Mass spectrometry digs deep into samples and helps scientists quantify the unexpected
Despite the seemingly complete trio of RNA—messenger, ribosomal and transfer—from the cellular biology classes of my generation, we keep finding new ones, and each shows us more about the complexity of pathways. Take microRNA, which is single-stranded, short (around 20 nucleotides), noncoding and impacts the expression of about one-third of a mammal’s genes that code for proteins. These strings of nucleotides come in more than 2000 forms in humans. Norman Chiu, associate professor of analytical chemistry at the University of North Carolina, Greensboro, and his colleagues joined with Robert “Chip” Cody, product manager at JEOL (Peabody, Mass.), to develop a protocol to sequence unknown microRNAs in samples with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS).1 Such techniques find use in basic research and applied studies, because microRNA can be used as diagnostic markers or potential drugs. This is just one place where scientists dealing with unknowns in samples turn to MS.
When asked where MS is usually applied to unknowns, Cody replies, “Everywhere!” Other experts agree with the breadth. Kirsten Craven, senior product manager for mass spectrometry at Waters (Milford, Mass.), says, “The identification of unknowns is common across many scientific disciplines, including pharmaceuticals, health science, food, environmental and fine and specialty chemicals.” She adds, “Generally, the need to identify unknowns occurs at the research end of that process or the research department of an organization where new compounds [are developed] or troubleshooting is performed.”
In the pharmaceutical industry, scientists use MS to characterize unknowns in small molecules and biological ones. “Often, the unknowns will be related to the product or the process being used to develop the product,” says Sean McCarthy, senior global market manager, biologics, at SCIEX (Framingham, Mass.).
In some areas, like pharmaceuticals, our lives depend on properly analyzing the unknowns.
How unknown is it?
To get started, says Cody, “we need to determine just how ‘unknown’ the molecules are.” Maybe the compound can be found in the literature, but the scientist didn’t know it was in a sample. Other compounds might be completely unknown—never before seen.
To find the unknowns, having a good idea of the likely known compounds helps. “Maybe you’ll have a list of the ions and compounds that you expect to find, which can help you see something new,” McCarthy explains. “An unknown might also co-elute with something else in chromatography, and being able to compare that to a standard can help with the identification.”
For any unknown, developing a method and interpreting the data create crucial challenges. “The analytical method needs to be broad enough to capture all the information about a sample,” Craven explains. “For example, what is the sample soluble in and which is the most appropriate ionization mode?” To interpret the data, an MS platform with a good software system really helps.
Scientists at Waters’ application laboratory in Wilmslow, U.K., explore various methods to analyze samples for unknowns with mass spectrometry. (Image courtesy of Waters.)
The depth of each step depends, too, on how much identification is required. “If I am trying to identify a material, whether it’s a commercial product or a wood sample, it might be sufficient to identify a ‘fingerprint’ pattern that is unique to that brand or species,” Cody says. “If we can identify that some typical components are probably present, that might be enough.” In some analyses, the right set of peaks is all a scientist may need.
If an application requires the exact structure of an unknown, chromatography plus MS is needed. “The combination of retention time together with mass spectrometry is much more specific than a mass spectrum alone,” Cody says. “We have been using comprehensive two-dimensional chromatography/mass spectrometry—GC×GC/ MS—for complex mixture analysis.” For example, Cody and his colleagues used this technology to analyze contaminants in an environmental sample.
SCIEX’s X500B QTOF system includes about half of the adjustments of the company’s previous quadrupole time-of-flight system, which makes it far easier to use. (Image courtesy of SCIEX.)
To improve an MS platform, sometimes you have to start from the ground up, and that’s just what SCIEX did with the X500B QTOF system. “In the X500B,” says McCarthy, “only about 5% of the pieces are reused from previous platforms.” This quadrupole time-of-flight (QToF) MS system uses a dual-spray source that, says McCarthy, “eliminates cross-contamination of the calibrant versus the analyte.” Changes in the geometry make the system compact enough for a benchtop. The X500B also includes about 50% fewer adjustments than its predecessors, which makes it easier to use.
Other manufacturers also keep improving their MS platforms in ways that benefit the analysis of unknowns. With the Waters Vion IMS QTof, scientists can use ion mobility to measure collision cross-section (CCS) values for each analyte in real time on an “ultraperformance”–liquid chromatography (UPLC) timescale as part of routine workflows. “A CCS measurement is a precise physicochemical property of an ion related to its size, shape and charge in the gas phase, revealing insights into a molecule’s unique chemical structure [and] providing a higher degree of specificity than mass-tocharge ratios alone,” Craven explains.
For Cody, almost every sample that he analyzes with JEOL’s AccuTOF-DART (direct analysis in real time) MS system is an unknown. “These could be unknown drugs—especially the new designer drugs—or unknown contamination on a part or in a formulation or food, an unknown material or an unknown compound from a plant, insect or animal,” he says. Consequently, Cody and his colleagues develop complementary methods to identify unknowns on the AccuTOF-DART system, including regular helium DART, argon DART and O2-adduct formation.
The sample determines the best method. “Thermal desorption and pyrolysis with BioChromato’s [San Diego, CA] IonRocket in combination with the AccuTOF-DART has been really effective for identifying materials such as polymers and other unknowns because it provides a temperature profile for desorbing compounds,” Cody explains.
Identification of one of the components in the DART mass spectrum of additives in an o-ring begins with high-resolution, accurate-mass measurements for the isotope peaks for that component to determine the elemental composition. By searching the chemical databases for compounds that have that elemental composition, candidate compounds are identified. A tentative assignment is made for one of the components that is a common polymer additive. Absolute confirmation of this assignment would require a fragment-ion mass spectrum and/or chromatography. (Image courtesy of JEOL.)
Not always enough
Even with today’s best MS options and additions, the world of unknowns can be too much to handle. “Not everything is MS compatible,” McCarthy says. “So, just relying on MS for unknowns won’t give us all the answers.”
To handle all of the unknowns, scientists must consult an entire toolbox. “You may have to use a number of different techniques to identify an unknown that could be organic or inorganic, a small molecule or a large biomolecule, or cationic or anionic,” Cody says. This might require nuclear magnetic resonance and other techniques. With enough tools, almost any unknown can be identified, and MS is often a great place to start.
1. Wambua, D.M.; Ubukata, M. et al. Bottomup mass spectrometric sequencing of microRNA. Anal. Meth. 2014; doi:10.1039/ c4ay01519c.
Mike May is a freelance writer and editor living in Florida. He can be reached at [email protected].