• Mass Spectrometry Letters
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• v.15 no.2
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• pp.107-119
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• 2024

We aimed to develop a suitable quantification method for detecting asparagine deamidation and aspartic acid isomerization in peptide mapping using LC-MS. Our assessment of its validity and suitability involved comparing its quantitative findings with those obtained from cation-exchange chromatography and capillary electrophoresis methods. By subjecting trastuzumab to rigorous conditions to induce these modifications, we validated the efficacy of this new analytical method in peptide mapping via LC-MS, evaluating both qualitative and quantitative aspects of asparagine deamidation and aspartic acid isomerization. Our investigation underscored the significance of enzyme selection and the presence of miss-cleaved or non-specific peptides in achieving accurate quantitative results. The experimental results demonstrated a strong correlation with results from cation-exchange chromatography and capillary electrophoresis analyses, confirming the reliability of the LC-MS based peptide mapping approach.

• Korean Journal of Food Science and Technology
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• v.27 no.5
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• pp.794-798
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• 1995

Deamidation by neutrase on glutaminyl and asparaginyl residues of protein was examined. The optimum pH and temperature for BSA(bovine serum albumin) deamidation by neutrase were 10 and $20^{\circ}C$, respectively. The incubation for 3 hrs under the optimum condition removed 24% of amide groups and hydrolyzed 2.9% of peptide bonds. Deamidation by neutrase was superior to that by pronase, bromelain, or ficin. Deamidation degrees of egg albumin, soy protein isolate and casein by neutrase under the optimum condition were about 20%, 14% and 14%, respectively. However, relatively high degree of peptide hydrolysis was accompanied with casein deamidation.

• Bulletin of the Korean Chemical Society
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• v.10 no.6
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• pp.600-604
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• 1989

In order to find the rational methods for improving the thermal stability of subtilisin Carlsberg, the mechanisms of irreversible thermoinactivation of the enzyme were studied at $90^{\circ}C.$ At pH 4, the main process was hydrolysis of peptide bond. This process followed first order kinetics, yielding a rate constant of $1.26\;{\times}\;10^{-1}h^{-1}$. Hydrolysis of peptide bond of PMS-subtilisin occurred at various sites, which produced new distinct fragments of molecular weights of 27.2 KD, 25.9 KD, 25.0 KD, 22.3 KD, 19.0 KD, 17.6 KD, 16.5 KD, 15.7 KD, 15.0 KD, 13.7 KD, and 12.7 KD. Most of the new fragments originated from the acidic hydrolysis at the C-side of aspartic acid residues. However 25.0 KD, 15.7 KD, and 13.7 KD which could not be removed in purification steps stemmed from the autolytic cleavage of subtilisin. The minor process at pH 4 was deamidation at asparagine and/or glutamine residues and some extend of aggregation was also observed. However, the aggregation was main process at pH 7 with a first order kinetic constant of $16 h^{-1}.$ At pH 9, the main process seemed to be combination of deamidation and cleavage of peptide bond.