Measuring 15N and 13C Enrichment Levels in Sparsely Labeled Proteins Using High-Resolution and Tandem Mass Spectrometry. J Am Soc Mass Spectrom 2024 Dec 04;35(12):2877-2889
Date
11/13/2024Pubmed ID
39530698Pubmed Central ID
PMC11622383DOI
10.1021/jasms.4c00237Scopus ID
2-s2.0-85211025791 (requires institutional sign-in at Scopus site)Abstract
Isotope labeling of both 15N and 13C 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 15N content of enriched amino acids or increase the 15N 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 15N and 13C 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 15N and 13C can be assigned unequivocally. Finally, tandem mass spectrometry can be used to confirm the location of heavy isotope labels in the peptides.
Author List
Roberts ET, Choi J, Risher J, Kremer PG, Barb AW, Amster IJAuthor
Paul G. Kremer Postdoctoral Researcher 1 in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceAmino Acids
Carbon Isotopes
Glycoproteins
HEK293 Cells
Humans
Isotope Labeling
Nitrogen Isotopes
Proteins
Tandem Mass Spectrometry
