Fractionation of serum components using nanoporous substrates.

Area of Science:

• Biochemistry
• Materials Science
• Analytical Chemistry

Background:

• Low molecular weight molecules in circulation offer insights into organismal pathophysiology.
• Proteomic signatures from these molecules are identified using mass spectrometry and bioinformatics.
• Many low molecular weight molecules bind to carrier proteins, presenting an opportunity for pre-analysis separation.

Purpose of the Study:

• To demonstrate a method for facile and reproducible fractionation and selective binding of serum-based biomarker material.
• To investigate the use of nanoporous substrates for isolating low molecular weight circulatory proteome components.
• To explore the potential of these materials in preserving labile and carrier-protein-bound molecules.

Main Methods:

• Utilized aminopropyl-coated nanoporous silicon to deplete serum proteins and alter the mass spectrometry (MS) profile.
• Employed aminopropyl-coated, nanoporous controlled-pore glass beads for selective binding and stringent elution of serum proteins.
• Analyzed eluted proteins using MS, gel electrophoresis, and differential peptide sequencing, varying nanopore sizes.

Main Results:

• Nanoporous silicon successfully depleted serum proteins, resulting in an altered MS profile.
• Nanoporous glass beads selectively bound and eluted a subset of serum proteins.
• Eluted proteins exhibited distinct MS and gel electrophoresis profiles, with peptide identities varying by nanopore size.

Conclusions:

• Nanoporous substrates offer a viable method for fractionating and selectively binding serum biomarkers.
• These materials can be used to harvest and preserve labile and carrier-protein-bound molecules in blood.
• The developed technique enhances the analysis of the low molecular weight circulatory proteome for diagnostic purposes.