Quantitative measurement of deamidation in lens βB2-crystallin and peptides by direct electrospray injection andfragmentation in a Fourier transform mass spectrometer

摘要

Purpose: Deamidation of lens crystallins and specific deamidationsites have been suggested to be associated with aging and cataracts.However, these studies have been hindered by the lack of suitablequantitative methods of measurement of protein deamidation. Wedemonstrate herein a method to quantitatively measure deamidation ofproteins and peptides without prior sample preparation or separation inorder to directly compare the amidated and deamidated forms. We havetested the hypothesis that the 19 mDa mass defect that distinguishesdeamidated peptides and proteins from the ordinary natural isotopicspecies can be utilized for quantitative measurement of their rate andextent of deamidation. The measurement technique used was ion cyclotronresonance Fourier transform mass spectrometry (FTMS), alone with noprior sample preparation or separation. The amidated and deamidatedspecies were recombinantly expressed human eye lens βB2-crystallinsand the peptides GlyIleAsnAlaGly and GlyAsnAsnAsnGly. FTMS measurementsof lens proteins from a 1-month-old human donor were also carried out.Methods: Wild type and mutant human eye lens βB2-crystallinswith Gln162 replaced by Glu162 were produced in bacteria,and GlyIleAsnAlaGly and GlyAsnAsnAsnGly were synthesized by Merrifieldsolid-phase peptide synthesis. The peptides were deamidated in pH 7.4,37.00 °C, 0.15 M Tris-HCl aqueous solution for 18 successive timeintervals before analysis. Mutant and wildtype βB2-crystallinsolutions at various compositional percentages were mixed and analyzed.The peptides were introduced by electrospray ionization and immediatelyanalyzed in the ion cyclotron resonance (ICR) Fourier transform massanalyzer. Two mass defect analysis procedures were demonstrated for theproteins. In the first, βB2-crystallin was introduced into the massspectrometer by electrospray ionization and the +29 isotopic group wasselectively introduced into the ICR mass analyzer, where 14 residue and18 residue laser-induced fragments were separated and the extent ofdeamidation determined by mass defect analysis. In the second, βB2-crystallin was introduced into the mass spectrometer by electrosprayionization and the entire sample was fragmented by collision ionizationbefore introduction into the ICR mass analyzer, where 14 residuefragments were separated and the extent of deamidation determined bymass defect analysis.Results: The βB2-crystallin mass spectra showed a goodquantitative dependence upon extent of deamidation. Direct injection byelectrospray ionization followed by ion selection and laserfragmentation or by collision fragmentation produced fragments ofamidated and deamidated βB2-crystallin that were appropriate forFTMS quantitative analysis. The two peptides exhibited the expected fourdeamidation rate curves with acceptable precision.Conclusions: Mass defect FTMS quantitative analysis of proteindeamidation, as reported for the first time herein and illustrated withβB2-crystallin, should prove quite useful. This procedure omits gelseparation, chromatography, enzymatic digestion, derivatization, andother procedures that currently add cost and time while degradingquantitative comparison of the amidated and deamidated forms. Massdefect FTMS is also well suited to quantitative deamidation rate studiesof peptides. The substantial potential significance of this technique isevident, as example, for lens crystallins where it makes possiblequantitative studies of age and disease-dependent deamidation that haveheretofore been very difficult. This technique should allow convenientand reliable identification and quantitative measurement of specificdeamidation sites that may play a role in aging and cataracts.