Expression, purification and identification of isotope-labeled recombinant cystatin C protein in Escheichia coli intended for absolute quantification using isotope dilution mass spectrometry

Abstract

Isotope-labeled proteins are expected to be used as internal standard proteins in the field of protein quantification by isotope dilution mass spectrometry (ID/MS). To achieve the absolute quantification of Cystatin C (Cys C) based on ID/MS, we aims to obtain ¹⁵N isotope-labeled recombinant Cys C (¹⁵N-Cys C) protein. Firstly, the Cys C gene was optimized based on the preferred codons of Escherichia coli, and inserted into the pET-28a(+) expression plasmid. Then, the plasmid was transformed into TOP10 and BL21 strains, and ¹⁵N-Cys C was expressed in M9 medium using ¹⁵N as the only nitrogen source. ¹⁵N-Cys C was detected by SDS-PAGE, protein immunoblotting and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The characteristic peptides obtained from ¹⁵N-Cys C were analyzed by a Q Exactive Plus MS system. Results showed that 53.06% of the codons were optimized. The codon adaptation index of the Cys C genes increased from 0.31 to 0.95, and the GC content was adjusted from 64.85% to 54.88%. The purity of ¹⁵N-Cys C was higher than 95%. MALDI-TOF MS analysis showed that the m/z of ¹⁵N-Cys C had changed from 13 449 to 14 850. The characteristic peptides showed that 619.79 m/z (M+2H)²⁺ was the parent ion of ¹⁵N-Cys C and that the secondary ions of ¹⁵N-labeled peptides from y⁺⁵ to y⁺⁹ were 616.27 m/z, 716.33 m/z, 788.39 m/z, 936.43 m/z, and 1052.46 m/z, respectively. In conclusion, we successfully expressed, purified and identified of ¹⁵N-Cys C protein in Escheichia coli intended for absolute quantification using ID/MS.

Keywords: Absolute quantitative protein; Escherichia coli; Human cystatin C; Isotope labeling.