This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Under physiological conditions, asparagine (Asn) residues can deamidate spontaneously, generating a mixture of aspartic (Asp) and isoaspartic acid (isoAsp) residues via a succinimide intermediate. In addition, isoAsp residue may also be formed through Asp isomerization, although this occurs at a much slower rate. Differentiation of Asp and isoAsp residues is important as the latter often causes more significant changes in protein conformation and functions, which has been linked to many protein misfolding diseases and other pathological processes. An electron capture dissociation (ECD)-MS/MS based method was established in this laboratory, where diagnostic c+57/z+-57 ions from isoAsp residues were used for the differentiation and relative quantification of the two isomeric forms. This method has been applied to study the Asn deamidation and Asp isomerization in several model systems as summarized below. Asn deamidation and Asp isomerization in A beta Amyloid beta (A beta) is a very hydrophobic 40-42 amino acid peptide that is the main constituent of amyloid plaques in the brain of Alzheimer's patients. There have been several reports on the isomerization of Asp residues in A beta in amyloid plaques at Asp(1), Asp(7), Asp(23) positions. IsoAsp formations have been associated with increases in beta-sheet structures that are found more abundantly in the filaments and plaques. The applicability of the ECD based method to the study of isoAsp formation in A beta was investigated (Sargaeva et al., 2009). ECD of the synthetic A beta peptide fragment 17-28 with isoAsp at position 23 showed the characteristic z6+-57 peak that defines the isoAsp location. Since artificial deamidation may also produce isoAsp during enzymatic digestion processes, particularly in a basic environment when using trypsin, it is sometimes desirable to analyze the whole protein with the top-down approach. A beta 1-40 with Asn substitution at position 7 was allowed to deamidate which generated a mixture of Asp and isoAsp residue. ECD of the deamidated A beta 1-40 produced 75% of the predicted fragments, including the z+-57/c+57 diagnostic peaks for the isoAsp form, clearly demonstrating the potential of extending this method to top-down analysis of intact A beta peptides. The next step is to apply this method to study isoAsp formation in biological samples from Alzheimer's patients. Electron ionization dissociation (EID) of the isomerized A beta 17-28 fragment was also performed, which showed abundant fragmentation and intense fragment ion peaks. However, in these higher energy studies, the diagnostic peaks were of low intensity, limiting the ability to detect the isomerized aspartate predicted in the Alzheimer's plaques, even though EID cleaved >97% of the inter-residue bonds. Nevertheless, this experiment demonstrated that EID may be used in isoaspartomics research alternative to ECD. This could be advantageous for analysis of singly charged ions where ECD is not applicable. Asn deamidation and Asp isomerization in Calmodulin The 148 amino acid Calmodulin (CaM) is a calcium-binding protein with multiple Asn-Gly and Asp-Gly sequences in its four EF-motif hands, which are labile deamidation and isomerization sites in absence of Ca2+. The ESI mass spectrum of the aged CaM (pH 8.0, 14 days, no Ca2+) showed a 2 Da mass shift, indicating the presence of deamidation at two Asn residues (Yao et al., 2009). Top-down analysis of the aged CaM with activated ion (AI)-ECD resulted in cleavages of 66 out of a total of 147 inter-residue bonds. The observation of the 1 Da mass shift in fragment ion z524+, the 2 Da mass shift in y969+, and no mass shifts in any N-terminal fragments smaller than c59 unambiguously identified the deamidation sites at Asn 60 and Asn 97. However, no diagnostic ions for isoAsp were observed, possibly due to the low ion abundance as the result of fragment ions being spread out over many dissociation channels. As an alternative, a bottom-up approach was employed to monitor the isoAsp formation in CaM, where the aged CaM was first digested by trypsin, followed by ECD analysis of the relevant peptides. One Da mass shift was observed for peptide (38-74) and peptide (91-106), identifying the Asn 60 and Asn 97 as the deamidation sites, in agreement with the top-down results. Isomerization of Asp has not been previously detected using the ECD method, presumably because of its much slower reaction rate compared with that of the Asn deamidation. In the current