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Fluorescence Detection of Tryptophan and Tyrosine Residues for Liquid Chromatography Peptide
Mapping of Therapeutic Proteins
J. Bongers, G. Balakrishnan, J. Fu, Y. Huang, L. Tao
Bristol-Myers Squibb Company
Purpose
Native fluorescence of tryptophan (Trp) and tyrosine (Tyr) can be a useful additional LC detection mode for peptide mapping of
proteins when used in-series with ultraviolet (UV) detection and mass spectrometry.
Methods
Excitation and emission wavelengths were chosen in order to obtain tyrosine-selective and tryptophan-selective peptide map
chromatograms (Figure 1).
Results
Several applications of native fluorescence detection for peptide mapping of therapeutic monoclonal antibodies and Fc-fusion proteins
are presented including the quantitation of minor sequence variants, charge variants, and degradation products. For example, Trp/Tyr
selective fluorescence detection was used in an accelerated degradation study of an IgG1 monoclonal antibody to monitor the kinetics
of degradation pathways at specific amino acid sequence sites including asparagine deamidation, aspartic acid isomerization to
isoaspartic acid, and methionine oxidation.
Conclusion
Trp/Tyr fluorescence detection generally results in a flatter baseline and improved signal-to-noise as compared to 280 nm UV
detection. Fluorescence detection of Trp/Tyr can be useful for obtaining peak areas in certain cases where peptides coelute and are not
resolved by UV detection and for obtaining better signal-to-noise for quantitation of low levels of modified Trp/Tyr peptides of
interest.