Identification and quantification of Nα-acetylated Y. pestis fusion protein F1-V expressed in Escherichia coli using LCMSE

N-terminal acetylation in E coli is a rare event catalyzed by three known N-acetyl-transferases (NATs), each having a specific ribosomal protein substrate. Multiple, gram-scale lots of recombinant F1-V, a fusion protein constructed from Y. Pestis antigens, were expressed and purified from a single stably transformed E. coli cell bank. A variant form of F1-V with mass increased by 42–43 Da was detected in all purified lots by electrospray orthogonal acceleration time-of-flight mass spectrometry (MS). Peptide mapping LCMS localized the increased mass to an N-terminal Lys-C peptide, residues 1–24, and defined it as +42.0308 ± 0.0231 Da using a LockSpray™ exact mass feature and a leucine enkaphalin mass standard. Sequencing of the variant 1–24 peptide by LCMS and high-energy collision induced dissociation (LCMSE) further localized the modification to the amino terminal tri-peptide ADL and identified the modification as Nα-acetylation. The average content of Nα-acetylated F1-V in five lots was 24.7 ± 2.6% indicating that a stable acetylation activity for F1-V was established in the E. coli expression system. Alignment of the F1-V N-terminal sequence with those of other known Nα-acetylated ectopic proteins expressed in E. coli reveals a substrate motif analogous to the eukaryote NatA′ acetylation pathway and distinct from endogenous E. coli NAT substrates.