Fractionation of serum components using nanoporous substrates.
Numerous previously uncharacterized molecules resident within the low molecular weight circulatory proteome may provide a picture of the ongoing pathophysiology of an organism. Recently, proteomic signatures composed of low molecular weight molecules have been identified using mass spectrometry combined with bioinformatic algorithms. Attempts to sequence and identify the molecules that underpin the fingerprints are currently underway. The finding that many of these low molecular weight molecules may exist bound to circulating carrier proteins affords a new opportunity for fractionation and separation techniques prior to mass spectrometry-based analysis. In this study we demonstrate a method whereby nanoporous substrates may be used for the facile and reproducible fractionation and selective binding of the serum-based biomarker material, including subcellular proteins found within the serum. Aminopropyl-coated nanoporous silicon, when exposed to serum, can deplete serum of proteins and yield a serum with a distinct, altered MS profile. Additionally, aminopropyl-coated, nanoporous controlled-pore glass beads are able to bind a subset of serum proteins and release them with stringent elution. The eluted proteins have distinct MS profiles, gel electrophoresis profiles, and differential peptide sequence identities, which vary based on the size of the nanopores. These material surfaces could be employed in strategies for the harvesting and preservation of labile and carrier-protein-bound molecules in the blood.
Geho
David
D
Laboratory of Pathology, FDA-NCI Clinical Proteomics Program, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Cheng
Mark Ming-Cheng
MM
Killian
Keith
K
Lowenthal
Mark
M
Ross
Sally
S
Frogale
Kristina
K
Nijdam
Jasper
J
Lahar
Nicholas
N
Johann
Don
D
Herrmann
Paul
P
Whiteley
Gordon
G
Ferrari
Mauro
M
Petricoin
Emanuel
E
Liotta
Lance
L
D004591:Electrophoresis, Polyacrylamide Gel; D005898:Glass; D006801:Humans; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D049329:Nanostructures / Q000737:chemistry* / Q000648:ultrastructure; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D044967:Serum / Q000737:chemistry*; D012825:Silicon / Q000737:chemistry; D013379:Substrate Specificity
D010455:Peptides; D012825:Silicon