Chapter 16 Analysis of Deamidation in Proteins

CHAPT ER

16 Analysis of Deamidation in Proteins Jason J. Cournoyer and Peter B. O’Connor

Contents

1. What is Deamidation? 2. How Does Deamidation Occur? 3. Biological Significance of Deamidation 3.1 Mass spectrometric methods for studying deamidation 3.2 Deamidation in monoclonal antibodies 3.3 Aging 3.4 Amyloid diseases 3.5 Autoimmune diseases: Lupus and celiac disease 3.6 Cancer 3.7 Cataracts 3.8 Anthrax vaccine 3.9 Other diseases 3.10 The PIMT repair enzyme 3.11 Chemical methods for detection of isoAsp 3.12 Deamidation as a sample-handling artifact 4. Non-MS Based Methods for Studying Deamidation 4.1 Proteolytic digestion 4.2 Isoaspartyl antibody 4.3 Reversed phase HPLC 4.4 Ion exchange chromatography 4.5 Electrophoresis 4.6 Nuclear magnetic resonance (NMR) 4.7 Edman degradation 4.8 The PIMT enzyme 5. Mass Spectrometry Based Methods for Studying Deamidation 5.1 Measuring deamidation by isotopic deconvolution and mass defect methods 5.2 The diagnostic bn1+H2O for isoaspartyl residues in CAD MS spectra 5.3 Aspartyl versus isoAspartyl fragment ion ratios in CAD spectra 5.4 Immonium ions of isoaspartyl residues 5.5 Liquid chromatography/mass spectrometry (LCMS) 5.6 Electron capture dissociation

Comprehensive Analytical Chemistry, Volume 52 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00216-X

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6. Quantitation of Deamidation and Its Products 6.1 HPLC combination methods 6.2 CAD methods without HPLC 6.3 ECD method without HPLC 7. Isotopic Labeling Methods 8. Summary References

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1. WHAT IS DEAMIDATION? Deamidation is the most common of all post-translational modifications (PTMs) in proteins, but it tends to be understudied for several reasons. First, since mass spectrometry (MS) is the most sensitive method for studying peptide PTMs, it is the obvious choice for study of deamidation; however, deamidation results in a +0.984 Da shift, and the ubiquitous ion trap mass spectrometer barely has the mass resolution and frequently does not have the mass accuracy to clearly define such a shift. Second, this +0.984 Da mass shift usually generates a deamidated product with isotopic peaks which overlap with the non-deamidated precursor. Third, the products of deamidation are isomeric, so even if the deamidation is noticed, the ratio of the two products is frequently unclear. Figure 1 shows the isotopic peak distributions of a peptide as it is exposed to high pH for 12 days. Before reaction, the peptide is primarily the native –NL– sequence, but has deamidated B50% within 3 days and is almost entirely deamidated within 12 days. The deamidated product is a mixture of Asp and isoAsp residues. Deamidation, technically, implies loss of an amide group — usually as a neutral ammonium molecule. However, traditionally, ‘‘deamidation’’ refers to replacement of an amide with a hydroxyl group. Thus, the true, net mass change is –NH2 (16.0188 Da)+OH (17.0027 Da) ¼ 0.9839 Da. Detailed discussion of the chemistry of this process is available in the next section, but the essential reaction (under basic conditions) is shown in Figure 2 for Asparagine residues and Figure 3 for Glutamine residues. Deprotonation of the backbone amide promotes nucleophilic attack of the side chain carbonyl, which results in loss of ammonium and formation of the cyclic succinimide intermediate. This intermediate is symmetric around the ?–C(O)–N–C(O)–? , and hydration on either side of this carbonyl will generate the two products. Whether the precursor residue is asparagine or glutamine, the two products are isomeric. Asparagine generates a mixture of aspartic and isoaspartic acid, also called a-aspartic (the natural aspartic acid) and b-aspartic acid. Glutamine generates a mixture of glutamic and g-glutamic acids. Furthermore, aspartic acid and isoaspartic acid can spontaneously isomerize similar to the glutamic acid variants, although this occurs at a much slower rate. In both cases, the two isomers are difficult to differentiate due to their similarity in chemical structures and reactivity. This chapter focuses primarily on

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Figure 1 A tryptic peptide from Cytochrome C undergoing harsh deamidation conditions (pH 11, 371C) for 2 weeks. The mass spectra of the doubly charged peptide ion shows systematic shift from Asn to a mixture of Asp/isoAsp.

mass spectrometric methods that can be used to distinguish them, but Section 4 also discusses non-mass spectrometric methods.

