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Japanese Team Achieves Attomole Levels of Sensitivity for Non-immunoenriched MRM-MS

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Using multiple-reaction monitoring mass spec without upfront immuno-enrichment, a team led by researchers at Japan's National Institute of Biomedical Innovation has quantified three APL1β plasma peptides at levels as low as several hundred attomoles per mL.

According to the researchers, this is one of the lowest levels of quantitation achieved to date for an endogenous peptide using such a workflow. Detailed in a study published last week in the Journal of Proteome Research, the assay is of additional interest in that one of the measured peptides, APL1β-28, has shown potential as an Alzheimer's biomarker, Takeshi Tomonaga, project leader in the NIBI's Laboratory of Proteome Research and author on the paper, told ProteoMonitor.

MRM-MS on triple quadrupoles has become a widespread technique for protein quantitation. The technique, however, is constrained by the sensitivity limitations of mass spec instrumentation. Typically, MRM assays offer reliable quantitation in the low nanogram-per-mL range, though approaches such as the Pacific Northwest National Laboratory's PRISM methodology have brought that down to the low picogram-per-mL range.

Immunoenrichment methods like SISCAPA or mass spectrometric immunoassay (MSIA), which use antibodies to enrich target peptides or proteins prior to mass spec analysis, are one of the main approaches used to improve the sensitivity of MRM assays. However, for many targets antibodies don't exist and generating them for specific assays can be expensive and time consuming. Thus, antibody-free methods like that presented by the NIBI researchers offer a useful alternative in cases where affinity reagents aren't readily available.

Key to the team's approach, Tomonaga said, was optimization of methods for upfront depletion of abundant plasma proteins. While a variety of depletion techniques exist, the optimal procedure will vary depending on the target peptide, he noted.

In the case of the three peptides targeted in the JPR study – APL1β-25, APL1β-27, and APL1β-28 – the researchers used a combination of Affi-Gel Blue albumin removal and acetonitrile extraction followed by C18 strong cation exchange multi-StageTip purification.

They hit upon this combination after initially attempting to measure the peptides using both ultrafiltration and acetonitrile extraction by themselves, finding in each case that the peptides were barely detectable – most likely, they noted, because the target peptides were depleted along with high-abundance proteins like albumin.

"The most important point of the procedure is to be able to deplete most of the plasma proteins without losing the target peptides," Tomonaga said, adding that beyond using a two-stage process this also required tinkering with the acetonitrile concentrations and StageTip buffer conditions to maximize the removal of non-target analytes and retention of the target peptides. Tracking levels of the APL1β-25, APL1β-27, and APL1β-28 peptides throughout the sample prep process, they found that they recovered, respectively, 64 percent, 50 percent, and 54 percent of these peptides after the combination of Affi-Gel Blue, acetonitrile, and StageTip treatments.

Starting with 800 μL of plasma, the researchers depleted their samples, then ran them on a 36-minute nano-LC gradient, following that with MRM analysis on a Thermo Fisher Scientific TSQ Vantage triple quad, ultimately achieving quantitation of the APL1β peptides at levels of 300 attomoles per mL.

Given that the Vantage is a relatively old instrument, it's possible that the assay sensitivity would be even greater were it run on a newer machine, Tomonaga noted. For instance, Thermo Fisher's TSQ Quantiva triple quad, which the company launched at last year's American Society of Mass Spectrometry annual meeting, offers a ten-fold improvement in sensitivity compared to the Vantage. Recent instruments from Agilent, AB Sciex, Waters, and Bruker offer similar advantages. Curiously, Tomonaga said, he and his colleagues were unable to detect the APL1β peptides when they performed the assays with an AB Sciex QTRAP 5500.

The researchers' interest in measuring the APL1β peptides in plasma stems from their investigations of APL1β-28 as a possible biomarker for Alzheimer's disease. In a 2009 paper in EMBO Molecular Medicine, a team of Japanese researchers including several authors from the JPR SRM study identified APL1β-28 as a potential surrogate marker for the production of the Alzheimer's biomarker β-amyloid-42, finding that this peptide and Aβ-42 were produced via similar processes.

"APL1β-28 is generated with the same mechanism as Aβ-42 generation and is secreted to CSF," Tomonaga said. "We previously found [in the EMBO study] that CSF APL1β-28 levels increase in Alzheimer's patients, and we are now examining if plasma APL1β-28 levels correlate to CSF APL1β-28 levels." Plasma markers are desirable for Alzheimer's research and diagnosis given the relative ease of obtaining blood samples compared to CSF samples, which typically require a lumbar puncture.

The NIBI researchers are also looking to apply their MRM workflow to other clinical questions, colorectal cancer in particular.

"We have identified several protein biomarker candidates in colorectal cancer tissue and verified the biomarkers using this methodology," Tomonaga said, noting that he and his colleagues plan to publish on this work in the near future.

He added that they are now applying their assays to quantify these markers in plasma, and have thus far managed to measure several successfully.

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