Analytical Cascades of Enzymes for Sensitive Detection of Structural Variations in Protein Samples.
Julia C HollerwegerIsabel J HoppeChristof ReglLorenz G StockChristian G HuberUrs LohrigHanno StutzKatharina HäussermannPublished in: Analytical chemistry (2018)
Protein function critically depends on structure. However, current analytical tools to monitor consistent higher-order structure with high sensitivity, as for instance required in the development of biopharmaceuticals, are limited. To complement existing assays, we present the analytical cascade of enzymes (ACE), a method based on enzymatic modifications of target proteins, which serve to exponentially amplify structural differences between them. The method enables conformational and chemical fingerprinting of closely related proteins, allowing for the sensitive detection of heterogeneities in protein preparations with high precision. Using this method, we detect protein variants differing in conformation only, as well as structural changes induced by diverse covalent modifications. Additionally, we employ this method to identify the nature of structural variants. Moreover, the ACE method should help to address the limited reproducibility in fundamental research, which partly relates to sample heterogeneities.
Keyphrases
- sensitive detection
- quantum dots
- protein protein
- amino acid
- binding protein
- angiotensin ii
- copy number
- liquid chromatography
- molecular dynamics simulations
- molecular dynamics
- mass spectrometry
- high throughput
- loop mediated isothermal amplification
- hydrogen peroxide
- gene expression
- nitric oxide
- genome wide
- dna methylation