2. HOW DOES DEAMIDATION OCCUR? While this chapter is not intended to be a treatise on deamidation, it is important to note the two primary, canonical reaction mechanisms that are involved. These mechanisms denote the ‘‘standard model’’ for deamidation which correctly implies that these models are not necessarily correct, but are useful for understanding and predicting products. A more detailed discussion is available elsewhere [1]. In particular, reaction rates as a function of the primary sequence of free, random-coil peptides has been extensively studied [2–4].

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Figure 2 The cannonical mechanism of deamidation involves cyclization of asparagines (Asn) to form the succinimide intermediate with loss of ammonium, followed by hydration at either amide bond to from a mixture of aspartic (Asp) and isoaspartic (isoAsp) acids. Deamidation is irreversible under physiological conditions, but the isomerization of the products occurs at a relatively slow rate.

Figure 3 Deamidation also occurs on glutamine, but at a substantially slower rate than on asparagines. The products, similarly, are a mixture of glutamic acid and g-glutamic acid.

Deamidation minimizes around pH 5–6, and there is a prominent basecatalyzed reaction at physiological pH and higher and an acid-catalyzed reaction below pH 4 or so. The base-catalyzed reaction is shown in Figure 4. This reaction is noticeable at pH 7 in the case of rapid deamidating sequences such as ?NG? and very rapid at pH 10 even for slow deamidating sequences such as ?NL? . Thus, it is significant under tryptic digestion conditions, which usually use ammonium bicarbonate buffer at pH B8. Figures 4 and 5 are drawn to involve asparagine, but similar reaction mechanisms can be drawn for glutamine. The base-catalyzed reaction is initiated by deprotonation of the backbone amide, with the resulting negatively charged amide reacting with the side chain carbonyl to cyclize and generate the succinimide intermediate. The limiting factor in the base-catalyzed reaction is deprotonation of the backbone amide, so this reaction is accelerated under basic conditions and is essentially prohibited when the C-terminal amino acid is proline. The succinimide intermediate has an axis of symmetry (Figures 2 and 3; dotted line) if one ignores the rest of the protein.

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Figure 4 Demidation at neutral or basic pHW6, starts by nucleophilic attack of the backbone amide nitrogen on the side chain carbonyl to form the succinimide.

Figure 5 Deamidation at acidic pH o 5, does not involve the cyclic intermediate, but instead involved direct hydrolysis on the side chain. Thus, deamidation under acidic conditions does not generate isomeric product mixture.

Hydration of the amide bond above or below of this axis of symmetry will generate the aspartic/glutamic acid and isoaspartic/g-glutamic acid products, respectively. A chemist’s immediate assumption, therefore, is that these two products should be formed in a 50:50 mixture. In reality, the rest of the protein and steric effects influence this product ratio with the isoaspartic acid form favored 3:1 on average; the glutamic acid variant is less studied, so there is no consensus about the standard product ratio for glutamine deamidation. The succinimide can also racemize the a-carbon position, to form a D-succinimide which, upon hydration, generates the D-amino acid forms. Distinguishing stereoisomers is sometimes possible with MS, but is beyond the scope of this chapter. The acid-catalyzed reaction is less important at physiological pH, but can become important once a sample is acidified with formic, acetic, or trifluoroacetic acid (TFA) for electrospray mass spectrometry. Under acidic conditions, the side chain carbonyl becomes protonated to a hydroxyl, which places a positive charge at the g-carbon/e-nitrogen making it highly susceptible to nucleophilic attack by water. This reaction results in only the aspartyl/glutamyl isoform; isoaspartyl/ g-glutamyl formation is not possible. A further reaction that is important at low pH is acid hydrolysis, which cleaves the backbone. Acid hydrolysis is dominant when the C-terminal amino acid is proline.

