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A method for the selective isolation and enrichment of carrier protein‐bound low‐molecular weight proteins and peptides in the blood
Author(s) -
Camerini Serena,
Polci Maria Letizia,
Liotta Lance A.,
Petricoin Emanuel F.,
Zhou Weidong
Publication year - 2007
Publication title -
proteomics – clinical applications
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.948
H-Index - 54
eISSN - 1862-8354
pISSN - 1862-8346
DOI - 10.1002/prca.200600618
Subject(s) - fractionation , proteome , chemistry , blood proteins , chromatography , denaturation (fissile materials) , peptide , biochemistry , gel electrophoresis , elution , nuclear chemistry
The low molecular weight (LMW) region of the circulatory proteome, thought to contain a rich source of biomarkers, resides in vivo, in a complexed state with larger, highly abundant resident proteins. Consequently, serum fractionation approaches that deplete the high‐abundance proteins under native conditions will remove much of the LMW proteome. We describe a new strategy to systematically collect, isolate and enrich the LMW molecules that would be otherwise eliminated during the depletion of high‐abundance circulatory proteins based on continuous elution electrophoresis. We employ strong denaturing conditions to disrupt association with the high‐abundance carrier proteins followed by fractionation and removal of SDS. Under denaturation, the LMW molecules were effectively stripped from the highly abundant carrier proteins. We then removed the SDS by ion exchange matrix sequestration and concentrated the fractions. The outcome is a series of SDS‐free fractions of LMW molecules. The isolated fractions were then analyzed by enzymatic digestion followed by LC‐MS/MS analysis. The yield of multiple peptide hits as well as the total number of identifications significantly increased (50%) compared to unfractionated serum. The method yielded a 30% higher number of low‐abundance serum proteins compared to direct sequencing of unfractionated serum.