| Autor: |
Roberts ET; Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States., Davis AR; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, United States., Risher JT; Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States., Barb AW; 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: |
Uniform enrichment of 15 N and 13 C in proteins is commonly employed for 2D heteronuclear NMR measurements of the three-dimensional protein structure. Achieving a high degree of enrichment of both elements is important for obtaining high quality data. Uniform labeling of proteins and glycoproteins expressed in higher organisms (yeast or mammalian cell lines) is more challenging than expression in Escherichia coli , a prokaryote that grows on simple, chemically defined media but does not provide appropriate eukaryotic modifications. It is difficult to achieve complete incorporation of both heavy isotopes, and quality control measures are important for quantitating the level of their enrichment. Mass spectrometry measurements of the isotopic distribution of the intact protein or its proteolytic fragments provide the means to assess the enrichment level. A mass accuracy of 1 ppm or better is shown to be required to distinguish the correct combination of 13 C and 15 N enrichment due to subtle shifts in peak centroids with differences in the underlying, but unresolved, isotopic fine structure. A simple computer program was developed to optimize the fitting of experimental isotope patterns to statistically derived distributions. This method can determine the isotopic abundance from isotope patterns and isotopologue masses in conventional MS data for peptides, intact proteins, and glycans. For this purpose, MATLAB's isotope simulator, isotopicdist, has been modified to permit the variation of 15 N and 13 C enrichment levels and to perform a two-dimensional grid search of enrichment levels of both isotopes. We also incorporated an alternate isotope simulator, js-emass, into MATLAB for use in the same fitting program. Herein we benchmark this technique on natural abundance ubiquitin and uniformly [ 15 N, 13 C]-labeled ubiquitin at both the intact and peptide level, outline considerations for data quality and mass accuracy, and report several improvements we have made to the previously reported analysis of the [ 15 N, 13 C]-enriched human IgG Fc domain, a glycoprotein that has been expressed in Saccharomyces cerevisiae . |