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Both the acid- and the base-catalyzed reactions ignore the influence of the protein higher-order structure. In general, proteins protect asparagine and glutamine residues from deamidation by keeping them hydrogen bonded in an alpha helix or beta sheet. However, there are many, well-established cases (see below) where specific Asn or Gln residues are exposed in a loop region or in the active site of a protein, where deamidation occurs and alters the protein function. Frequently, this is related to aging, unfolding, and turnover of the protein, but it also can be related to disease.

3. BIOLOGICAL SIGNIFICANCE OF DEAMIDATION Deamidation of asparagine (and to a lesser extent glutamine) residues is the most common of all PTMs [1,5]. Because the deamidation products, aspartic acid and isoaspartic acid, accumulate in the body over time, it is strongly implicated in many diseases (see below). Currently, about 100 papers/year are published in biological journals regarding Asp/isoAsp formation in proteins, but to date only one systematic study has been published regarding the ratios of the rates of formation of these two products over time [6–8]. This study was a heroic effort which involved high-resolution High-performance liquid chromatography (HPLC) separation of the two aspartic acid isomers as well as synthesis of each individual peptide in both isoforms to verify their peak elution times. While deamidation itself is relatively easy to detect (due to a charge shift and a +0.984 Da mass shift), its products are difficult to analyze, in general, because of the mass spectral interference and similarity in chemical reactivity. The standard model for the deamidation reaction (Figures 2 and 3) involves nucleophilic attack of the backbone amide nitrogen on the side chain carbonyl with loss of ammonia and formation of a cyclic succinimide [1,3,5,9–14]. Deprotonation of the backbone amide nitrogen facilitates this reaction which is why it is accelerated under basic conditions and polar solvent systems [15]. The succinimide is unstable in water and, due to the symmetry around the ring nitrogen, can hydrate to form a mixture of aspartic acid and isoaspartic acid, usually in a B3:1 ratio in favor of isoaspartic acid [16–18], although this ratio is peptide sequence and conformation dependent [19–21]. The structural dependence of deamidation (for crystal structures, see Catazano et al. [22]) has been a matter of intense study over the last 3–4 decades (recently reviewed by Robinson and Robinson [1]). However, very little has been done in studying the structural dependence on the formation of the two deamidation products, aspartic acid and isoaspartic acid.

3.1 Mass spectrometric methods for studying deamidation MS has been extensively used to study deamidation as conversion of asparagine to aspartic acid and conversion of glutamine to glutamic acid involves a +0.984 Da mass shift. However, isotopic interference usually complicates the assignment requiring fitting of the experimental isotopic distribution to a sum of two model isotopic distributions to quantify them [3] (which prohibits quantitative analysis

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of low abundance Asp/isoAsp products), although this fitting method is not necessary if sufficient resolution is available [23,24]. Furthermore, MS has difficulty distinguishing aspartic acid from isoaspartic acid residues because they are isomeric and, thus, have zero mass shift. Many attempts have been made to distinguish these isomers with varying success. An old method using fast atom bombardment relied on signature immonium ions [25–27], the abundance ratio of b/y fragments at Asp versus isoAsp varies widely but unpredictably [28,29], and one group showed that b+H2O peaks were observed with isoAsp residues [28], although this effect appears to be sequence dependent [30]. A new method for distinguishing Asp and isoAsp residues in peptides [31,32] and proteins [33] has potential for clearly distinguishing these isomers. The most effective methods to date utilize liqiud chromatography (LC) or capillary electrophoresis (CE) combined with MS and Tandem mass spectrometry (MS/MS) [34–36]. CE and polyacrylamide gel electrophoresis (PAGE) are particularly useful for separating asparagine from Asp/isoAsp containing peptides and proteins due to the charge shift [36,37], and these separations can be followed up by MS, albeit with caveats described below.

