Toward real-time protein profiling in biofluids using discriminative Raman spectral markers

Area of Science:

• Biophotonics
• Spectroscopy
• Biomarker Discovery

Background:

• Simultaneous protein analysis in liquid biopsies is crucial for disease diagnosis but challenging due to protein similarity.
• Mass spectrometry and immunoassays are common, but label-free Raman spectroscopy offers a rapid, non-invasive alternative.

Purpose of the Study:

• To demonstrate label-free Raman spectroscopy's ability to discriminate clinically relevant proteins (albumins and globulins) in complex biological matrices.
• To identify a robust Raman spectral marker for multiplexed protein analysis.

Main Methods:

• Utilized label-free Raman spectroscopy to analyze single, binary, and tertiary mixtures of human serum albumin (HSA), bovine serum albumin (BSA), and γ-globulin.
• Analyzed protein mixtures in phosphate-buffered saline (PBS), artificial urine, and human serum.
• Calculated the intensity ratio of phenylalanine (622 cm^-1) and tyrosine (643 cm^-1) bands (I₆₂₂/I₆₄₃) as a spectral marker.
• Performed statistical validation using two-tailed Welch's t-tests.

Main Results:

• Reliably distinguished albumins (HSA, BSA) from γ-globulin in various mixtures and concentrations.
• Identified the I₆₂₂/I₆₄₃ ratio as a consistent marker differentiating albumin-rich from globulin-dominant samples.
• Observed strong matrix dependence, with significant discrimination in artificial urine and preserved significance in serum.
• Demonstrated the potential for distinguishing proteins with high structural similarity without Raman enhancement.

Conclusions:

• The I₆₂₂/I₆₄₃ ratio serves as a quantitative Raman spectral marker for multiplexed protein analysis.
• Label-free Raman spectroscopy shows translational potential for liquid biopsy-based diagnostics.
• The phenylalanine-to-tyrosine ratio is a relevant biomarker for disease states.