Rapid mass spectrometric analysis of 15N-Leu incorporation fidelity during preparation of specifically labeled NMR samples.
Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific (15)N-amino acid incorporation is a powerful tool for reducing spectral overlap and attaining reliable sequential assignments. However, scrambling of the label during protein expression is a common problem. We describe a rapid method to evaluate the fidelity of specific (15)N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The (15)N incorporation is determined by analyzing the isotopic abundance of the peptides in the mass spectra using the program DEX. This analysis determined that expression with a 10-fold excess of unlabeled amino acids relative to the (15)N-amino acid prevents the scrambling of the (15)N label that is observed when equimolar amounts are used. MALDI TOF-TOF MS/MS data provide additional information that shows where the "extra" (15)N labels are incorporated, which can be useful in confirming ambiguous assignments. The described procedure provides a rapid technique to monitor the fidelity of selective labeling that does not require a lot of protein. These advantages make it an ideal way of determining optimal expression conditions for selectively labeled NMR samples.
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla 92093-0378, California, USA.
Stephanie M E
SM
Truhlar
Carla F
CF
Cervantes
Justin W
JW
Torpey
Magnus
M
Kjaergaard
Elizabeth A
EA
Komives
D000595:Amino Acid Sequence; D017089:Ankyrin Repeat / Q000235:genetics; D004577:Electron Probe Microanalysis; D006868:Hydrolysis; D051550:I-kappa B Kinase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007553:Isotope Labeling; D007930:Leucine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000032:analysis*; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D013997:Time Factors
D009587:Nitrogen Isotopes; D010455:Peptides; D051550:I-kappa B Kinase; D007930:Leucine