Measuring <sup>15</sup>N and <sup>13</sup>C Enrichment\nLevels in Sparsely Labeled Proteins Using High-Resolution and Tandem\nMass Spectrometry

Isotope labeling of both <sup>15</sup>N and <sup>13</sup>C in selected\namino acids in a protein, known as sparse labeling, is an alternative\nto uniform labeling and is particularly useful for proteins that must\nbe expressed using mammalian cells, including glycoproteins. High\nlevels of enrichment in the selected amino acids enable multidimensional\nheteronuclear NMR measurements of glycoprotein three-dimensional structure.\nMass spectrometry provides a means to quantify the degree of enrichment.\nMass spectrometric measurements of tryptic peptides of a selectively\nlabeled glycoprotein expressed in HEK293 cells revealed complicated\nisotope patterns which consisted of many overlapping isotope patterns\nfrom intermediately labeled peptides, which complicates the determination\nof the label incorporation. Two challenges are uncovered by these\nmeasurements. Metabolic scrambling of amino groups can reduce the <sup>15</sup>N content of enriched amino acids or increase the <sup>15</sup>N in nontarget amino acids. Also, undefined, unlabeled medium components\nmay dilute the enrichment level of labeled amino acids. The impact\nof this unexpected metabolic scrambling was overcome by simulating\nisotope patterns for all isotope-labeled peptide states and generating\nlinear combinations to fit to the data. This method has been used\nto determine the percent incorporation of <sup>15</sup>N and <sup>13</sup>C labels and has identified several metabolic scrambling\neffects that were previously undetected in NMR experiments. Ultrahigh\nmass resolution is also utilized to obtain isotopic fine structure,\nfrom which enrichment levels of <sup>15</sup>N and <sup>13</sup>C\ncan be assigned unequivocally. Finally, tandem mass spectrometry can\nbe used to confirm the location of heavy isotope labels in the peptides.