Nanodiamond Solid-Phase Extraction and Triton X-114 Cloud Point Separation for Robust Fractionation and Shotgun Proteomics Analysis of the Human Serum Proteome.

Abstract

Human serum is one of the most attractive specimens in biomarker research. However, its overcomplicated properties have hindered the analysis of low-abundance proteins by conventional mass spectrometry techniques. This work proposes an innovative strategy for utilizing nanodiamonds (NDs) in combination with Triton X-114 protein extraction to fractionate the crude serum to six pH-tuned fractions, simplifying the overall proteome and facilitating protein profiling with high efficiency. A total of 663 proteins are identified and evenly distributed among the fractions along with 39 FDA-approved biomarkers─a remarkable increase from the 230 proteins found in unfractionated crude serum. In the low-abundance protein section, 88 proteins with 7 FDA-approved biomarkers are detected─a marked increase from the 15 proteins (2 biomarkers) observed in the untreated sample. Notably, fractions at pH 11, derived from the aqueous phase of detergent separation, suggest potential applications in rapid and robust serum proteome analysis. Notably, by outlining the excellent properties of NDs for proteomic research, this work suggests a promising extraction protocol utilizing the great compatibility of NDs with streamlined serum proteomics and identifies potential avenues for future developments. Finally, we believe that this work not just improves shotgun proteomics but also opens up studies on the interaction between NDs and the human proteome. Data are available via ProteomeXchange with the identifier PXD029710.