Screening antibody and immunosorbent selectivity by two-dimensional liquid chromatography-MS/MS (2-D LC-MS/MS).

Selectivity of both peptide- and glycan-targeting antibodies was examined by 2-D LC-MS/MS. Proteins selected from biological extracts immunospecifically in a first chromatography dimension using antibodies immobilized by either covalent coupling or adsorption to protein G were desorbed with a denaturing mobile phase and transferred to a 1.5 microm nonporous particle RP chromatography (NP-RPC) column in a second dimension. Protein peak capacity of the NP-RPC column was approximately 50. Peaks collected from the RPC column were tryptic digested and the peptide fragments were identified by MALDI-MS/MS. The objective of this analytical strategy was to discriminate between protein antigens and nonantigens through identification of their peptides, leading to an evaluation of the selectivity of antibodies and immunosorbents. Quantification of the relative amount of antigen and nonantigen species captured by immunosorbents was achieved by absorbance, along with the likely capture mechanism. A limitation of the approach was in discriminating between isoforms of an antigen in which neither the antibody nor the LC-MS system targeted the differentiating feature in the isoforms.