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Merck Team Develops Immunoaffinity Mass Spec Assay for Quantifying CSF Tau in Alzheimer's Patients

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Merck researchers have developed an immunoaffinity mass spec-based assay for quantifying the Alzheimer's biomarker tau in human cerebrospinal fluid.

Detailed in a paper published last week in Clinical Chemistry, the assay offers comparable performance to conventional tau immunoassays while also having the potential to improve analysis of different tau isoforms, Thomas McAvoy, a senior research biochemist at Merck and author on the paper, told ProteoMonitor.

More broadly, the assay is an example of a move within pharma towards mass spec for protein analysis, particularly, McAvoy said, in cases where existing immunoassays are lacking.

"In our group, protein analysis by mass spectrometry has seen increased demand, primarily in special cases where immunoassays are not available or are not sufficiently adequate," he said.

Indeed, at the Mass Spectrometry Applications to the Clinical Laboratory annual meeting this week, Michael Lassman, principal scientist at Merck and co-author on the Clinical Chemistry paper, presented on the advantages of mass spec compared to immunoassays, noting the former's improved selectivity and multiplexing capabilities. He added that, while mass spec assays are typically less sensitive than immunoassays, approaches like immunoaffinity enrichment and low-flow chromatography have made them competitive on this front, as well.

In the case of tau, which along with β-amyloid-42 is one of the primary protein biomarkers currently used in Alzheimer's research, a mass spec-based assay was desirable due to the protein's numerous isoforms – a factor that, McAvoy said, calls into question the selectivity of existing immunoassays.

"There are six different isoforms of tau," he said. "In addition, tau is known to be heavily post-translationally modified. In light of this diversity, the selectivity of immunoassays is often questioned."

For instance, existing immunoassays may have difficulty distinguishing one form of tau from another, returning simply a measure of a sample's overall tau content. Alzheimer's research has suggested, however, that specific tau modifications may be related to disease progression, making it desirable to monitor specific forms of the protein.

"What mass spectrometry brings to the table is its inherent selectivity," McAvoy said. "It allows us to get a better understanding of what exactly is measured in the assay."

In the Clinical Chemistry study, McAvoy and his colleagues focused on measuring total tau, developing an immunoenrichment method that targeted a tau peptide common to all isoforms of the protein. The assay had a limit of quantification of .25 picomoles per liter and intra-assay CVs of between 3.2 percent and 8.1 percent and interassay CVs of between 7.8 percent and 18.9 percent.

Applying the assay to a cohort of 50 healthy controls and 50 Alzheimer's patients, the researchers found that the results of their mass spec assay showed correlation of .95 to a total-tau immunoassay from Meso Scale Discovery. Both assays were able to measure tau concentrations in all 46 samples. The IA-MS assay also identified a significant difference in tau levels between the two populations, with Alzheimer's patients averaging 29 pmol/L and controls averaging 17 pmol/L..

Immunoenrichment mass spec experiments can enrich targets either at the peptide level, as in SISCAPA, or at the protein level, as in Thermo Fisher Scientific's MSIA methodology. For their tau assay, the Merck researchers chose to enrich at the protein level, which, McAvoy said, allowed them to more easily control for variability in the enrichment and trypsin digestion steps by adding isotope labeled protein internal standards at the beginning of the experiment.

One disadvantage of this approach is the cost and development time required to create these full-length protein standards, McAvoy said. However, he noted, "as protein immunoenrichment becomes more commonplace, we expect vendor capabilities to increase and cost to decrease."

The researchers performed the assay using a Waters Xevo-TQS mass spec and microflow LC conditions on a Waters nanoAcuity system combined with Waters' Trizaic nanoTile device. A middle ground between nanoflow and standard flow LC, microflow, McAvoy noted, offers some of the sensitivity gains of the former technology with much of the improved robustness and throughput of the latter.

In particular, he said, "the value of microflow LC is optimally realized following enrichment," as it allows the researchers to reduce "column over-loading issues associated with smaller diameter columns."

"Using this [microflow] methodology, we can inject the same amount of sample as with a higher flow system, but realize a dramatic increase in sensitivity," he said.

McAvoy also cited workflow improvements offered by the Trizaic system, which integrates chromatography in a microfluidic chip with the mass spec emitter, allowing for more effective interfacing of the LC and mass spec. Use of the system, he said, offered an additional boost in sensitivity, while maintaining the method's speed and robustness.

In addition to the quantitative SRM assay, the Merck team also used high-resolution mass spec on a Thermo Fisher Orbitrap Velos to investigate the different tau peptides generated by digestion of the protein, identifying 34 unique tau peptides in a single sample. Among these 34 were the three peptides they identified in silico as likely total-tau surrogates. Their analysis found that two of these peptides however, were poor choices due to problems with trypsin digestion and oxidation, respectively. The third peptide – which was, in fact, the peptide they had selected for their SRM assay – demonstrated good performance, exhibiting high peak intensity and only minimal phosphorylation, they wrote.

Several of the authors including McAvoy noted in the paper that they had patent applications pending related to the work. McAvoy declined to comment on what aspects of the work those applications might cover.

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