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In a breakthrough that offers new possibilities for mass spectrometry (MS) in clinical labs, researchers have developed a top-down “dilute and infuse” tandem MS (MS/MS) approach that identifies hemoglobin 7(Hb) variants and thalassemias, two common genetic disorders, within just 3 minutes for data acquisition. A study published in Clinical Chemistry describes this technology’s application in Hb variant de novo sequencing and in diagnosing β thalassemias and its prospects for future use in clinical settings.

Two of the most common screening methods for these blood disorders, high-performance liquid chromatography and electrophoresis (EP) have limited resolving power often yielding ambiguous results that require further confirmation via genetic tests. While MS has been explored as an alternative for Hb variant testing, current techniques have their own limitations.

Most protein sequencing is based on bottom-up MS/MS, a process in which proteins are separated on a two-dimensional electrophoresis gel, then proteolyzed into fragments. The gel is then cut into pieces, each of which is analyzed by on-line liquid chromatography MS/MS. “However, sequence coverage and the identity and site(s) of post-translational modifications (PTMs) are typically incomplete,” Alan G. Marshall, PhD, the study’s corresponding author, professor of chemistry and biochemistry at Florida State University, and founding director and chief scientist at the Ion Cyclotron Resonance Program at the National High Magnetic Field Laboratory (MagLab), told CLN Stat. Work performed at the MagLab is supported by National Science Foundation Cooperative Agreement No. DMR-1644779 and the state of Florida.

Marshall and his colleagues tested a new “top-down” MS/MS approach using a custom-built 21-T FT-ICR (fourier transform ion cyclotron) mass spectrometer with a high-field superconducting magnet. Inside, a charged protein molecule rotates at a cyclotron frequency, enabling molecular mass measurements at 100 parts per billion accuracy (i.e. 10 to 100 times higher than other mass analyzers). “It is the world’s highest resolution mass spectrometer, and thus especially useful for analysis of complex mixtures,” he said.

Under this approach, “each intact hemoglobin subunit is ionized directly, automatically ensuring complete sequence coverage, as well as the identity of each “proteoform” (i.e., a given set of genetic mutations and PTMs),” Marshall explained. The identity and location of point mutations in hemoglobin can be determined definitively in a few minutes, from one microliter of human red blood cells.

In a blind analysis of 18 samples, the research team was able to correctly identify Hb variants using the 21-T FT-ICR MS/MS tool. In one important discovery, they were able to identify the first homozygous Hb Himeji variant. “Moreover, in blind analysis, we also unequivocally diagnosed β-thalassemia based on relative abundances of δ and α subunits,” Marshall said.

FT-ICR MS “provides the baseline information to make rational decisions about the capabilities of other, more widely available, lower-resolution mass analyzers ... for routine clinical laboratory testing, as well as how best to design the most efficient experimental protocols while at the same time improving quality and turnaround time,” wrote the researchers.

“The possibility of sequencing proteins and readily identifying fine variations in protein structure, not only sequence changes but also post-translation modifications … could open new fields of investigation for disease diagnosis and prognosis,” Didia Coelho Graça and Pierre Lescuyer wrote in a related editorial.

Some technical issues remain before this new MS tool has practical implications for assessing Hb variants or thalassemias. “First, the top-down MS analysis is performed on purified proteins. Extraction of hemoglobin from whole blood is not difficult, but the analysis of lower abundance proteins in biological samples certainly represents another challenge,” wrote the editorialists. While it’s a fairly easy task to achieve high sequence coverage with hemoglobin subunits, “the work becomes much trickier as proteins of higher molecular weight are analyzed. Third, the transferability of such an analytical approach on mass spectrometers available in clinical laboratories will have to be demonstrated.”

Readers of this study won’t find its take-home message through a formal analysis of the data, Coelho Graça and Lescuyer offered. “It is rather in the prospects they are opening up. This work shows how far it is actually possible to go in the analysis of a protein by MS,” they wrote, adding, “who could have imagined, when the human genome project launched, what is now regularly achieved with next-generation sequencing?”

Marshall essentially agreed with this assessment. The new method is rapid in comparison with DNA sequencing and also detects post-translational modifications—which DNA cannot, he said. Going forward, its main value will be in identifying new Hb mutants—heterozygous ones in particular—and in providing a gold standard to guide analyses with lower resolution instruments.

Read the latest issue of Clinical Chemistry to get the details on this new MS/MS technology for identifying inherited blood disorders.