NEW YORK (GenomeWeb) – A new study from Waters on mass spectrometry instrumentation suggests that tandem quadrupole instruments and microfluidics separation could be a useful setup for large-cohort translational research looking for specific peptide analytes.
Along with scientists from the UK's University of Leicester, the Waters scientists compared several different instrument setups to find one that might work well in a pilot clinical study of heart failure patients. They published their results this week in Proteomics.
Comparing tandem quadrupole and time-of-flight mass spectrometers in combination with different chromatography separation techniques, the scientists found that the Xevo TQ-S tandem quadrupole with microfluidics-based separation yielded both specificity and sensitivity required for translational clinical studies. That platform has recently been awarded the CE-IVDD mark , approved by the US Food and Drug Administration as a Class I medical device, and approved by the Chinese FDA as an in vitro diagnostic device.
"We were interested to see in a representative sample of plasma proteins, which [instruments] give us the best sensitivity and the best coefficient of variation to see what a given compound is," Waters Director of Health Sciences James Langridge told GenomeWeb. "Surprisingly, the tandem quadrupole did very well. We showed that if you use good chromatographic separations — that's the key to this, providing discrete peptides to the system — then the performance of the systems, in broad terms, is roughly equivalent." That good separation came from micro-scale, rather than nano-scale, chromatography, which was more robust, Langridge said.
The researchers then used the Xevo TQ-S micro instrument in a pilot study of heart failure and healthy control tissue samples using an established set of biomarkers for that disease. Using a combination of three biomarkers, they were able to distinguish diseased patients from the controls. While there were two different types of heart failure patients, the biomarkers were not able to successfully distinguish between the two disease states.
Still, the study offers proof of concept that mass spectrometry can be used in larger translational studies that require high specificity and high-throughput techniques.
"Traditionally, mass spectrometers have fallen into two categories," Langridge said. "For quantitative applications where you're trying to get an accurate measure of a known, targeted compound then you've traditionally used tandem quadrupole mass spec. They're very sensitive, but they can be prone to interferences because of the fact that they're not working at high resolution."
For qualitative applications, time-of-flight (TOF) and orbitrap instruments have often enabled higher resolution, he said. "You can resolve compounds away from each other so you're very sure of what you're measuring. They haven't been as widely adopted for quantitative studies and are primarily used for qualitative, structural elucidation."
The problem for clinical studies is that they require quantification of particular analytes, but the biological samples are often complex and provide interference.
To find the right instrument for the second, clinical part of the study, the Leicester and Waters scientists tried out a couple instruments. In addition to the Xevo TQ-S tandem quadrupole mass spectrometers, they also used the Xevo GS-XS quadrupole TOF and the Synapt G2-Si hybrid quadrupole orthogonal acceleration time-of-flight platforms.
"We kind of expected perhaps the tandem quad would be the most sensitive, but in complex mixtures there would be a lot interferences," Langridge said. But based on the signal-to-noise ratio and the percentage coefficient of variation, the tandem quad came out on top, especially when used with micro-scale chromatography.
"Proteomics has typically used nanoscale chromatography," such as 75-micron columns, Langridge said, likely because researchers were limited in the amount of sample they had to analyze. "By our standards, we aren't sample limited. Moving up in chromatography, it's more robust and the throughput is better. We showed there that in terms of robustness and the accuracy of the quantization, it was better on a microfluidics platform," he said, important for the type of study they imagined the platform being used for.
"If you look at this space in general, there's a huge amount of literature for known [disease] markers that people have previously described," Langridge said. "That's interesting from the perspective of being able to take those markers to see if they have the requisite specificity and sensitivity to distinguish these disease states."
The pilot study the researchers embarked on using the Xevo TQ-S micro involved an existing set of 15 biomarkers for heart failure. The study looked at 20 patient samples and 20 controls. The 20 heart failure samples were heterogeneous, comprising two types of heart failure, tagged as either heart failure with preserved ejection fraction (a measure of how much blood is being pumped by the left ventricle, by convention) and heart failure with reduced ejection fraction.
"What we really looked to do was get down to a panel of just three proteins which we could use in a multiplexed fashion," Langridge said. "The advantage of mass spec is we can look at a multiplex panel in a fairly straightforward fashion," compared to, say, an immunoassay.
While using any one marker on its own could roughly differentiate the heart failure samples from the controls, it didn't approach the specificity or sensitivity a clinical assay would require. Using three biomarkers, specifically apolipoprotein A-1 (APOA), plasma protease C1 inhibitor (SERPING1), and C-reactive protein (CRP), provided a useful assay, though it couldn't discriminate between the two types of heart failure in the sample size provided.
Langridge envisions that mass spec has a role to play in spurring many more of these types of studies. "All the work has been done generating markers," he said. "Can we build assays to be used to look at larger patient cohorts?"
"The nice thing about mass spec is that you can apply it in relatively high throughput to these types of samples," he said, noting that the recent study didn't apply any depletion or extensive fractionation which would help speed things along. The micro-scale separation alone helps reduce processing time. Separation in complex mixtures can often take 90 minutes. In the study, the researchers were able to cut that in half and Langridge said they can get it down to 20 to 30 minutes.
The Leicester and Waters scientists wrote in their paper that they would be moving to test their multiplex assay in a larger cohort of heart failure patients.