Minimization of side reactions during Lys Tag derivatization of C-terminal lysine peptides |
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Authors: | Olav Mjaavatten Gyrid Nygaard Frode S. Berven Frode Selheim |
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Affiliation: | Proteomics Unit (PROBE), Department of Biomedicine, University of Bergen, Jonas Lies Vei 91, N-5009 Bergen, Norway |
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Abstract: | Several issues need to be considered concerning chemical labeling strategies in proteomics. Some of these are labeling specificity, possible side reactions, completeness of reaction, recovery rate, conserving integrity of sample, hydrolysis of peptide bonds at high pH, and signal suppression in mass spectrometry (MS). We tested the effects of different reaction conditions for 2-methoxy-4,5-dihydro-1H-imidazole (Lys Tag) derivatization of the ?-amine group of lysine (K) residues. By using nanoflow LC–electrospray ionization-MS (LC–ESI-MS) and MS/MS in combination with MSight 2-D image analysis, we found that standard Lys Tag derivatization processes and conditions induce side reactions such as (i) Lys Tag labeling of the N-terminus, (ii) methylation of internal aspartic acid (D), glutamic acid (E) and C- and N-peptide termini and (iii) deamidation of asparagine (N) and glutamine (Q). We found temperature and pH to be the main variables to control side reactions. Lowering the reaction temperature from 55 °C to room temperature reduced deamidation from 22.8 ± 1.4% (SEM) to 7.7 ± 5.5% (SEM) and almost totally blocked methylation (7.0 ± 1.2% (SEM) to 0.4 ± 0.4% (SEM) of the internal acidic amino acids (D and E) at high pH. We conclude that lowering the reaction temperature minimizes undesired side reactions during Lys Tag derivatization in solution. |
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Keywords: | Chromatography Chemical labeling Nanoflow LC&ndash ESI-MS MS/MS MSight 2-D image analysis |
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