study, diagnostic c+57/z+-57 ions were observed in several tryptic peptide ECD spectra, indicating the formation of isoAsp not only at residues Asn 60 and Asn 97 as the result of deamidation, but also at Asp 22, Asp 24 and Asp 95 due to Asp isomerization. The presence of Ca2+ during incubation appeared to inhibit the deamidation process. Aging of CaM at pH 8 for 2 weeks with Ca2+ produced far less deamidations. This is evident in the ESI spectra of the resulted tryptic peptide (91-106), with its isotopic pattern indicating that less than 20% of Asn 97 were converted to acidic products. Amino acid residues 91-106 are in domain III of CaM, with its -DGNG- sequence serving as the Ca2+ binding motif. Asn deamidation in HGH Not only does deamidation play an important role in many diseases, it also affects the potency and shelf-life of therapeutic drugs. The 191 amino acid protein human growth hormone (HGH) is the first biotechnology drug that is used for stimulation of growth and cell reproduction in humans and other animals. HGH is known to deamidate in the pharmaceutical formulations that are sold. We have recently initiated a collaboration with a local start-up pharmaceutical company to study modifications of HGH, including deamidation (Cui et al., 2009). The disulfide bonds in HGH were reduced and alkylated before the ESI-MS and MS/MS analysis, which resulted in a shift towards higher charge states. Collision-activated dissociation (CAD) at 25 eV of the mass selected 18+ charge state of the native HGH yielded 14 y-type and 7 b-type as well as numerous internal fragment ions;while the 21+ charge state generated 39 y-type and 8 b-type fragments. Increasing the collision energy from 25 eV to 35 and 45 eV did not produce more sequence specific ions for the 21+ charge state, instead, more internal fragment ions were observed. Top-down analysis of the aged HGH using CAD and ECD is ongoing. Bottom-up study of the aged HGH has also been carried out. After the reductive alkylation, HGH was allowed to incubate in 0.1 M ammonium bicarbonate buffer solution (pH ~8) at 37oC for 5 days. The aged HGH was then digested by trypsin for one hour, and the resulted peptide mixture was purified by self-packed POROS column and analyzed by ESI-MS and ECD. One peptide (147-FDTNSHNDDALLK-159) was observed with a mass shift of +1 Da, indicative of deamidation occurring at one or both of the two Asn residues. In the ECD spectrum of this peptide, z7 ion displayed a significant shift in its isotopic pattern, indicating that Asn 153 is partially deamidated. Because the intra-complex hydrogen transfer may also lead to a +1 Da mass shift to the z ion isotopic pattern (z+ to z2), it was difficult to quantify the percentage of deamidation at Asn 153 site, or to determine if Asn 150 also deamidated. High resolution tandem MS experiment should help to differentiate these two effects, as the deamidation leads to a +0.984 Da mass shift, while the z2 ion formation leads to a +1.008 Da shift. No diagnostic ions for the isoAsp form were observed, which was a little surprising, but not completely unexpected. Since the deamidation was carried out prior to enzymatic digestion, the tertiary structure of the HGH might have prevented the formation of isoAsp residue when the succinimide intermediate underwent hydrolysis. However, there also existed another possibility that the diagnostic ion formation was suppressed in this acidic residue rich peptide due to abundant intramolecular interactions. It is thus imperative to carry out the aging study after the HGH is digested. Since isoAsp formation would be expected to be favored during the deamidation process of a random coil peptide, ECD of the resulted deamidated peptide should be able to discern the two possibilities. This experiment will be performed next. We are currently extending this work to beta-amino acids and to glutamine deamidation studies. Research progresses made in this area are reported below. Characterization of beta-peptides using ECD Isoaspartic acid is a beta-linked amino acid, whose amino group is bonded to the ? carbon rather than the ? carbon. With the sole exception of ? alanine, other beta amino acids rarely appear in nature. Because of this, ? peptide based antibiotics are being explored as ways of evading antibiotic resistance. ? peptides may exist in two different forms: a ?2 linked peptide has its side chain connected to the ? carbon, and a ?3 linked peptide has its side chain connected to the ? carbon. Since ECD has been implemented successfully to identify isoAsp residues, based on the diagnostic ions generated via C?