New Way of Approaching Tear Test May Identify Protein Biomarkers of Sjögren’s
Researchers have developed a quick, noninvasive way of collecting tear fluid and analyzing the proteins in it — an approach that could eventually be used to identify biomarkers that help to diagnose diseases affecting the eyes, including Sjögren’s syndrome.
“We have successfully identified a sensitive approach for discerning the proteomic [protein] profile of human tear fluid,” the scientists wrote. This approach “may be adopted in future studies of human tear fluid to improve the diagnosis of ocular diseases and discern their underlying mechanisms.”
The study, “Comparison of Different Mass Spectrometry Workflows for the Proteomic Analysis of Tear Fluid,” was published in the International Journal of Molecular Sciences.
Tears are the body’s way of lubricating, protecting, and providing nutrients to the eyes. In Sjögren’s, tear glands are unable to produce enough tears, leading to chronically dry eyes that are vulnerable to infection.
Given that tears contain a high concentration of proteins that may reflect eye health and can be collected noninvasively, they have the potential to be used as a source of biomarkers for Sjögren’s and other diseases affect the eyes. But collecting enough tears for analysis remains a significant challenge, especially among people with dry-eye conditions.
Researchers at Augusta University in Georgia have come up with a way of bypassing this limitation.
To do this, they partly leveraged the use of Schirmer strips. The Schirmer test is commonly used to help in diagnosing Sjögren’s by measuring tear production. It involves placing a small piece of filter paper, called a Schirmer strip, under the lower eyelid of each eye to collect tears.
Schirmer strips were used to collect tears from 11 healthy people. The researchers then tested four approaches to analyze the protein content in these tear samples.
Broadly, protein analysis involves first digesting, or breaking down, proteins in a sample. Those proteins are then further fragmented and analyzed using a technique called mass spectrometry. The team tested combinations of two protein digestion techniques and two fragmentation methods to determine the one yielding the most sensitive analysis.
Digestion techniques included in-strip or post-extraction digestion. In-strip digestion means that the proteins were digested while still on the strip, whereas post-extraction involves removing the proteins before digestion.
“You have to have confidence that you are extracting every single protein, that the fluid does not remain on the strip,” Shruti Sharma, PhD, a vascular and endothelial biologist and the study’s senior author, said in a press release.
Proteins were then fragmented with either collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD). While both use kinetic energy — that generated through motion — to cause molecules to collide and break apart, HCD uses a higher energy level to generate smaller protein fragments. This approach is though to make it easier to identify protein modifications that may occur due to disease.
Results showed the digestion method was the most important factor for determining how many proteins could be identified, with in-strip digestion having the greatest yield: an average of 666 proteins per patient sample when combined with CID and 678 when combined with HCD.
In contrast, post-extraction digestion identified an average of 489 proteins per sample with CID and 496 with HCD.
A combination of in-strip digestion and HCD appears to be the most effective approach, the researchers noted, with this method identifying 3,370 unique proteins across all the samples.
Among the most abundant proteins were immunoglobulins and complement proteins, which are involved in mediating immune responses and protecting against infection. Keratins, which are involved in forming a protective covering for the eye, were also abundant.
“Our findings further our understanding of the tear film by identifying pathways and protein families associated with the healthy tear film and its barrier integrity,” the researchers wrote.
The team now aims to build a large database of proteins present in human tears that will be freely available to other scientists. Building a database that includes samples from 1,600 people will likely take about a year, the researchers said.
“We are trying to create a healthy profile of these proteins: What is the range,” said Ashok Sharma, PhD, director of the university’s genomic medicine graduate program and a study author.
Eventually, the researchers hope the technique can be adapted to test for dry eye diseases like Sjögren’s. Since the study’s publication, they report having used their technique in examining tear fluid of 82 people, half of whom have dry eye disease, with a goal of establishing disease biomarkers.
“Our proposed workflow, including our in-strip protein digestion method, can be used in future studies attempting to identify tear proteomic biomarkers in ocular diseases,” the researchers wrote.
“This is the first step in developing diagnostic and prognostic assays for clinical use,” they noted.
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