversity of MS instruments on the market today, each has strengths and weakness. The preferred HRMS instrument depends on the application and what capabilities are needed to best solve a laboratory’s analytical problems.

References

1 Breitkopf, SC, et al. “A relative quantitative positive/negative ion switching method for untargeted lipidomics via high resolution LC-MS/MS from any biological source,” Metabolomics 13:30, 2017. [PMID: 28496395]

2 Ghaste, M, et al. “Applications of Fourier transform ion cyclotron resonance (FT-ICR) and Orbitrap-based high resolution mass spectrometry in metabolomics and lipidomics,” Int. J. Mol. Sci. 17:E816, 2016. [PMID: 27231903]

Image: High-resolution mass spectrometry provides enough mass accuracy and resolution to explore new areas, such as abnormal lipid biochemistry in prostate cancer and other diseases. (Image courtesy of Ji-Xin Cheng, Purdue University Center for Cancer Research, National Cancer Institute, National Institutes of Health.)