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Article

Detecting Deamidation Products in Proteins by Electron Capture Dissociation

Jason J. Cournoyer,^†^‡ Cheng Lin,^† and Peter B. O'Connor*^†

Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine, 715 Albany Street, R806, Boston, Massachusetts 02118, and Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215

Anal. Chem., 2006, 78 (4), pp 1264–1271

DOI: 10.1021/ac051691q

Publication Date (Web): December 24, 2005

†

Boston University School of Medicine.

‡

Boston University.

To whom correspondence should be addressed. Telephone: (617) 638-6705. Fax: (617) 638-6761. E-mail, poconnor@bu.edu.

Abstract

A nonenzymatic posttranslational modification of proteins and peptides is the spontaneous deamidation of asparaginyl residues via a succinimide intermediate to form a varying mixture of aspartyl and isoaspartyl residues. The isoaspartyl residue is generally difficult to detect particularly using mass spectrometry because isoaspartic acid is isomeric with aspartic acid so that there is no mass difference. However, electron capture dissociation has demonstrated the ability to differentiate the two isoforms in synthetic peptides using unique diagnostic ions for each form; the c_r^• + 58 and z_l_-_r − 57 fragment ions for the isoAsp form and the Asp side chain loss ((M + nH)⁽ⁿ^-¹⁾^+• − 60) for the Asp form. Shown here are three examples of isoaspartyl detection in peptides from proteins; a deamidated tryptic peptide of cytochrome c, a tryptic peptide from unfolded and deamidated ribonuclease A, and a tryptic peptide from calmodulin deamidated in its native state. In all cases, the c_r^• + 58 and z_l_-_r − 57 ions allowed the detection and localization of isoaspartyl residues to positions previously occupied by asparaginyl residues. The (M + nH)⁽ⁿ^-¹⁾^+• − 60 ions were also detected, indicating the presence of aspartyl residues. Observation of these diagnostic ions in peptides from proteins shows that the method is applicable to defining the isomerization state of deamidated proteins.