-C? bond cleavage, it may also be used to differentiate the ?2 and ?3 linked peptides. An effort to extend the ECD method for the characterization of other beta amino acid residues produced mixed results (Sargaeva et al., 2008, 2009). Electrospray ionization (ESI) of the Q06 peptide (V?2A?2L?2V?3A?3L?3) generated predominantly singly charged precursor ions, which are not suitable for ECD analysis. Three methods have been applied to increase the charge state of the Q06 peptide. Addition of p-nitrobenzyl alcohol (p-NBA) to the ESI solution often led to an up-shift of the charge state distribution;although, in the case of the Q06 peptide, the resulted doubly charged precursor ions were still of too low an abundance to be analyzed by ECD. Covalent attachment of cholamine to the carboxylic acid is much more efficient in producing doubly charged ions, but ECD of these ions produced mostly losses of the cholamine tag and b/y cleavages, with just one N-C? and no C?-C? cleavages. Finally, divalent metal ion (Ca2+) adduction also produced abundant doubly charged Q06 ions. However, no C?-C? cleavages were produced in ECD of these metal adducted ions either. Since the addition of a fixed charge group or a metal ion may alter the ECD fragmentation behavior, a substance P analogue with the Gln 5 and Leu 10 replaced by beta-homoGln and beta-homoLeu, respectively, was synthesized and analyzed by ECD. Surprisingly, the doubly protonated beta-substance P generated neither N-C? nor C?-C? cleavage at sites containing beta amino acid residues. The disparity between the ECD fragmentation pattern of the isoAsp peptides and that of other beta-linked peptides demonstrated the importance of side-chain groups in ECD. It suggested that the adjacent carboxyl group might be instrumental in stabilizing the radical formed in ECD of isoAsp containing peptides. Such stabilization effect is, in general, absent in other beta-linked peptides. Differentiation of Glutamic and ?-glutamic acid residues Although Asn deamidation is the most commonly observed PTM in proteins, glutamine (Gln) may also deamidate under physiological conditions to generate a mixture of glutamic acid (Glu) and ?-glutamic acid (?-Glu) acid. Gln deamidates at a much slower rate, about two orders of magnitude slower compared with its Asn counterpart. Gln deamidation is usually observed in proteins with long turn-over time, such as in eye lens crystallins. Crystallins are highly soluble structure proteins and comprise 90% of lens proteins, which undergo little turnover during their life spans, allowing accumulation of many kinds of modifications. Among these, deamidation is one of the most prevalent, which decreases crystallin solubility, alters lens transparency, and is also involved in cataract formation, a leading cause of blindness. Extensive Gln deamidation has been observed in crystallin proteins. In this study (Li et al., 2009), we developed an ECD-based method to differentiate the Glu and ?-Glu residues, based on the knowledge from the isoAsp research. A set of synthetic crystallin peptide fragments containing either Glu or ?-Glu residues were analyzed by ECD. Similar to the c+57/z+-57 diagnostic ions observed in isoAsp containing peptides, characteristic c+72/z+-72 ions were also observed in ?-Glu peptides. Some z+-72 ions were also present in the ECD spectra of Glu containing peptides, although they never appeared at cleavage sites immediately preceding the Glu residues. These ions likely resulted from the charge remote fragmentation initiated by the radical site on the z+ ion, which is a subject currently being investigated. Since the z+-72 ions in ECD of ?-Glu peptides are formed only when the initial N-C? bond cleavage occurred on the N-terminal side of the ?-Glu residue, the z+-72 ions can be used for differentiation of Glu and ?-Glu residues, provided that the sequence of peptide is known. N-terminal diagnostic ions, c+57, c+59 and c+72 ions were also observed in ECD spectra of ?-Glu containing peptides, but not in those of Glu containing peptides. It appears that the ring opening of proline may be involved in the formation of these N-terminal diagnostic ions, although the number of systems investigated here is too small to make a conclusive statement. The exact mechanism to generate these ions is not yet clear, but is expected to involve radical rearrangements similar to that which generates the isoaspartic acid diagnostic ion series.
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