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Researchers have developed a novel application of histological staining to screen for ancient proteins in situ—within the fossil's microstructure—advancing the field of paleoproteomics by addressing long-standing challenges of contamination.

A research team led by Okayama University of Science (OUS) has successfully adapted a widely used histological technique to visualize the presence and distribution of ancient proteins directly within fossilized tissues, preserving the crucial microstructural context. This method provides a vital tool for verifying the authenticity of endogenous (originating from the ancient organism) proteins before complex analyses, significantly enhancing the reliability of paleoproteomic studies.

The findings are in the Journal of Proteome Research.

The field of paleoproteomics utilizes the fact that proteins are chemically more stable than DNA and can survive in fossils for millions of years. Analyzing these ancient proteins can reveal the evolutionary relationships and biology of extinct organisms. However, conventional methods typically require crushing fossil samples into powder to extract proteins. This process eradicates valuable structural information—making it impossible to determine the exact location of the proteins within the tissue—and increases the risk of contamination from modern sources or microbes.

"A persistent challenge in paleoproteomics is the problem of contamination—the question of whether the detected proteins are truly ancient," explained Hayato Inaba, lead author of the study, Education and Outreach Specialist at the Dinosaur Division of Tamba City, and a graduate student at OUS.

Adapting histological staining for in situ detection of collagen in nondemineralized fossils

To overcome these limitations, the OUS team focused on applying histological staining techniques directly to thin sections of nondemineralized fossils. Unlike methods requiring demineralization (dissolving minerals with acid), which can alter tissue morphology and increase contamination risk, this approach preserves the in situ structural information.

The researchers tested several staining methods on Pleistocene-aged elephant fossils (tens of thousands of years old) recovered from the Seto Inland Sea, Japan. They identified Van Gieson's staining—a common method in modern biology—as the most effective technique for visualizing Type I collagen, the primary protein in bone.

This method vividly stains preserved collagen bright red, allowing the team to directly map its distribution within the fossil matrix. Professor Hidetsugu Tsujigiwa, a specialist in regenerative medicine at OUS and a senior author of the study, adapted this staining method for application to fossils.

Mass spectrometry supports staining results and highlights tissue-specific preservation

To validate that the stained material was indeed ancient collagen, the team employed state-of-the-art mass spectrometry on the areas that tested positive. The results confirmed the presence of Type I collagen sequences matching those of extinct elephants (Proboscidea), providing strong evidence that the proteins were endogenous.

The study also revealed significant differences in protein preservation between different types of fossilized tissues from the same animals. While bone showed excellent collagen preservation, dentin (the material making up tusks/ivory) showed almost none.

The researchers attribute this difference to the microstructure of the tissues. Dentin contains countless microscopic channels called dentin tubules, which expose the matrix more directly to the environment, accelerating the degradation and leaching of collagen over time. Bone, being denser, tends to preserve proteins better.

"This finding provides an important guideline for future paleoproteomic research," said Dr. Kentaro Chiba, Lecturer at OUS's Department of Dinosaur Paleontology and corresponding author of the study. "Understanding which tissues are more likely to retain proteins helps us select the best samples for analysis."

A powerful screening tool for analyzing ancient proteins, including dinosaurs

This new application of Van Gieson's staining offers a simple yet powerful screening tool. By visualizing collagen preservation in situ, researchers can prioritize samples for expensive and complex mass spectrometry analysis and be more confident in the authenticity of the results.

The research group is now looking to apply this method to older fossils, including dinosaurs, which date back over tens of millions of years.

"Extracting proteins from dinosaur fossils is vastly more challenging due to the minute amounts remaining," noted Professor Tsujigiwa. "However, if we can analyze the amino acid sequences of their proteins, we move beyond classifying dinosaurs solely by morphology. We hope to analyze dinosaurs as living organisms and get closer to understanding their physiological functions."

Inaba concluded, "I will never forget the excitement of seeing collagen from tens of thousands of years ago stain a vivid red right before my eyes. I hope this technology will help open the door to future discoveries, such as proteins from dinosaurs that no one has ever seen."