Probing site-specific ¹³C/¹⁵N-isotope enrichment of spider silk with liquid-state NMR spectroscopy Xiangyan Shi1

Solid-state nuclear magnetic resonance (NMR) has been extensively used to elucidate spider silk protein structure and dynamics. In many of these studies, site-specific isotope enrichment is critical for designing particular NMR methods for silk structure determination. The commonly used isotope analysis techniques, isotope-ratio mass spectroscopy and liquid/gas chromatography-mass spectroscopy, are typically not capable of providing the site-specific isotope information for many systems because an appropriate sample derivatization method is not available. In contrast, NMR does not require any sample derivatization or separation prior to analysis. In this article, conventional liquid-state 1H NMR was implemented to evaluate incorporation of ¹³C/ ¹⁵N-labeled amino acids in hydrolyzed spider dragline silk. To determine site-specific ¹³C and ¹⁵N isotope enrichments, an analysis method was developed to fit the 1H-¹³C and 1H- ¹⁵N J-splitting (JCH and JNH) 1H NMR peak patterns of hydrolyzed silk fiber. This is demonstrated for Nephila clavipes spiders, where [U- ¹³C3,¹⁵N]-Ala and [1-¹³C,¹⁵N]-Gly were dissolved in their water supplies. Overall, contents for Ala and Gly isotopomers are extracted for these silk samples. The current methodology can be applied to many fields where site-specific tracking of isotopes is of interest.