HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article ACCEPTED PREPRINT Full Text (PDF) Supplemental Research Data All Versions of this Article: ps.036418.108v1 17/9/1636    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Truhlar, S. M. E. Articles by Komives, E. A. PubMed PubMed Citation Articles by Truhlar, S. M. E. Articles by Komives, E. A. Social Bookmarking                   What's this?

For the Record

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

¹ University of California, San Diego;
² University of Denmark;
³ University of California San Diego

(RECEIVED May 12, 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: Mass Spectrometry; NMR Spectroscopy

⁴ E-mail: ekomives{at}ucsd.edu

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?