| Autor: |
Roberts ET; Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States., Choi J; Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States., Risher J; Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States., Kremer PG; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, United States., Barb AW; Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States.; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, United States.; Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, United States., Amster IJ; Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States. |
| Abstrakt: |
Isotope labeling of both 15 N and 13 C in selected amino acids in a protein, known as sparse labeling, is an alternative to uniform labeling and is particularly useful for proteins that must be expressed using mammalian cells, including glycoproteins. High levels of enrichment in the selected amino acids enable multidimensional heteronuclear NMR measurements of glycoprotein three-dimensional structure. Mass spectrometry provides a means to quantify the degree of enrichment. Mass spectrometric measurements of tryptic peptides of a selectively labeled glycoprotein expressed in HEK293 cells revealed complicated isotope patterns which consisted of many overlapping isotope patterns from intermediately labeled peptides, which complicates the determination of the label incorporation. Two challenges are uncovered by these measurements. Metabolic scrambling of amino groups can reduce the 15 N content of enriched amino acids or increase the 15 N in nontarget amino acids. Also, undefined, unlabeled medium components may dilute the enrichment level of labeled amino acids. The impact of this unexpected metabolic scrambling was overcome by simulating isotope patterns for all isotope-labeled peptide states and generating linear combinations to fit to the data. This method has been used to determine the percent incorporation of 15 N and 13 C labels and has identified several metabolic scrambling effects that were previously undetected in NMR experiments. Ultrahigh mass resolution is also utilized to obtain isotopic fine structure, from which enrichment levels of 15 N and 13 C can be assigned unequivocally. Finally, tandem mass spectrometry can be used to confirm the location of heavy isotope labels in the peptides. |
