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BRIMS, MGH Scientists Partnering to Identify Protein Biomarkers Linked to Stroke

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Researchers at Thermo Fisher Scientific's BRIMS Center are working with scientists at Massachusetts General Hospital to identify protein biomarkers linked to stroke.

The work, which uses a new biomarker-discovery workflow under development at BRIMS, aims to find markers to improve stroke patients' treatment options and shed light onto the biological mechanisms underlying the disease, said project co-leader MingMing Ning, a scientist at MGH's Clinical Proteomics Research Center.

In particular, the research is focused on exploring the relationship between strokes and patent foramen ovale. Around one-quarter of the world's population is born with this congenital heart defect, which, during gestation, causes a passage between the left and right atria of the heart to remain open.

Though normally this opening closes in the first three months of life, a resulting phenotype could allow a blood clot to travel from an extremity to the brain and potentially trigger a stroke.

The collaboration also aims to identify protein markers to guide doctors' use of tissue plasminogen activator in stroke treatment. The therapy, which acts much like an anticoagulant, dissolves blood clots and is therefore effective against ischemic, or blocked, strokes.

However — and also because it mimics an anticoagulant — the treatment can be fatal to patients suffering hemorrhagic, or bleeding, strokes.

Because it can be difficult to distinguish between the two, currently only 5 percent of stroke patients receive tPA treatment.

Although strokes primarily affect the brain, "it's not just the brain itself that's the problem," Ning told ProteoMonitor. Key to better understanding the disease is to "better understand the signaling between the heart and the brain," she said. MGH researchers hope the BRIMS biomarker work will help them better understand this cardioneural signaling.

"We always center on what happens to the brain, but to really understand in the blood how [the heart and brain] are talking to each other is very important," Ning said. "That's a new concept in treating stroke because the brain can be injured by a lot of factors in the blood, so [the BRIMS biomarker] discovery approach is [helping us] to understand for the first time the underlying patho-physiology of what's happening."

As part of the project, BRIMS researchers developed a new label-free MS/MS biomarker-discovery process that decouples marker identification from quantitation — a change from typical discovery workflows that do identification and quantification in a single step.

By first looking for differentially expressed features and then trying to identify them during a second MS scan, the researchers were able to "capture all of the differentially expressed features, whether or not they produced useful MS/MS spectra that could lead to identification" during the initial scan, said BRIMS Center Director Mary Lopez.

She noted that this allowed the researchers "to dig deeper into the proteome and get beyond the high-abundance markers that usually come up when you do these types of experiments."

The discovery work, which was done on a Thermo Fisher Orbitrap Velos machine with samples from five clinical foramen ovale patients, identified 263 proteins, 128 of which were differentially expressed. To winnow that number down, the BRIMs team subjected the 128 putative markers to a variety of statistical analysis tools, including the center's new ROCstar algorithm, which generates receiver operating curve AUCs for panels of multiple biomarkers, allowing researchers to stratify putative markers according to their potential usefulness.

Running the highest-scoring markers through pathway-analysis software revealed a number of proteins tied to coagulation pathways and other processes linked to stroke. The BRIMS researchers now plan to take the most promising of these markers and translate them into targeted single-reaction monitor mass-spec assays that will allow for "very high-throughput interrogation" of them in clinical samples to generate "the proper clinical statistics," Lopez told ProteoMonitor.

Working with the MGH scientists, Lopez's team has also been using such targeted SRM assays to investigate apolipoproteins that could be used to determine which stroke patients should receive tPA treatment. This portion of the work, which is being done on a Thermo Fisher TSQ Vantage triple quadrupole, has examined roughly 300 patient samples, identifying several proteins – ApoC3, ApoC1, and ApoA1 in particular – that appear promising.

The research "really helps with understanding specific questions like 'Is this a [hemorrhagic] or an ischemic stroke," Ning said, "because the treatment is completely different. If you give [tPA] to a bleeding patient, they will surely die, and vice-versa. So to have that kind of rapid, targeted information to help us make clinical decisions, that's what we're looking for."

While CT scans, the current gold standard for such diagnoses, are good at detecting hemorrhagic strokes, they are less reliable for detecting ischemic strokes, Lopez said. Ischemic strokes comprise roughly 80 percent of stroke cases.

The BRIMS researchers are also studying the various isoforms of the markers they've identified, Lopez said, noting that "because many of these proteins exist in various forms and each of these isoforms may have clinical significance, it's very important to understand not just what the protein is, but what the isoforms are and what their relative quantities are in the blood."

For this work, the team is using a mass-spec immunoassay platform made by Intrinsic Bioprobes that employs a high-throughput, high-binding-capacity microcolumn activated with antibodies to isolate low-abundance proteins in complex samples. It can then quantitate the protein variants via SRM mass spec.

Thermo Fisher and IBI signed an agreement in July to develop and market a test on the platform for parathyroid hormone. That deal covered only the PTH assay, but at the time IBI CEO Dobrin Nedelkov told ProteoMonitor that the two companies planned to discuss future assays that they would work on "jointly" (PM 07/16/2010).

The BRIMS researchers are now working with Ning to evaluate which of the biomarkers are "of clinical significance," Lopez said, and the MGH group, which is receiving funding for the project from the National Institutes of Health and the National Institute of Neurological Disorders and Stroke, plans to continue supplying the center with patient samples for proteomic analysis.

The researchers have submitted a paper on the stroke biomarker work to the journal Clinical Chemistry that is currently under review.

"We'd like to continue working with [the MHG researchers] for as long as it’s fruitful," Lopez said. "So we haven't put any kind of end date on this from our side. It's still very early days."

"There are a lot more things we need to understand" about stroke, Ning said. "I personally think we're just scratching the surface."


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