3.2 Deamidation in monoclonal antibodies Stability of monoclonal antibody based therapeutics is becoming a critical concern for the US Food and Drug Administration and for the biotechnology and pharmaceutical companies that are developing these products, and one of the primary modifications affecting the stability of monoclonal antibodies is deamidation and isomerization of aspartic acid residues [38]. The current methods generally involve LCMS and LCMS/MS [30,39,40], which is slow but relatively effective in detecting Asn, Asp, isoAsp, and sometimes the succinimide residues [30]. For example, Chelius et al. [30] did a detailed study of deamidation of a Human Immunoglobulin Gamma antibody, identified four deamidation sites of the –NG– or –NN– motif, and found that b/y intensity ratios were an unreliable indicator of isoaspartic acid. Overall, LCMS and LCMS/MS methods rely entirely on isoaspartate containing peptides eluting before aspartate residues in the chromatographic trace to differentiate them; there is, in general, no reliable mass spectrometric confirmation of this assignment. Deamidation also affects other biotech products. For example, the hematopoietic growth factor known as stem cell factor or SCF shows a 50-fold decrease in activation if an isoAsp is formed at Asn10, but a slight increase in activity if Asp is formed there [41]. A potential thrombosis inhibitor, RGD, which is a small pseudopeptide including one aspartic acid residue, has been shown to isomerize causing instability in its activity over time [29].

3.3 Aging Increased deamidation is observed extensively in aged proteins, and presumably these deamidation sites are converted to a mixture of aspartic acid and isoaspartic acid. The ratio of the two products is not generally known. One of the many aging

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hypotheses, therefore, is that accumulation of deamidated proteins leads to cell death [4]. Several nice examples include erythrocyte aging where the membrane protein 4.1b deamidates in vivo with a half-life of 41 days [42], which roughly correlates with erythrocyte lifespan. Overexpression of the protein L-isoaspartyl methyltransferase (PIMT) repair enzyme (see below) in drosophila increased their lifespan [43,44], and PIMT knockout mice die quickly and can be rescued by PIMT gene therapy experiments [45–50].

3.4 Amyloid diseases Deamidation (and presumably the formation of isoaspartic acid residues) is considered as a possible initiation event for Amyloid diseases like Alzheimer’s disease (AD) [51–53] and Type 2 diabetes mellitus [54]. While the jury is still out on this hypothesis, there is substantial supporting information. Aspartic acids 1 and 7 of the a-beta peptide are known to be isomerized (and racemized) in Amyloid plaques with B70% isoAsp content [55]. Deamidation of insulin leads to amyloid fibril formation [56]. Formation of isoAsp residues is correlated with increased formation of beta-sheet structures [11,57,58] and deamidation is suppressed when the asparagine is constrained within an alpha helix [59]. The hypothesis that amyloid diseases, which are characterized by misfolding of proteins followed by aggregation and formation of fibrils, could be caused by formation of isoaspartic acid is very reasonable at this point.

3.5 Autoimmune diseases: Lupus and celiac disease Lupus is an autoimmune disease that is correlated with a high level of isoAsp formation on Histone H2B, and knockdown studies of the isoaspartic acid repair enzyme PIMT (see below) have shown that histone H2B will accumulate B1% isoAsp per day without PIMT. It is speculated that the isoAsp containing H2B generates the immune response which causes the disease. In a systematic and fundamental study, McAdam et al. [60] showed that T-cells will recognize deamidated hen egg lysozyme, but not native peptides thus demonstrating that immune response can be activated by deamidation. Mazzeo et al. [61] showed that the protein transglutaminase could deamidate 19 sites in alpha-gliadin. These modifications are likely responsible for the autoimmune response noted in celiac disease. Sjostrom et al. [62] have also correlated deamidation with celiac disease.

