Identification of Deamidated Peptides in Cytokine-Exposed MIN6 Cells through LC-MS/MS Using a Shortened Digestion Time and Inspection of MS2 Spectra

J Proteome Res. 2021 Feb 5;20(2):1405-1414. doi: 10.1021/acs.jproteome.0c00801. Epub 2020 Dec 29.


Enzymatic deamidation, the conversion of glutamine (Gln) into glutamic acid (Glu) residues, mediated by tissue transglutaminase enzymes, can provoke autoimmunity by generating altered self-epitopes, a process well-known in celiac disease and more recently also described in type 1 diabetes (T1D). To identify deamidated proteins, liquid chromatography-tandem mass spectrometry is the method of choice. However, as nonenzymatic deamidations on asparagine (Asn) and to a minor extent on Gln are frequently induced in vitro during proteomics sample preparation, the accurate detection of in vivo deamidation can be hampered. Here we report on the optimization of a method to reduce in vitro generated deamidation by 70% using improved trypsin digestion conditions (90 min/pH 8). We also point to the critical importance of manual inspection of MS2 spectra, considering that only 55% of the high quality peptides with Gln deamidation were assigned correctly using an automated search algorithm. As proof of principal, using these criteria, we showed a significant increase in levels of both Asn and Gln deamidation in cytokine-exposed murine MIN6 β-cells, paralleled by an increase in tissue transglutaminase activity. These findings add evidence to the hypothesis that deamidation is occurring in stressed β-cell proteins and can be involved in the autoimmune process in T1D.

Keywords: artifactual deamidation; autoimmunity; enzymatic deamidation; type 1 diabetes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amides
  • Animals
  • Asparagine
  • Chromatography, Liquid
  • Cytokines*
  • Digestion
  • Mice
  • Peptides
  • Tandem Mass Spectrometry*


  • Amides
  • Cytokines
  • Peptides
  • Asparagine