"de-novo" amino acid sequence elucidation of protein Ge by combined "top-Down" and "bottom-Up" mass spectrometry

Yelena Yefremova, Mahmoud Al-Majdoub, Kwabena F.M. Opuni, Cornelia Koy, Weidong Cui, Yuetian Yan, Michael L. Gross, Michael O. Glocker

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)

Abstract

Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein G with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α-N-gluconoylation and α-N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G< comprised 241 amino acids and resulted in a molecular mass of 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G (185 amino acids), we named this protein "protein Ge." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α-N-gluconoylations, was confirmed with 100% sequence coverage. After the protein Ge sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein Ge in E. coli. A dissociation constant (K d ) value of 9.4 nM for protein G<e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins. [Figure not available: see fulltext.]

Original languageEnglish
Pages (from-to)482-492
Number of pages11
JournalJournal of the American Society for Mass Spectrometry
Volume26
Issue number3
DOIs
Publication statusPublished - Mar 2015
Externally publishedYes

Keywords

  • Amino acid sequencing
  • Bottom-up MS
  • ESI-MS
  • FTICR MS
  • K value
  • MALDI ToF MS
  • PTM analysis
  • Thermophoresis
  • Top-down MS

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