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

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.