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

Area of Science:

• Biochemistry
• Analytical Chemistry
• Immunology

Background:

• Antibody selectivity is crucial for accurate biological analysis.
• Evaluating immunosorbent performance requires robust analytical methods.
• Distinguishing specific targets from background is a common challenge in proteomics.

Purpose of the Study:

• To assess the selectivity of peptide- and glycan-targeting antibodies.
• To develop and validate a 2-D LC-MS/MS strategy for evaluating antibody and immunosorbent performance.
• To discriminate between protein antigens and nonantigens captured by immunosorbents.

Main Methods:

• Utilized two-dimensional liquid chromatography coupled with tandem mass spectrometry (2-D LC-MS/MS).
• Employed immunospecific capture in the first dimension using immobilized antibodies (covalent coupling or protein G).
• Analyzed desorbed proteins on a nonporous particle reversed-phase chromatography (NP-RPC) column, followed by tryptic digestion and MALDI-MS/MS peptide identification.

Main Results:

• Achieved protein peak capacity of approximately 50 on the NP-RPC column.
• Successfully identified peptide fragments to discriminate between captured antigens and nonantigens.
• Quantified relative antigen and nonantigen amounts via absorbance, providing insights into capture mechanisms.

Conclusions:

• The developed analytical strategy effectively evaluates antibody and immunosorbent selectivity.
• The method allows for the discrimination of antigens from nonantigens based on peptide identification.
• A limitation exists in differentiating antigen isoforms when the distinguishing feature is not targeted by the antibody or LC-MS system.