Applicability of the alkylation chemistry for chemical C-terminal protein sequence analysis.
We have evaluated the alkylation chemistry first described some years ago by Boyd et al. which is now routinely applied in a commercial instrument. We have found that the low repetitive yields observed during these analyses are due to the formation of a major side product when alkylating the C-terminal thiohydantoin. This side product, resistant to the chemical cleavage methods currently used, was characterized by NMR experiments in solution. We further demonstrate that chemical C-terminal sequence analysis of proteins using the alkylation chemistry is feasable with low picomole amounts of material. High-sensitivity C-terminal sequencing allows a complementary approach by which a protein is first subjected to N-terminal Edman degradation followed by C-terminal sequence analysis, limiting the amount of material necessary for the characterization of the protein under study. This limited C-terminal sequence information is often sufficient to solve problems that cannot be solved by applying any other analytical method commonly used today.
Samyn
B
B
University of Gent, Department of Biochemistry, Physiology, and Microbiology, Belgium.
Hardeman
K
K
Van der Eycken
J
J
Van Beeumen
J
J
D000478:Alkylation; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D020539:Sequence Analysis, Protein / Q000379:methods*