title: Reduction of the concentration difference of proteins in biological liquids using a library of combinatorial ligands.
abstract: The discovery of polypeptides and proteins with relevance to a particular biological state is complicated by their vast number and concentration range in most biological mixtures. Depletion methodologies are frequently used to remove the most abundant species; however, this removal not only fails significantly to enrich trace proteins, it may also nonspecifically deplete them due to their interactions with the removed high-abundance proteins. Here we report a simple-to-use methodology that reduces the protein concentration range of a complex mixture like whole serum through the simultaneous dilution of high-abundance proteins and the concentration of low-abundance proteins. This methodology utilizes solid-phase ligand libraries of immense diversity, generated by "split, couple, recombine" combinatorial chemistry, that are used for affinity-based binding to the proteins of a given mixture. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that many peptides or proteins that are undetectable by classical analytical methods become easily accessible. The reduction in the dynamic range of unfractionated serum is specifically described along with treatment of other proteomes such as extracts from Escherichia coli, chicken egg white and cell culture supernatant. Mono- and bi-dimensional electrophoresis (1-DE and 2-DE respectively) and surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-TOF-MS) technology demonstrate the reduction in protein concentration range. Combining this approach with additional fractionation methods further increased the number of detectable species.
authors.affiliation: Ciphergen Biosystems, Fremont, CA 94555, USA.
authors.forename: Vanitha
authors.initials: V
authors.lastname: Thulasiraman
authors.forename: Shanhua
authors.initials: S
authors.lastname: Lin
authors.forename: Liliana
authors.initials: L
authors.lastname: Gheorghiu
authors.forename: Julia
authors.initials: J
authors.lastname: Lathrop
authors.forename: Lee
authors.initials: L
authors.lastname: Lomas
authors.forename: David
authors.initials: D
authors.lastname: Hammond
authors.forename: Egisto
authors.initials: E
authors.lastname: Boschetti
mesh_terms: D000327:Adsorption; D000818:Animals; D001798:Blood Proteins / Q000302:isolation & purification; D005591:Chemical Fractionation / Q000379:methods; D002645:Chickens; D002852:Chromatography, Ion Exchange / Q000379:methods; D003470:Culture Media / Q000737:chemistry; D004529:Egg White / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry; D009842:Oligopeptides / Q000737:chemistry; D019151:Peptide Library; D011506:Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001798:Blood Proteins; D003470:Culture Media; D008024:Ligands; D009842:Oligopeptides; D019151:Peptide Library; D011506:Proteins