Abstract
Purpose::
Deamidation has long been known to occur in the lens and has been reported to be one of the major post-translational modifications. Recently, it was confirmed that deamidation is significantly increased in the insoluble proteins (Wilmarth et al 2006). However, the role of deamidation as a trigger for protein insolubilization is highly controversial. Key to determining the role of deamidation in the lens is quantitating deamidation. This has been hampered by the current methods of enzymatically digesting proteins and summing levels at different sites of deamidation. We present data here quantitating total deamidation of a whole protein.
Methods::
The high resolution of Fourier transform mass spectrometers (FTMS) allows for the deconvolution of the deamidated form of a protein from the isotopic envelope of the amide form. This method is routinely used for quantitating deamidation in peptides, but can be used to quantitate deamidation in whole proteins with FTMS. For a protein with +30 charges, the mass/charge isotopic peaks are separated by only 0.03 Da, but can be resolved by FTMS. Mixtures of wild type betaB2 and deamidated betaB2 at glutamine 162 were made to mimic different extents of deamidation. Next, protein lens extracts from a 52-year-old donor were compared to samples from a 1-month-old donor. Eyes were obtained anonymously from the Lions Eye Bank of Oregon.
Results::
The experimental envelope deconvolution percentages were computed and then averaged for the six charge states, +27 through +32, of betaB2. These percentages were determined by comparison to predicted isotopic distributions and choosing the percentage most closely matching. The difference between the predicted value and actual data was 4%. Using this method, a protein with a mass matching that of gamma-S from a 52 year-old lens sample showed 30% total deamidation. Fragmentation by collisionally activated dissociation identified the primary site of deamidation as glutamine 120.
Conclusions::
While deamidation is routinely measured on peptides, high resolution FTMS makes it possible to measure deamidation on whole proteins. When directly injected into the instrument, differential loss or ionization of the sample common to previous methods is avoided. Accurately measuring the extent of deamidation will be the first step in determining the significance of this modification.
Keywords: proteomics • protein modifications-post translational • aging