Deamidation: Differentiation of aspartyl from isoaspartyl products in peptides by electron capture dissociation.
Journal: 2005/July - Protein Science
ISSN: 0961-8368
Abstract:
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(beta) 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(l-n)-57 fragment ions (n, position of Asp; l, 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(alpha)-C(beta) 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.
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Protein Sci 14(2): 452-463

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

Department of Chemistry, Boston University, Boston, Massachusetts 02215, USA
Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA
University of Michigan, VA Medical Center, Ann Arbor, Michigan 48105
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: ude.ub@ronnocop; fax: 617-638-6761.
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: ude.ub@ronnocop; fax: 617-638-6761.
Received 2004 Aug 20; Revised 2004 Oct 27; Accepted 2004 Oct 27.

Abstract

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 Cβ 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 cn•+58 and zℓ−n-57 fragment ions (n, position of Asp; ℓ, 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 Cα—Cβ 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
Abstract

An important in vivo modification of proteins is the deamidation of asparaginyl and isomerization of aspartyl residues, through a common cyclic intermediate, to aspartyl or isoaspartyl residues (Clarke 1987; Radkiewicz et al. 1996; Robinson and Robinson 2001a; Robinson et al. 2001; Fig. 1), wherein the formation of the isoaspartyl residue is believed to be partly responsible for the inactivation, aggregation, and aging of proteins in tissue because the backbone is lengthened by one methylene unit (—CH2—) (Roher et al. 1993; Kim et al. 1997; Aswad et al. 2000; Shimizu et al. 2000; Robinson and Robinson 2001a; Robinson et al. 2001; Ritz-Timme and Collins 2002; Reissner and Aswad 2003). The conversion from asparaginyl to aspartyl or isoaspartyl residues (monoisotopic masses 114.0429 and 115.0269 Da, respectively) results in a change of mass of 0.9840 Da, a difference which, aside from isotopic interferences, can easily be distinguished in peptides and proteins by most present-day mass spectrometers, but the conversion from aspartyl to isoaspartyl residues occurs with no detectable mass change.

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Isomerization of aspartic acid and deamidation of asparagine to isoaspartic acid via a succinimide intermediate.

The common, and largely accepted, model for this non-enzymatic, post-translational modification is that it spontaneously occurs under physiological conditions through a succinimide intermediate whose rate is affected by both its amino acid sequence and three-dimensional structure. Asparaginyl residues undergo ammonia loss (deamidation) to form the succinimide intermediate while aspartyl residues lose water (dehydration) to form the same intermediate at a rate ~40 times slower than deamidation (Clarke 1987). Amino acids on the carboxyl side adjacent to either the asparaginyl or aspartyl residue influence the rate of succinimide formation more so than those on the amino side (Clarke 1987; Robinson and Robinson 2001a; Robinson et al. 2001). It has been shown that this rate increases for amino acids with less bulky and highly polar side chains, such as glycine and histidine, respectively (Robinson and Robinson 2001a). The less bulky side chain of an adjacent residue lowers the steric hindrance facilitating the nucleophilic attack of the peptide nitrogen on the γ-carbonyl of either the asparagine or aspartic acid side chain while a more polar side chain helps to stabilize the succinimide intermediate. The deamidation rate increases 10-fold when replacing leucine with histidine in position X of the model pentapeptide GGNXG, and 100-fold when replaced with glycine (Robinson and Robinson 2001a). The three-dimensional structure affects both succinimide formation as well as the final products. A dihedral Ψ angle of −120° and χ angle of 120° offers the most favorable position (distance of 1.89 Å) for nucleophilic attack of the peptide nitrogen on the γ-carbonyl to form the succinimide intermediate (Clarke 1987). However, such configurations are uncommon in proteins, suggesting that protein tertiary structure mitigates this modification by placing these residues where succinimide formation is hindered. For example, deamidation of an asparaginyl residue within the α-helix of rabbit muscle aldolase experienced a 15-fold slower half-life than that of its linear tetrapeptide model (Robinson and Robinson 2001a). The addition of water opening the succinimide ring to form either an aspartyl or isoaspartyl residue (pathways 1 and 2 of Fig. 1, respectively) depends on which of the amide bonds of the intermediate is hydrolyzed. This choice is partially governed by the three-dimensional structure of the peptide or protein (Clarke 1987; Kossiakoff 1988; Robinson and Robinson 2001a, b; Robinson et al. 2001; Athiner et al. 2002), which effects the accessibility of each of the bonds to attack by water. Typically, >60% of the products resulting from succinimide hydrolysis are isoaspartyl residues (Athiner et al. 2002; Reissner and Aswad 2003).

