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FOR THE RECORD

Rapid mass spectrometric analysis of ¹⁵N-Leu incorporation fidelity during preparation of specifically labeled NMR samples

¹ Department of Chemistry and Biochemistry, University of California San Diego, La Jolla 92093-0378, California, USA

(RECEIVED May 12, 2008; FINAL REVISION June 16, 2008; ACCEPTED June 17, 2008)

Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific ¹⁵N-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 ¹⁵N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The ¹⁵N 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 ¹⁵N-amino acid prevents the scrambling of the ¹⁵N label that is observed when equimolar amounts are used. MALDI TOF-TOF MS/MS data provide additional information that shows where the "extra" ¹⁵N 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.

Keywords: MALDI mass spectrometry; tandem mass spectrometry; specific ¹⁵N-amino acid labeling; proteolytic digestion; NMR spectroscopy

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