Nano-LC-MS/MS Tracks Saliva in Multiple Sclerosis

Multiple sclerosis (MS) is a long-term disease in which the immune system attacks the brain and spinal cord. Doctors need simple, painless tests that can pick up changes in the body linked to the disease and to how patients respond to treatment.

To help with this, researchers studied all the proteins in the saliva of people with the most common form of MS, relapsing-remitting (RR) MS, in which symptoms flare up and then ease for a time. They collected saliva from 66 patients before they started treatment and again from 40 of them after six months on medication designed to slow the disease. They compared these samples with saliva from 31 healthy people. To identify the proteins, they used nano-liquid chromatography-high-resolution tandem mass spectrometry (nano-LC-HRMS/MS), a highly sensitive laboratory technique that separates the components of very small samples and then identifies and measures them precisely. The team published their findings in the journal Biomolecules.¹

What Is Multiple Sclerosis, and Why Are New Biomarkers Needed to Monitor It?

MS is a long-term disease in which the immune system attacks the brain and spinal cord, and it is one of the most common causes of nerve-related disability in young adults. The damage it causes includes inflammation that strips away the protective coating around nerves, injury to the nerve fibers themselves, scarring, and gradual loss of nerve tissue. These problems can occur together to different degrees, varying from one patient to another and as the disease progresses.^2,3

This variation matters especially in RRMS, in which symptoms flare up and then ease for a time. Doctors rely on these flare-ups and on MRI brain scans to keep track of the disease, but these only give part of the picture. They do not fully show what is happening at the molecular level as the disease develops or how patients are responding to treatment.^3,4

The number of treatments available for MS has grown a great deal in recent decades. These medications, designed to slow the disease, work in different ways, for example by calming parts of the immune system, stopping certain immune cells from traveling into the brain and spinal cord, or blocking inflammation signals. While they have helped control the disease and improved patients’ long-term outlook, people still vary widely in how well they respond. Because of this, there is a growing need for markers in the body that can add to what doctors learn from examining patients and looking at scans. Such markers could help sort patients into groups that are likely to respond in similar ways and reveal the biological changes that happen while patients are on treatment. ^5,6

What Did the Study Reveal About How Saliva Proteins Differ in People with MS Before and After Treatment?

The researchers identified 1305 proteins in the saliva samples, and 209 of them differed between the groups being compared. By comparing the groups in different ways, including tracking the same patients over time, they found four kinds of patterns. Some protein changes linked to MS were present both before and after treatment. Others were present before treatment but became less pronounced afterward, moving closer to the levels seen in healthy people. Some differences appeared only after six months of treatment, and others showed up only when the same patients were compared over time. The researchers stress that these patterns only describe what they observed and cannot be taken as proof that the medication caused them or that patients were responding to treatment.¹

A machine-learning analysis supported the idea that MS leaves its own distinct mark on saliva proteins. Using a set of 50 proteins, it was able to sort the samples into healthy people, patients before treatment, and patients after treatment. Almost all these proteins (96%) also showed clear differences when looked at one by one. When the team checked what these proteins do in the body, they found most were involved in the body’s first line of immune defense, the release of substances by a type of white blood cell, cell death, chemical signaling between immune cells, and how proteins fold into their proper shape.¹

“These findings,” write the authors of the paper,¹ “indicate that six months of therapy was associated with substantial salivary proteome changes and support saliva as a candidate biofluid for longitudinal molecular monitoring and non-invasive detection of treatment-associated salivary proteome variations in RRMS.”

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References

1. Guadalupi, G.; Contini, C.; Piras, C. et al. Salivary Proteomics Reveals Disease- and Therapy-Associated Molecular Patterns in Relapsing-Remitting Multiple Sclerosis. Biomolecules 2026, 16 (10), 1524. DOI: 10.3390/biom16101524
2. Jakimovski, D.; Bittner, S.; Zivadinov, R. et al. Multiple Sclerosis. Lancet 2024, 403 (10422), 183-202. DOI: 10.1016/S0140-6736(23)01473-3
3. Lassmann, H. Pathogenic Mechanisms Associated With Different Clinical Courses of Multiple Sclerosis. Front Immunol. 2019, 9, 3116. DOI: 10.3389/fimmu.2018.03116
4. Lublin, F. D.; Reingold, S. C.; Cohen, J. A. et al. Defining the Clinical Course of Multiple Sclerosis: The 2013 Revisions. Neurology 2014, 83 (3), 278-286. DOI: 10.1212/WNL.0000000000000560
5. Faissner, S.; Gold, R. Efficacy and Safety of Multiple Sclerosis Drugs Approved Since 2018 and Future Developments. CNS Drugs 2022, 36 (8), 803-817. DOI: 10.1007/s40263-022-00939-9
6. Kuhle, J.; Kropshofer, H.; Haering, D. A. et al. Blood Neurofilament Light Chain as a Biomarker of MS Disease Activity and Treatment Response. Neurology 2019, 92 (10), e1007-e1015. DOI: 10.1212/WNL.0000000000007032