3.6 Cancer Deamidation of two specific asparagine residues in Bcl-XL is a critical apoptotic switch in a range of tumor cells [63,64]. Normal cells suppress the apoptotic signal by suppression of deamidation of Bcl-XL, which is an apoptosis inhibitor. DNA damage induced removal of the two proteins p53 and Rb in cancer cells (which are the deamidation suppressors of Bcl-XL) therefore leads to cell death. Some

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tumor cells resist apoptosis by reducing Bcl-XL deamidation rate or by increasing Bcl-XL production [65,66].

3.7 Cataracts Cataracts are often associated with PTMs in crystallins in the eye. One of the most common of these PTM’s is deamidation [67–73]. For example, Lapko et al. [68] found five Asn and nine Gln deamidation sites in gS crystalline and correlated their positions with solvent exposure. Harms et al. [70] noted that deamidation of Gln146 in bB1-crystallin resulted in greater aggregation. Interestingly, crystallins seem to show more glutamine deamidation than other proteins leading to speculation that an enzyme may be involved as was shown above with transglutaminase. Another protein associated with cataracts, Aquaporin, showed two sites of deamidation and one site of aspartic acid isomerization [74]. In the latter case, the b/y pattern found by Gonzalez et al. [28] was corroborated and peptides were synthesized to confirm the assignment of Asp versus isoAsp residues.

3.8 Anthrax vaccine A newly developed bacillus anthracis vaccine is primarily composed of a recombinant 83 kDa protein called Protective Antigen (rPA) [75–79]. This protein deamidates readily at 7 of 68 asparagine residues, increasing isoform complexity in electrophoretic separations, and this complexity increases with in vitro aging experiments. Aged rPA has marked decrease in activity.

3.9 Other diseases Deamidation has been observed in Osteopontin, a sialic acid rich glycophosphoprotein, which is implicated in many bone diseases and in the formation of calcium deposits [80]. Similarly, calbindin’s calcium homeostasis capacity in the central nervous system is modulated by its deamidation state [81]. Beutow et al. [82] have noted using a series of mutation and loop deletion studies that cytotoxic necrotizing factor 1 (CNF1), which is implicated in urinary tract infections and neonatal meningitis, allows Escherichia coli entrance to the cell by specifically deamidating one Gln residue in a series of skeletal proteins RhoA, Rac1, and Cdc-42. This result has also been observed by other groups [83–87]. Erythrocyte aging has been shown to be associated with increase in isoaspartic acid which is also associated with oxidative stress using mild oxidants such as H2O2 [88,89]. A disease associated with weakened red blood cell walls, hereditary spherocytocis, is correlated with enhanced methylation (presumably at isoAsp or D-Asp) on membrane proteins [90]. And finally, a number of other biochemical effects are noticed that are dependent on deamidation and aspartic acid isomerization. For example, cAMP-dependent protein kinase activity is dependent on Asn–Asp conversion [91], serine hydroxymethyltransferase deamidates and isomerizes in vivo [92], and human phenylalanine hydroxylase deamidation at Asn32

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appears to have a regulatory function and increases the rate of phosphorylation of this enzyme [93].

3.10 The PIMT repair enzyme One of the most important developments to date in the field of isoaspartic acid analysis is the discovery of PIMT (also sometimes called protein carboxyl methyltransferase, PCMT) [94,95]. This enzyme selectively methylates L-isoaspartyl residues which promotes their cyclization back to the succinimide with the loss of methanol [10,96]. Over time it converts isoAsp residues (and D-Asp residues) to L-Asp residues making it effectively an isoAsp repair enzyme [9,97]. Due to its selectivity for isoAsp residues, when this enzyme is combined with a radioactive methyl donor, the radioactivity can be used to trace formation of isoaspartic acid. This is the principle of the IsoQuant kit sold by Promega [98]. PIMT is naturally occurring in almost all mammalian tissues, and studies in the literature concerning its substrates are too numerous to cite, but for example, calmodulin aged at 7.4 pH and 371C accumulates 1.2 mols of methylation sites per mol of protein with one NG site as the major contributor but two DG sites and one NG sites as additional prominent contributors with seven more aspartic acid sites showing isomerization [99]. Particularly interesting, however, were a set of studies involving knockdown of PIMT levels in Rat PC12 cells using PIMT inhibitors [100,101] and PIMT-deficient knockout mice [45,102]. In the former case, isoAsp accumulated when PIMT was knocked down and resulted in immune response on Histone H2B which may be the cause of autoimmune diseases such as lupus. The knockout case was even more dramatic with KO mice suffering epileptic seizures and death within 1–2 months. In these cases isoAsp levels in brain tissue extracts were B5–10x higher than their heterozygous littermates (which could produce PIMT). Gene therapy techniques used to replace PIMT in knockout mice partially improved the symptoms, but only partially repaired the isoAsp residues in damaged proteins which implies that once isoAsp forms, some proteins cannot be repaired by PIMT [48]. Clearly PIMT is a critical protein in central nervous system function. PIMTbased assays (including one using LC to detect a non-radioactive methanol [103]) are currently used extensively for checking for the presence of isoAsp, but they can only determine the presence, but not the position of isoAsp residues.

