Identification of four novel types of in vitro protein modifications

Gang Xing, Junmei Zhang, Yue Chen, Yingming Zhao

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29 Scopus citations


In vitro chemical modifications in proteins, introduced during sample preparation, can complicate mass spectra and increase the potential for false-positive identifications. While several in vitro protein modifications have been described previously, additional types of such modifications may exist. Here, we report discovery of four types of in vitro protein modifications, identified by HPLC/ MS/MS analysis and nonrestrictive protein sequence alignment by PTMap, an algorithm recently developed in our laboratory. These novel in vitro modifications included ethylation of aspartate and glutamate (+28 Da), esterification of aspartate and glutamate by glycerol (+74 Da), loss of 19 Da from lysine, and addition of 108 Da to cysteine. We confirmed that these modifications occurred in vitro and not in vivo in control experiments designed to avoid conditions likely to induce the modifications. We propose a plausible molecular mechanism for the -19 Da modification of lysine. Our study therefore conclusively identifies several novel in vitro protein modifications, suggests ways to avoid these modifications, and highlights the possibility of misidentification of peptides because of in vitro modifications.

Original languageEnglish (US)
Pages (from-to)4603-4608
Number of pages6
JournalJournal of Proteome Research
Issue number10
StatePublished - Oct 2008


  • Automated database searching
  • PTMap
  • Protein modifications


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