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J. Biochem, 2003, Vol. 134, No. 1 129-135
© 2003 Japanese Biochemical Society

BIOCHEMISTRY

A Method for the Detection of Asparagine Deamidation and Aspartate Isomerization of Proteins by MALDI/TOF–Mass Spectrometry Using Endoproteinase Asp-N

Daisuke Kameoka+,, Tadashi Ueda§, and Taiji Imoto,

Graduate School of Pharmaceutical Sciences, Kyushu University, 3-3-1 Maidashi, Higashi-ku, Fukuoka 812-8582

A method was established for evaluating Asn deamidation and Asp isomerization/racemization. To detect the subtle changes in mass that accompany these chemical modifications, we used a combination of enzyme digestion by endoproteinase Asp-N, which selectively cleaves the N-terminus of L-{alpha}-Asp, and MALDI/TOF-mass spectrometry. To achieve better resolution, we employed digests of 15N-labeled protein as an internal standard. To demonstrate the advantages of this method, we applied it to identify deamidated sites in mutant lysozymes in which the Asn residue is mutated to Asp. We also identified the deamidation or isomerization site of the lysozyme samples after incubating them under acidic or basic conditions.

+ Present address: Chugai Pharmaceutical Co. Ltd., Tokyo 171-8545.

§ To whom correspondence should be addressed. Tel: +81-92-642-6662, Fax: +81-92-642-6667, E-mail: ueda{at}phar.kyushu-u.ac.jp

Present address: Faculty of Engineering, Sojo University, Kumamoto 860-0082.


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