Measurement of deamidation of intact proteins by isotopic envelope and mass defect with ion cyclotron resonance Fourier transform mass spectrometry.
After synthesis and folding, proteins undergo many post-synthetic modifications, including cleavage, oxidation, glycosylation, methylation, racemization, phosphorylation, and deamidation. Of these modifications, non-enymatic deamidation is the most prevalent. Each asparaginyl and glutaminyl residue in a protein is a miniature molecular clock that deamidates with a genetically determined half-time. These half-times vary from a few hours to more than a century, depending on a primary, secondary, tertiary, and quaternary structure near the amide residue. It has been suggested that these clocks regulate many biological processes. A few such processes have been discovered. These discoveries have been difficult because deamidation is inconvenient to measure. While most post-synthetic changes are easily measured by mass spectrometry, deamidation increases molecular mass by only one nominal Dalton, so the deamidated isotopic envelope overlaps the undeamidated isotopic envelope. While peptide deamidation rate determination through deconvolution of these envelopes has been accomplished for several hundred peptides, deconvolution becomes more difficult as the molecular weight increases. In high-resolution mass spectrometers, this deconvolution is possible for larger molecules and an alternative method based on the 19 mDa mass defect between the deamidated envelope and the isotopic envelope of protein fragments can also be utilized. We herein report a comparison of the envelope deconvolution and the mass defect methods for measurement of deamidation in human eye lens crystallins, with special emphasis on betaB2 crystallin and gammaS crystallin. Measurement of extent of deamidation of betaB2 crystallin in a 7 Tesla ion cyclotron resonance Fourier transform mass spectrometer is found to be accurate to a relative standard deviation in a single measurement of about 4% for each method. The envelope deconvolution method is further illustrated by detection of deamidation in intact gammaS crystallin, a 20 904 Da protein, and discovery of the principal gammaS deamidation site.
Robinson
Noah E
NE
Oregon Institute of Science and Medicine, 2251 Dick George Road, Cave Junction, OR 97523, USA. noah@oism.org
Zabrouskov
Vlad
V
Zhang
Jennifer
J
Lampi
Kirsten J
KJ
Robinson
Arthur B
AB
D000577:Amides / Q000032:analysis*; D017357:Cyclotrons; D005583:Fourier Analysis; D007477:Ions; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*
D000577:Amides; D007477:Ions; D011506:Proteins