Microscale epitope mapping by affinity capillary electrophoresis-mass spectrometry.
Using beta-endorphin as a model system, a new microscale solution-based approach for linear epitope mapping based on affinity capillary electrophoresis-mass spectrometry (ACE-MS) is demonstrated. Tryptic peptides are separated in a neutral coated capillary and monitored by ultraviolet absorbance and electrospray mass spectrometry. Then, following injection of the peptide digest mixture, anti-beta-endorphin antibody is injected. The peptide, which binds to the antibody, is captured and disappears from its migration time. Following this subtraction-screening procedure, the binding of the individually synthesized or isolated immunoreactive peptide is examined by the ACE-MS procedure to confirm that the epitope resides on the peptide. A series of truncated peptides can then be made and the precise epitope determined by ACE-MS. The method utilizes low femtomole amounts of antibody and peptide digest per run and is rapid and easily automatable.
Lyubarskaya
Y V
YV
Barnett Institute, Northeastern University, Boston, Massachusetts 02115, USA.
Dunayevskiy
Y M
YM
Vouros
P
P
Karger
B L
BL
D000906:Antibodies / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D018604:Epitope Mapping / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000737:chemistry; D001615:beta-Endorphin / Q000737:chemistry / Q000276:immunology
D000906:Antibodies; D001615:beta-Endorphin; D014357:Trypsin