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Deamidation: Differentiation of aspartyl from isoaspartyl products in peptides by electron capture dissociation

¹ Department of Chemistry, Boston University, Boston, Massachusetts 02215, USA
² Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA
³ University of Michigan, VA Medical Center, Ann Arbor, Michigan 48105

(RECEIVED August 20, 2004; FINAL REVISION October 27, 2004; ACCEPTED October 27, 2004)

Deamidation of asparaginyl and isomerization of aspartyl residues in proteins proceed through a succinimide intermediate producing a mixture of aspartyl and isoaspartyl residues. Isoaspartic acid is an isomer of aspartic acid with the C incorporated into the backbone, thus increasing the length of the protein backbone by one methylene unit. This post-translation modification is suspected to contribute to the aging of proteins and to protein folding disorders such as Alzheimer’s disease, so that differentiating the two isomers becomes important. This manuscript reports that distinguishing aspartyl from isoaspartyl residues in peptides has been accomplished by electron capture dissociation (ECD) using a Fourier transform mass spectrometer (FTMS). Model peptides with aspartyl residues and their isoaspartyl analogs were examined and unique peaks corresponding to c_n•+58 and z_–n-57 fragment ions (n, position of Asp; , total number of amino acids in the peptide) were found only in the spectra of the peptides with isoaspartyl residues. The proposed fragmentation mechanism involves cleavage of the C—C backbone bond, therefore splitting the isoaspartyl residue between the two fragments. Also, a complementary feature observed specific to aspartyl residues was the neutral loss of the aspartic acid side chain from the charge reduced species. CAD spectra of the peptides from the same instrument demonstrated the improved method because previously published CAD methods rely on the comparison to the spectra of standards with aspartyl residues. The potential use of the top-down approach to detect and resolve products from the deamidation of asparaginyl and isomerization of aspartyl residues is discussed.

Keywords: Deamidation; protein aging; isoaspartic acid; mass spectrometry; electron capture dissociation

Abbreviations: ECD, electron capture dissociation • ETD, electron transfer dissociation • ICR, ion cyclotron resonance • MS, mass spectrometry • FT-ICR-MS or FTMS, Fourier transform mass spectrometry • Asp or D, aspartic acid • isoAsp or D, isoaspartic acid • G_2D, ,-deuterated glycine • Da, Daltons • CAD, collisionally activated dissociation • SORI, sustained off-resonance irradiation • , dihedral psi angle • , chi angle; PIMT, L-isoaspartyl-O-methyltransferase • AdoMet, S-adenosyl-L-methionine • OE, odd electron • EE, even electron • HMAP, high mass methyl-accepting protein • ESI, electrospray ionization • qQq, triple quadrupole • MS/MS, tandem mass spectrometry • HPLC, high-performance liquid chromatography • NMR, nuclear magnetic resonance spectrometry • Q₁ or Q₂, quadrupoles

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041062905.

Reprint requests to: Peter B. O’Connor, Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine, 715 Albany St., R806, Boston, MA 02118, USA; e-mail: poconnor{at}bu.edu; fax: 617-638-6761.

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