3.11 Chemical methods for detection of isoAsp Edman degradation fails at isoaspartic acid residues which can be used to assign isoAsp positions. Also the Asp-N digestion enzyme fails at isoaspartic acid [104] so that different peptide patterns produced by this enzyme can be used to differentiate a- from b-aspartic acid. Also formation of the succinimide can be taken as an advantage for cleavage of the peptide backbone [105]. While such chemical methods work, they are time consuming, usually require at least microgram quantities of proteins, and are subject to side reactions. Newer methods which are faster, more reliable, and more sensitive will greatly advance the field.

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3.12 Deamidation as a sample-handling artifact While deamidation, as discussed, has many in vivo consequences, it can also be formed in vitro during sample handling and analysis if care is not taken [106]. Deamidation is frequently observed as an artifact of tryptic digestion since ammonium bicarbonate buffers are often used at pH 8. Chelius et al. [30] observed that overnight digestion at pH 8 yielded 30% deamidation of labile sites in an antibody, while a 4-h digestion yielded no detectible deamidation. Use of deglycosylation enzymes [107] and use of beta elimination reactions [108] both cause deamidation, and separation of proteins on PAGE gels can sometimes cause deamidation as well [93]. Liu et al. [109] showed that deamidation in 18O labeled water could be used to detect ‘‘real’’ deamidation from artifacts because it results in a +3 Da shift instead of the expected +1 Da shift. Finally, racemization can occur as well during deamidation [110]. Clearly deamidation can occur if proteins or peptides with labile sites are exposed to basic environments for any period of time. It is, therefore, imperative that care be taken to minimize artifactual deamidation and develop methods which allow distinguishing deamidation products that are real from those that are formed during sample handling.

4. NON-MS BASED METHODS FOR STUDYING DEAMIDATION 4.1 Proteolytic digestion Kameoka used endoproteinase Asp-N and MS to detect deamidation of asparagine and isomerization of aspartyl residues in a protein [111]. Endoproteinase Asp-N is a residue specific protease that cleaves on the N-terminal side of an L-aspartyl residue and not at D-aspartyl or D/L-isoaspartyl residues. Lysozyme with an isomerized aspartyl and a deamidated asparagine residue were mixed separately with N15 labeled lysozyme, digested with Asp-N and analyzed by matrix assisted laser desorption-ionization time-of-flight (MALDI-TOF) MS. Modified sites were identified by the presence of new peptides (asparagine deamidation) or the lack of expected peptides (aspartyl isomerization) when compared to those generated from digestion of the N15 labeled protein. Although most mass spectrometers can detect deamidation (+0.984 Da mass shift), protein digestion with Asp-N is a useful and simple technique for detecting aspartyl isomerization. Also, Asp-N could be used to differentiate aspartyl and isoaspartyl peptides since one of the two forms is an acceptable substrate for the enzyme. Other proteases have been used to detect isoaspartyl residues in proteins and peptides due to the uncommon peptide linkage associated with the form. The tryptic digest of the separated aspartyl and isoaspartyl forms of ribonuclease A showed differences in their HPLC peptide maps [112,113]. The differences were due to an apparent missed cleavage in the isoaspartyl chromatogram. The isoaspartyl residue, D67, was found to be adjacent to the missed cleavage site (?KD67G?). Therefore, the abnormal linkage associated with the isoaspartyl was presumed to be the reason why the K66 site was resistant to cleavage by trypsin.

