Shotgun proteomics analysis of protein purifications from human plasma using antibodies and affimers

Antibodies are widely used to purify protein from complex mixtures like human plasma. Assessing the specificity of anntibodies, or any protein binder, is essential for the development of a protein biomarker assay. In addition to antibodies, novel protein binders like affimers are developed with a high specificity against the target protein and without batch-to-batch variability. In this study, we compared the specificity of affimers and commercially available antibodies against two non-related proteins (IL-37 and proinsulin). We conducted protein capture experiments with antibodies coupled to magnetic protein G beads and (biotinylated) affimers coupled to streptavidin magnetic beads. Binding capacity of anti-IL-37 and anti-proinsulin antibodies and affimers was investigated via these magnetic-bead captures of their recombinant protein targets in human plasma. The purified fractions were transferred to an SDS-PAGE, followed by in-gel digestion of the visualized protein bands and subsequently analyzed with western blot or shotgun proteomics using LC-MS/MS. From the proteomics experiments we evaluated the amount of background proteins that were co-purified during the protein purification procedure. In addition we determined the relative abundance of the target protein relative to other non-related proteins.