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

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Roel Tans  

LAB HEAD: Alain van Gool

PROVIDER: PXD016435 | Pride | 2020-05-29

REPOSITORIES: Pride