title: High-affinity recombinant antibody fragments (Fabs) can be applied in peptide enrichment immuno-MRM assays.
abstract: High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays.
authors.affiliation: Fred Hutchinson Cancer Research Center , 1100 Fairview Avenue North, Seattle, Washington 98109, United States.
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Whiteaker
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.forename: Christian
authors.initials: C
authors.lastname: Frisch
authors.forename: Francisco
authors.initials: F
authors.lastname: Ylera
authors.forename: Stefan
authors.initials: S
authors.lastname: Harth
authors.forename: Achim
authors.initials: A
authors.lastname: Knappik
authors.forename: Amanda G
authors.initials: AG
authors.lastname: Paulovich
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D001798:Blood Proteins / Q000737:chemistry; D002846:Chromatography, Affinity; D005240:Feasibility Studies; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D007140:Immunoglobulin Fab Fragments; D010455:Peptides; D011994:Recombinant Proteins