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

Simultaneous analysis of multiple proteins in liquid biopsies is essential for accurate disease diagnosis and monitoring yet remains challenging in complex biological matrices when proteins exhibit high structural similarity. While mass spectrometry and immunoassays dominate biomarker discovery and validation, label-free Raman spectroscopy offers a rapid, non-invasive alternative for protein profiling. Here, we demonstrate the capability of label-free Raman spectroscopy to discriminate clinically relevant proteins-albumins and globulins-in artificial urine and human serum without employing any Raman enhancement strategies. Human serum albumin (HSA) and bovine serum albumin (BSA), despite differing by only a single tryptophan residue, were reliably distinguished from γ-globulin across single, binary, and tertiary mixtures at both high and clinically relevant concentrations. Analysis of multiplexed samples identified a robust Raman spectral marker defined by the intensity ratio of the phenylalanine (622 cm^-1) and tyrosine (643 cm^-1) bands (I₆₂₂/I₆₄₃), which consistently differentiated albumin-rich from globulin-dominant compositions across matrices of PBS, artificial urine and serum. Statistical validation using two-tailed Welch's t-tests revealed strong matrix dependence, with highly significant discrimination in artificial urine and preserved, though weaker, significance in serum due to increased biochemical complexity. Given the known biological relevance of phenylalanine-to-tyrosine biomarker in several disease states, these findings establish the I₆₂₂/I₆₄₃ ratio as a quantitative Raman spectral marker for multiplexed protein analysis and highlight the translational potential of label-free Raman spectroscopy for liquid biopsy-based diagnostics.