Upon hydrolysis to the isoaspartyl form, the aspartyl or asparaginyl position in the protein backbone is lengthened by one methylene unit, resulting in a modification that has been correlated with protein inactivation and misfolding and whose physiological importance has been illustrated in experiments involving the repair enzyme L-isoaspartyl-O-methyltransferase (PIMT) (Roher et al. 1993; Kim et al. 1997; Aswad et al. 2000; Shimizu et al. 2000; Ritz-Timme and Collins 2002; Reissner and Aswad 2003). PIMT is a highly conserved enzyme that uses S-adenosyl-L-methionine (AdoMet) as a methyl donor to convert isoaspartyl to aspartyl residues, partially restoring the function of enzymes affected by deamidation or isomerization (Johnson et al. 1987). The importance of the PIMT enzyme and the negative effects of the isoaspartyl modification have been shown in several experiments such as seizures and early death experienced by PIMT knockout mice (Kim et al. 1997) and the extension of life by 30% for Drosphilia with overexpression of PIMT (Chavous et al. 2001). This experimental evidence has led researchers to suspect aspartate isomerization as a possible contributor to Alzheimer’s disease since the isoaspartyl modification alters the fundamental structure of the protein backbone and that the highest level of PIMT activity was located in the brain. The cerebral plaque samples of Alzheimer patients have shown evidence of aspartyls isomerized to isoaspartyls at residue positions 1, 7, and 23 of the β-amyloid peptide, where the isoaspartyl content was the highest at position D7 (75%) (Roher et al. 1993). Other affected proteins include those long-lived proteins important for the structure of tooth, skin, lens of the eye, and bone in humans (Ritz-Timme and Collins 2002). Some proteins such as HMAP (high mass methyl-accepting protein), found in the mammalian brain, contain a high percentage of isoaspartyl residues which is suspected to modulate its activity, and may present an example of a beneficial modification (Reissner and Aswad 2003). Whatever role is ultimately attributed to this modification, developing an analytical technique to reliably and easily differentiate aspartyl from isoaspartyl residues is critical to the biological assessment of how certain proteins aggregate, age, and regulate their own activity.

Since the initial publication in 1998 (Zubarev et al. 1998), electron capture dissociation (ECD) has helped to drive mass spectrometry (MS) to the forefront of proteomics and other areas related to the structural analysis of important biological molecules. ECD is used in conjunction with Fourier transform mass spectrometry (FT-ICR-MS or FTMS) (Marshall and Verdun 1990) where multiply charged positive ions ([M+nH], n is number of H) are trapped in the ion cyclotron resonance (ICR) cell and irradiated with low energy electrons to form odd (OE) and even (EE) electron fragment ions (Fig. 2). More recently, a related technique, electron transfer dissociation (ETD), has demonstrated similar fragmentation on the far more ubiquitous ion trap instruments (Syka et al. 2004). Analysis by ECD requires a charge state of at least 2+ for the precursor ion so that a positive charge remains for detection of fragment ions. ECD is unique because it uses free radical chemistry to cleave the peptide backbone (Leymarie et al. 2003) creating c and z• ions as opposed to b and y ions (Fig. 3) that are typical of fragmentation techniques such as CAD (collisionally activated dissociation) (Gauthier et al. 1991; Senko et al. 1994). For ECD, capture of a 0.2 eV electron by the precursor ion produce fragmentation lending to c and z ions while capture of 9.0 eV electrons (hot ECD) produces additional ions such as b, y, a, v, w, and d fragment ions due to ion-electron inelastic collisions (Fig. 3; Kjeldsen et al. 2002). The fragmentation mechanism of ECD (Fig. 2) involves capture of an electron by the positively charged species, thus neutralizing one charge site and producing enough energy to initiate the homolytic cleavage of the N—Cα bond (either c• and z or c and z• fragments) of a peptide in the vicinity of capture. This type of cleavage is useful for peptide and protein sequence analysis (Tsybin et al. 2004) because fragmentation occurs almost without regard to amino acid composition (with the exception of proline) (Leymarie et al. 2003), causing a more uniform cleavage pattern. This type of fragmentation is contrary to standard MS/MS methods such as CAD (Kruger et al. 1999) where collisions with a neutral gas excite the many vibrational modes of the peptide or protein producing spectra dominated by fragments resulting from the cleavages of the most labile bonds (such as the peptide bonds adjacent to proline and aspartic acid) (Gu et al. 2000), making complete sequencing difficult. In addition to sequencing, ECD has been shown to be useful for the analysis of post-translational modifications of peptides and proteins (Kelleher et al. 1999b; Mirgorodskaya et al. 1999; Stensballe et al. 2000; Hakansson et al. 2001; Shi et al. 2001), distinguishing isomeric (Kjeldsen et al. 2003) and enantiomeric structures (Adams et al. 2004) and revealing gas phase protein conformation (Horn et al. 2001; Breuker et al. 2002; Oh et al. 2002). For example, ECD can preserve labile protein modifications such as phosphorylation, which may account for ~30% of the ~300 known modifications (Qian et al. 2003). Also, side-chain cleavage by ECD has been used to help define the amino acid residues (Cooper et al. 2002; Haselmann et al. 2002; Leymarie et al. 2003) in peptides and proteins including differentiating between isoleucine and leucine (Kjeldsen et al. 2003). With all these attributes, ECD is a useful technique not only for routine sequencing but also for studying biological molecules with structural ambiguities that have previously been difficult to discern by typical mass spectrometric techniques.

