Linchpin-directed precise labeling of lysine in native proteins, purification, and analysis.
Methods Enzymol
; 675: 383-396, 2022.
Article
in En
| MEDLINE
| ID: mdl-36220278
ABSTRACT
The proteins are critical building blocks of living systems and serve as a tool for their investigation and intervention. Their precision engineering enables its tuning and expands the functional landscape. Among various proteinogenic amino acids, high-frequency lysine offers a promising bioconjugation target. However, it is also among the most challenging candidates for homogeneous single-site modification. The linchpin-directed modification (LDM) addresses this concern by offering chemoselective, site-selective, and modular protein bioconjugation. The protocol outlines a general method for single-site modification of a native protein. At first, the selected LDM reagent constructs a stable bioconjugate through acylation of the Lys side chain. Subsequently, its chemically orthogonal handle creates an opportunity to install desired probes directly. Alternatively, the same group enables bioconjugate enrichment through ordered immobilization. The subsequent release, coupled with probe installation, renders analytically pure single-site tagged protein bioconjugate. The analysis of these constructs involves intact MS of protein bioconjugate, peptide mapping, and MS-MS for the site of modification and homogeneity.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Proteins
/
Lysine
Language:
En
Journal:
Methods Enzymol
Year:
2022
Document type:
Article