title: One-step fractionation of complex proteomes enables detection of low abundant analytes using antibody-based microarrays.
abstract: Antibody-based microarray is a novel technology with great promise within high-throughput proteomics. The tremendous complexity of all proteomes will, however, pose major technological challenges, especially when targeting low-abundant analytes that remains to be resolved. In this paper, we have shown that antibody microarrays readily could be used for screening of low-abundant low molecular weight analytes in complex proteomes by optimizing the sample format. Focused antibody microarrays, based on human recombinant single-chain Fv anti-cytokine antibodies on Ni2+-NTA functionalized glass slides or black polymer Maxisorp substrates, and crude cell supernatants from activated dendritic cells, containing low levels of secreted cytokines, was used for evaluation. The proteome was pre-fractionated based on size in a simple one-step procedure using centrifugal filter devices of various molecular weight cutoffs. The results showed that the generation of a nondiluted low molecular weight (LMW) fraction, corresponding to less than 2% of the original protein content, was critical for the successful screening of cytokines in the sub pg/mL range. The reduced complexity of the LMW fraction significantly improved the assay sensitivity, by improving the fluorescent tagging step and/or reducing the nonspecific binding to the substrates.
authors.affiliation: Department of Immunotechnology, Lund University, P.O.Box 7031, SE-220 07 Lund, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Ingvarsson
authors.forename: Malin
authors.initials: M
authors.lastname: Lindstedt
authors.forename: Carl A K
authors.initials: CA
authors.lastname: Borrebaeck
authors.forename: Christer
authors.initials: C
authors.lastname: Wingren
mesh_terms: D005591:Chemical Fractionation; D016207:Cytokines / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification*; D005898:Glass / Q000737:chemistry; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D008970:Molecular Weight; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry; D040081:Protein Array Analysis; D020543:Proteome / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*
chemical_list: D016207:Cytokines; D007135:Immunoglobulin Variable Region; D009942:Organometallic Compounds; D020543:Proteome; C088321:nickel nitrilotriacetic acid; D009571:Nitrilotriacetic Acid