Quantitative measurement of deamidation in lens betaB2-crystallin and peptides by direct electrospray injection and fragmentation in a Fourier transform mass spectrometer.
Bibliographic record
Abstract
PURPOSE: Deamidation of lens crystallins and specific deamidation sites have been suggested to be associated with aging and cataracts. However, these studies have been hindered by the lack of suitable quantitative methods of measurement of protein deamidation. We demonstrate herein a method to quantitatively measure deamidation of proteins and peptides without prior sample preparation or separation in order to directly compare the amidated and deamidated forms. We have tested the hypothesis that the 19 mDa mass defect that distinguishes deamidated peptides and proteins from the ordinary natural isotopic species can be utilized for quantitative measurement of their rate and extent of deamidation. The measurement technique used was ion cyclotron resonance Fourier transform mass spectrometry (FTMS), alone with no prior sample preparation or separation. The amidated and deamidated species were recombinantly expressed human eye lens betaB2-crystallins and the peptides GlyIleAsnAlaGly and GlyAsnAsnAsnGly. FTMS measurements of lens proteins from a 1-month-old human donor were also carried out. METHODS: Wild type and mutant human eye lens betaB2-crystallins with Gln162 replaced by Glu162 were produced in bacteria, and GlyIleAsnAlaGly and GlyAsnAsnAsnGly were synthesized by Merrifield solid-phase peptide synthesis. The peptides were deamidated in pH 7.4, 37.00 degrees C, 0.15 M Tris-HCl aqueous solution for 18 successive time intervals before analysis. Mutant and wildtype betaB2-crystallin solutions at various compositional percentages were mixed and analyzed. The peptides were introduced by electrospray ionization and immediately analyzed in the ion cyclotron resonance (ICR) Fourier transform mass analyzer. Two mass defect analysis procedures were demonstrated for the proteins. In the first, betaB2-crystallin was introduced into the mass spectrometer by electrospray ionization and the +29 isotopic group was selectively introduced into the ICR mass analyzer, where 14 residue and 18 residue laser-induced fragments were separated and the extent of deamidation determined by mass defect analysis. In the second, betaB2-crystallin was introduced into the mass spectrometer by electrospray ionization and the entire sample was fragmented by collision ionization before introduction into the ICR mass analyzer, where 14 residue fragments were separated and the extent of deamidation determined by mass defect analysis. RESULTS: The betaB2-crystallin mass spectra showed a good quantitative dependence upon extent of deamidation. Direct injection by electrospray ionization followed by ion selection and laser fragmentation or by collision fragmentation produced fragments of amidated and deamidated betaB2-crystallin that were appropriate for FTMS quantitative analysis. The two peptides exhibited the expected four deamidation rate curves with acceptable precision. CONCLUSIONS: Mass defect FTMS quantitative analysis of protein deamidation, as reported for the first time herein and illustrated with betaB2-crystallin, should prove quite useful. This procedure omits gel separation, chromatography, enzymatic digestion, derivatization, and other procedures that currently add cost and time while degrading quantitative comparison of the amidated and deamidated forms. Mass defect FTMS is also well suited to quantitative deamidation rate studies of peptides. The substantial potential significance of this technique is evident, as example, for lens crystallins where it makes possible quantitative studies of age and disease-dependent deamidation that have heretofore been very difficult. This technique should allow convenient and reliable identification and quantitative measurement of specific deamidation sites that may play a role in aging and cataracts.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.001 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".