Automated Assignment\nof <sup>15</sup>N And <sup>13</sup>C Enrichment Levels in Doubly-Labeled\nProteins

Uniform enrichment of <sup>15</sup>N and <sup>13</sup>C in proteins\nis commonly employed for 2D heteronuclear NMR measurements of the\nthree-dimensional protein structure. Achieving a high degree of enrichment\nof both elements is important for obtaining high quality data. Uniform\nlabeling of proteins and glycoproteins expressed in higher organisms\n(yeast or mammalian cell lines) is more challenging than expression\nin <i>Escherichia coli</i>, a prokaryote that grows on simple,\nchemically defined media but does not provide appropriate eukaryotic\nmodifications. It is difficult to achieve complete incorporation of\nboth heavy isotopes, and quality control measures are important for\nquantitating the level of their enrichment. Mass spectrometry measurements\nof the isotopic distribution of the intact protein or its proteolytic\nfragments provide the means to assess the enrichment level. A mass\naccuracy of 1 ppm or better is shown to be required to distinguish\nthe correct combination of <sup>13</sup>C and <sup>15</sup>N enrichment\ndue to subtle shifts in peak centroids with differences in the underlying,\nbut unresolved, isotopic fine structure. A simple computer program\nwas developed to optimize the fitting of experimental isotope patterns\nto statistically derived distributions. This method can determine\nthe isotopic abundance from isotope patterns and isotopologue masses\nin conventional MS data for peptides, intact proteins, and glycans.\nFor this purpose, MATLAB’s isotope simulator, isotopicdist,\nhas been modified to permit the variation of <sup>15</sup>N and <sup>13</sup>C enrichment levels and to perform a two-dimensional grid\nsearch of enrichment levels of both isotopes. We also incorporated\nan alternate isotope simulator, js-emass, into MATLAB for use in the\nsame fitting program. Herein we benchmark this technique on natural\nabundance ubiquitin and uniformly [<sup>15</sup>N,<sup>13</sup>C]-labeled\nubiquitin at both the intact and peptide level, outline considerations\nfor data quality and mass accuracy, and report several improvements\nwe have made to the previously reported analysis of the [<sup>15</sup>N,<sup>13</sup>C]-enriched human IgG Fc domain, a glycoprotein that\nhas been expressed in <i>Saccharomyces cerevisiae</i>.