Abstract
Selectivity of both peptideβ and glycanβtargeting antibodies was examined by 2βD LCβMS/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 1.5βΞΌm nonporous particle RP chromatography (NPβRPC) column in a second dimension. Protein peak capacity of the NPβRPC column was approximately 50. Peaks collected from the RPC column were tryptic digested and the peptide fragments were identified by MALDIβMS/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 LCβMS system targeted the differentiating feature in the isoforms.