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Transferrin Assay Points to Clinical Potential of MALDI Mass Spec

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NEW YORK (360Dx) – A team led by researchers at the Czech Academy of Sciences' Institute of Microbiology have developed a MALDI mass spectrometry-based assay for measuring carbohydrate-deficient transferrin (CDT), a protein biomarker used for detecting excessive alcohol use.

Described in a study published last month in Clinical Chemistry, the assay could prove useful for monitoring patients in alcohol treatment programs or with chronic liver disease. The effort also provides an example of MALDI mass spec's potential as a clinical technology.

The researchers are collaborating with Czech research and diagnostics firm BioVendor to develop similar MALDI-based assays for the clinic, said Petr Pompach, a scientist at the Czech Academy of Sciences and senior author on the study. Pompach is also the founder and director of a company, AffiPro, that is developing functionalized MALDI targets for purposes including in situ digestion or enrichment of proteins.

The CDT assay used MALDI targets functionalized with anti-transferrin antibodies, which allowed the researchers to enrich transferrin from patient serum and then measure levels of the carbohydrate-deficient forms using mass spec. Transferrin typically contains a pair of glycosylations at its C-terminus, but excessive alcohol consumption can cause the protein to lose one or both of these modifications. Transferrin lacking these glycosylations is termed CDT and, the authors note, is potentially a useful marker for monitoring alcohol use "because of its clinical specificity, high clinical sensitivity, and half-life of two to four weeks following abstinence."

Currently, HPLC and capillary electrophoresis are most commonly used for quantifying patient CDT levels. However, these approaches can struggle with hemolyzed samples or patient serum containing other glycosylated forms of transferrin, while the MALDI assay is less susceptible to these issues.

The researchers used the method to measure CDT levels in 186 patients, identifying 44 as positive for excessive alcohol consumption and 142 as negative. A CE assay using the Sebia Capillarys CE system identified 44 as positive, 135 as negative, and was unable to analyze 7 samples due to interference from lipemia.

Pompach said he is exploring the possibility of commercializing a kit for MALDI-based transferrin testing with BioVendor, whose CEO, Viktor Ruzicka, was a coauthor on the Clinical Chemistry paper.

The work is part of a larger effort by Pompach and his colleagues to develop clinical assays using the combination of functionalized MALDI targets and MALDI mass spectrometry. The researchers, Pompach noted, have also developed MALDI assays for different glycoforms of the protein haptoglobin, which have shown potential as markers for colorectal and liver cancer.

MALDI-based systems like Bruker's MALDI Biotyper and BioMériuex's Vitek MS have dominated the clinical microbiology market, but otherwise clinical mass spec development work has primarily focused on LC-MS technology. Researchers like Pompach, however, have been exploring the use of MALDI for clinical testing, drawn by the technology's speed and simplicity.

For instance, last year, Dobrin Nedelkov, formerly a group leader at the Arizona State Biodesign Institute and the CEO of clinical proteomics firm Intrinsic Bioprobes, launched a new company, Isoformix, that plans to develop clinical tests using MALDI to measure protein isoforms linked to disease.

Like Pompach and his colleagues have done in their transferrin and haptoglobin work, Nedelkov is using antibody enrichment to pull intact proteins of interest out of patient samples followed by MALDI to assess the levels of the relevant isoforms. In addition to the simplicity provided by MALDI, which does not use an upfront LC separation, these assays are streamlined by their use of intact proteins, meaning no trypsin digestion step is required.

Such an approach likely offers a lower level of performance than a high-end LC-MS workflow, but it has the advantage of high throughput and ease of use, both of which are key in a clinical environment.

While LC-MS systems are common in large clinical labs, they require expert users.

"And that is time and cost consuming," Pompach said. "You have to pay for a specialist who will run and take care of that instrumentation, and it's not so easy."

"With MALDI, we have what we think is a really easy, simple system," he said. "Everyone can use it. Just push the button and that's it — that's how we want to make it, a very, very simple way to analyze samples."

To further simplify the approach, Pompach and his colleagues have developed a range of functionalized MALDI targets like those used in the transferrin assay, which allows for in situ protein digestion or enrichment. To functionalize the targets, they use the ambient ion soft landing method developed by Purdue University researcher Graham Cooks, which combines electrospray ionization with passage through a heated metal drying tube to select and deposit molecules on a surface.

AffiPro, the firm Pompach launched to sell functionalized MALDI targets offers targets featuring a range of functional molecules, including proteases for protein digestion, lectins for glycan and glycopeptide enrichment, titanium oxide for phosphopeptide enrichment, and antibodies to proteins including transferrin and haptoglobin.

Christopher Borchers, director of the University of Victoria-Genome BC Proteomics Centre, suggested that the Clinical Chemistry study (which he was not involved with) and the launch of firms like Isoformix indicates growing clinical interest in MALDI mass spec.

"I think people are realizing that this technology has great potential and, hopefully, more and more people will show how it can be applied, since conceptually it is quite easy," he said.

Borchers is senior author on a commentary in Clinical Chemistry that accompanied the transferrin paper in which he and his coauthors noted the MALDI assay had an "impressively high" level of correlation with the conventional CE assays and a low total coefficient of variation of 4.6 percent. They added that the MALDI assay had the advantage of being faster and less affected by interference than the CE and HPLC approaches currently used.

Another potential advantage of MALDI in clinical work is the fact that MALDI instruments have already been taken through the US Food and Drug Administration 510(k) clearance process as part of moving Bruker's Biotyper and BioMérieux's Vitek MS into the clinic.

This means new MALDI assays "can be built on [a platform] that is already there," Borchers said.

Pompach likewise cited this as a potential advantage though he noted that to date he had not seen much collaboration between the clinical chemistry labs where a test like his group's transferrin assay would be run and the clinical microbiology labs that typically house MALDI-based clinical microbiology systems.

Borchers agreed that the mass spec in clinical microbiology labs will likely remain locked down for that purpose, but he said he believed their presence in those labs would help clinical chemists become more familiar with the instrumentation.

Borchers added that he was currently working with the clinical chemistry lab at Montreal's Jewish General Hospital to add MALDI instrumentation. "They are very keen to bring MALDI-TOF into the clinical chemistry lab," he said.

Borchers and his colleagues are also active in MALDI test development. MRM Proteomics, a proteomics firm he cofounded and of which he is chief scientific officer, offers immuno-MALDI assays that combine antibody-based enrichment of target proteins or peptides with MALDI mass spec analysis.

He said he saw uptake of MALDI as particularly strong at the moment for clinical research applications where developing an ELISA to a target of interest would take too long. He declined to specify when he thought his lab would see its first MALDI-based assay in the clinic, but he said he believed MALDI assays in general were close to clinical implementation.

"It is coming," he said. "People are picking up on this technology, and I won't be surprised if in a year or two we have a new MALDI-based assay in the clinic."