N-terminal modification of eGFP by LC-MSMS

ABSTRACT: We used an in-depth analysis of the characteristics of eGFP as a proof-of-principle analyte to establish a robust assay for the determination of degradation rates on the single-cell level using microinjection and live-cell microscopy. We aimed to determine the influence of the exposed n-terminal aminoacid on the proteins degradation. The high sensitivity of our single-cell analysis in combination with the controlled starting conditions of microinjection provides an unique advantage to examine differences in rates with an improved resolution and to compare the influence of different n-terminal aminoacids on eGFP-degradation without accounting for differences during translation. We produced XS-eGFP, with X being substituted by different proteinogenic amino acids, from E.coli and extensively purified it by a three column process. We could show pronounced differences in degradation rates for the constructs with differing N-terminal aminoacids. We also chemically acetylated the N-terminal α amino groups of methionine, alanine, threonine, valine and serine that would normally occur co translationally, in vitro before, microinjection to analyze the influence of acetylation on target degradation. Acetylation at the N-terminus was confirmed by LC-MS-MS. Acetylation reduced degradation of the respective construct. Finally we purified eGFP by anion-exchange from eGFP expressing HEK293 cells to elucidate modifications of eGFP after eukaryotic expression. We could show that eGFP is likely cleaved during HEK293 expression and n-terminally acetylated, which leads to a starkly reduced degradation rate. The degradation rate was similarly reduced for a N-terminally shortened eGFP produced in E.coli and acetylated in-vitro.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human) Escherichia Coli

TISSUE(S): Cell Culture

SUBMITTER: Dorothea Winkelvoss  

LAB HEAD: Andreas Plückthun

PROVIDER: PXD042569 | Pride | 2025-05-06

REPOSITORIES: Pride