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4.2 Isoaspartyl antibody Antibodies raised against isoaspartyl containing peptides using the MAP procedure (multiple antigen peptide system [114]) have been successful in identifying racemized aspartyl residues and deamidated asparaginyl residues in peptides and proteins, particularly for the Ab peptide associated with AD [115–117]. The antibodies raised are epitope specific so each suspected isoaspartyl residue requires a specific antibody and thus synthetic peptide for the MAP procedure. The antibodies raised against the synthetic peptides Ab1-42(isoD7) and Ab142(isoD23) were used to immunostain the brain tissue from six AD brains and non-AD brains that were used as control samples [116]. The Ab1-42(isoD7) antibody failed to be of use because it stained both AD samples and control samples, but the Ab1-42(isoD23) antibody was found to preferentially stain highly aggregated forms of Ab1-42 in the amyloid-bearing vessels and the core of mature plaques. Therefore, isomerized D23 was suggested to play a role in Ab1-42 sedimentation. In another example, the distribution of isoaspartyl residues in the postmortem brain of a 65-year-old patient with cerebral amyloid angiopathy (CAA) was studied with the Ab1-42(isoD7) and Ab1-42(isoD23) antibodies [117]. The patient had a D23N mutation within their Ab sequence (Iowa-type) that was suspected to undergo deamidation to the isoD23 form more easily than D23 form, possibly triggering an early onset of fibrillogenesis in blood vessels. Tissue immunostained with anti-isoaspartyl detected isoD7 in vascular and parenchymal deposits but isoD23 was detected only in vascular deposits suggesting that deamidation at the mutation site could have played a part in premature deposition of the Ab peptide in this case.

4.3 Reversed phase HPLC Separation of the asparaginyl, aspartyl, and isoaspartyl forms of peptides can be accomplished by RP-HPLC. Chromatography is an appealing methodology for separation because it can be used in combination with other techniques that assist in identifying the separated species. The elutant can be infused directly into a mass spectrometer or fractions can be collected so that a more detailed analysis by MS, Edman degradation, or PIMT methylation can be performed. Many studies have shown that the deamidation products elute in the following order; isoaspartyl, aspartyl, and then the aminosuccinyl form of the peptide [30,33,40,54,91,118–121]. This trend has been used to identify each peptide form [30] yet subsequent analysis by other techniques provides a more dependable result. When developing a method for separation, many factors need to be considered including the type of gradient, mobile phase composition, and column selection. Typically, a linear gradient on a RP-HPLC platform equipped with a C18 column, and a mobile phase system that consists of an acidified aqueous phase (mobile phase A), such as 0.1% TFA, and an organic phase (mobile phase B), such as acetonitrile, is used and separation is achieved by varying the gradient. This approach was useful for measuring the deamidation rates of peptides and the enzyme kinetics and repair rates associated with the protein isoaspartyl

