Targeted protein pullout from human tissue samples using competitive elution

Abstract

One commonly used strategy to gain information on the proteins in a cell is to isolate the proteins of interest by specific binders, often antibodies. Not only the specificity of the capturing antibodies but also the washing and elution conditions are crucial to avoid false-positive protein identifications. Eluting the target protein from the matrix, while avoiding the release of unrelated background proteins, should both provide more correct information on the target protein and its interaction partners, and minimize the effort to perform downstream analyses through the reduced number of eluted proteins. In this study, a novel approach for selective protein pullout is presented. Monospecific antibodies were used to selectively pullout target proteins from a complex biosample. Subsequently, the target proteins were competitively eluted from the affinity media with the recombinant antigen. To deplete the antigen from the eluted sample, IMAC spin columns were utilized to bind the N-terminal His-tag of the antigens. The competitive elution method was applied both to a model system, and for the extraction of a native human target protein. In the model system the recombinant target protein BBC7 was spiked into a protein extract of human liver, whereas an endogenously expressed target protein, cTAGE5, was extracted from the liver extract directly. SDS-PAGE analysis and mass spectrometry confirmed affinity isolation of expected target proteins. More selective elution was obtained using the competitive procedure as compared to elution at low pH. Competitive elution has thus been shown to offer an effective approach for wide-scale pullout experiments where proteins and their interaction partners are to be studied.

Investigating the Applicability of Antibodies Generated within the Human Protein Atlas as Capture Agents in Immunoenrichment Coupled to Mass Spectrometry

“…The sample was incubated at 4 °C for 30 min before cell debris was removed by centrifugation at 13 000 rpm for 15 min and subsequent filtration through a 0. 45…”

“…An appealing solution is therefore to combine the specificity of MS with an antibody capture step to increase the sensitivity. This approach has been applied in different setups, where the antibody capture step is placed either before tryptic digestion to capture full-length proteins − or after digestion to enrich tryptic peptides − or using both protein and peptide immunoenrichment in a two-step setup. , For capture of full-length proteins, the following tryptic digestion can either be done directly on the beads, in solution after an elution step, or in-gel after electrophoretic separation. The latter option is convenient, as analyzing only a small part of the gel can decrease the degree of contaminating proteins.…”