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ECD fragmentation mechanism. Upon electron capture at the positively charged site, OH bond formation with a carbonyl oxygen initiates the cleavage of the Cá—N bond producing (A) c and z• ions or (B) c• and z ions.

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Structure of the observed b, y, a•, c, and z• ions and d, w, and v side-chain fragment ions. R2′ and R3′ represent partial side-chain loss (Roepstorff et al. 1984; Biemann et al. 1990).

Aspartyl or isoaspartyl residues, resulting from the deamidation of asparaginyl and isomerization of aspartyl residues, are difficult to differentiate by mass spectrometric means because their masses are essentially identical (Schindler et al. 1996; Gonzalez et al. 2000; Lehmann et al. 2000; Luu et al. 2004). Presently, the most efficient way to detect the modification is by immunological methods (Reissner and Aswad 2003), PIMT assays with labeled AdoMet (Roher et al. 1993; Aswad et al. 2000) and Edman degradation (Roher et al. 1993). Other analytical techniques such as HPLC and NMR both have their drawbacks. For example, HPLC can separate the two isoforms (Roher et al. 1993) but they remain indistinguishable from one another, and NMR requires more sample than can realistically be expected from most biological experiments. Several MS methods have been developed to indirectly detect the presence of isoaspartyl residues in peptides based on the relative abundance of fragment ions (Lehmann et al. 2000), modifications to isoaspartyl terminal fragments (Schindler et al. 1996; Gonzalez et al. 2000), and the specific sequence of neutral losses experienced by isoaspartyl residues (Luu et al. 2004). Although useful for their particular studies, these mass spectrometric methods require control samples in order to clearly differentiate between aspartyl and isoaspartyl residues in peptides, and therefore may yield ambiguous results when applied to real biological samples for which there is no control sample. Data presented in this paper demonstrates that direct differentiation of aspartyl from isoaspartyl residues in peptides can be accomplished by ECD. Under ECD conditions, peptides with aspartyl residues underwent neutral loss of the aspartic acid side chains that was not observed in the isoaspartyl analogues. Also, the data yielded the observation of a unique fragmentation pattern that accounts for peaks observed only in the ECD spectra of peptides with isoaspartyl residues that do not appear in the spectra of the peptides with aspartyl residues. These fragments can be used to unambiguously determine the presence of isoaspartyl residues in biological samples without the need for control samples.

Acknowledgments

We thank Dr. Bogdan Budnik and Raman Mathur for helpful discussions and Dr. Henriette Remmer for synthesizing some of the model peptides. This work was supported in part by Federal funds from the National Center for Research Resources under grant P41-RR10888 (C.E.C.), the National Heart, Lung, and Blood Institute under contract HHSN268200248178C (C.E.C.), a grant from the ACS Petroleum Research Fund (P.B.O.), a Research Collaborative Agreement with MDS Sciex (P.B.O.), and NIH Grant GM35533 (to L.W.).

Acknowledgments

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 Dα, aspartic acid

  • isoAsp or Dβ, isoaspartic acid

  • G2D, α,α-deuterated glycine

  • Da, Daltons

  • CAD, collisionally activated dissociation

  • SORI, sustained off-resonance irradiation

  • Ψ, dihedral psi angle

  • χ, 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

Abbreviations

Notes

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

Notes
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