Novel comprehensive approach for accessible biomarker identification and absolute quantification from precious human...

Area of Science:

• Biochemistry
• Proteomics
• Biomarker Discovery

Background:

• Identifying biomarkers from limited human pathological lesions is challenging.
• Accessible proteins (cell membrane-associated, extracellular) are crucial for diagnosis and targeted therapy.
• Existing methods struggle with limited sample sizes.

Purpose of the Study:

• To develop a novel, comprehensive, and efficient method for identifying and quantifying accessible proteins from precious, limited tissue samples.
• To enhance the discovery of potential diagnostic and therapeutic biomarkers.
• To demonstrate the method's utility on human breast cancer tissues.

Main Methods:

• A sequential three-step approach combining biotin labeling of accessible proteins and glycopeptide isolation.
• Utilized chemically modified biotin for ex vivo protein labeling.
• Integrated in silico analysis with glycopeptide and non-glycopeptide analysis for confident glycoprotein identification.

Main Results:

• The method identified over 400 potentially accessible proteins per sample.
• Significantly increased the number of therapeutically and diagnostically valuable proteins compared to individual methods (≥30% and ≥50%).
• Identified 93 differentially modulated proteins in breast cancer, including novel potential biomarker CD276, which showed differential expression via immunohistochemistry.

Conclusions:

• The developed sequential method is efficient for identifying and quantifying accessible proteins from limited tissue samples.
• This approach significantly enhances the discovery of valuable diagnostic and therapeutic protein biomarkers.
• The technology is broadly applicable to various pathologies, accelerating biomarker discovery.