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methyltransferase. A linear gradient of 0–40% B in 40 min was adequate to separate the isoforms of the peptide VYPDGA, corresponding to residues 22–27 of deamidated ACTH, wherein the isoaspartyl form was discovered to be an excellent substrate for PIMT [118]. Also, the system was used to follow the repair process of the small peptide, WMisoDF, to the aspartyl version by PIMT via time course plots based on the abundance of the intermediates and by-products in the reaction mixture [119]. All five products involved in the repair process (D/L-aspartyl, D/L-isoaspartyl, and succinimide versions of WMDF) were separated by a gradient 20–40% in 40 min. Both amino acid composition and length of the peptide affect the gradient necessary for separation of their isomers, each to varying degrees. For example, although the peptide GFDLDGGGVG contained twice as many residues as VYPDGA, they required the same chromatographic conditions to separate their isoforms [118,121]. Therefore, the unexpected behavior of peptides makes predicting RP-HPLC conditions almost impossible and often times results in having to develop custom gradients for every set of peptides. A general method can be developed but may require an extremely long gradient so that all possible deamidation sites are sufficiently separated. The products of three deamidation sites in a recombinant monoclonal antibody were separated on one HPLC run using a gradient of 0–65% B in 195 min [30]. Other types of gradients used for separation include step and concave gradients and even isocratic conditions. Although C18 is a popular choice for isoaspartyl/aspartyl separations, C8 columns can also separate the peptide isoforms although a shallower gradient may be necessary [99]. Finally, acidic modifiers such as TFA, formic acid, and acetic acid provide adequate retention and peak shape, but changing the pH of the mobile phase can help shift the retention times of interfering species in the chromatogram. For example, using a mobile phase system at pH 6 helped separate four forms of a peptide that was both deamidated and isomerized [99]. Since the aspartyl/isoaspartyl forms are ionized at pH 6, the native, non-deamidated form is shifted to a higher retention time away from the ionized forms therefore providing a less complex chromatogram.

4.4 Ion exchange chromatography Ion exchange chromatography is useful for separating species based on their ionic charge and therefore can be used to separate the native from the deamidated form of a protein since asparagine/glutamine is converted to their ionizable acid homologues. Mobile phase systems used for separation should have a pH that ensures deprotonation of the acidic group so that the overall charge of the native and deamidated forms show differential binding to the stationary phase. Bound proteins are eluted with gradual increase in concentration of a counter ion, i.e. salt. Both cation exchange and anion exchange can be used to differentiate the two forms. The deamidated form of the protein binds more strongly for anion exchange chromatography therefore eluting later than the native form, and vice versa for cation exchange analysis. Cation exchange chromatography has been used to separate the forms of a partially deamidated proteins [41,122] including ribonuclease A [113,123–124] and a monoclonal antibody [40] and anion exchange has been used for gS-crystallin [68] and protective antigen [75]. Separation of

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isoglutamyl/isoasapartyl and glutamyl/aspartyl by ion exchange is difficult, since the difference between the pKa of the isomers is so similar. The kinetics of isoaspartyl and aspartyl formation from deamidating ribonuclease A was measured using a very shallow KCl gradient on a cation exchange system [6].

4.5 Electrophoresis Gel electrophoresis can be used to analyze proteins affected by deamidation since both the isoelectric point and shape are altered. Isoelectric focusing (IEF) can separate based on the change in charge and native gel electrophoresis can discriminate protein forms based on the change in shape. The more traditional method, SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is not used since the molecular weight resolution on gels is insufficient to discriminate between the native and deamidated forms. IEF is performed on a pH gradient gel that allows proteins to migrate based on their isoelectric point (pI) in the presence of an applied electric field. Performing separations using buffers with pH greater than the pKa of the aspartyl or isoaspartyl side chain allows separation of the deamidated from non-deamidated forms since their respective pIs are different. The method is especially useful for separating multiple forms of a protein due to several deamidations; the multiple forms of stem cell factor [41], protective antigen [76], and phenylalanine hydroxylase [93] were separated by IEF. Native gel electrophoresis is also a useful electrophoretic technique for analyzing deamidated proteins since it can also separate the isomeric products of deamidation. The rationale for separation is that formation of aspartyl/isoaspartyl residues affects the shape of the protein when compared to the native form. For example, the three forms of partially deamidated calmodulin (N97 to isoD/D97) were separated by native gel electrophoresis [125]. The isoD97 form migrated the slowest followed by the D97 and then N97 form. The larger molecular radii for isoD97 and D97 account for their slower migration; formation of isoaspartyl affects the shape of the protein more than the aspartyl form. Other proteins separated by native gel electrophoresis include the multiple deamidated forms of calbindin [81] and protective antigen [75]. CE has been used to separate the deamidation products and their respective stereoisomers. In CE, analytes are separated by their charge and interaction with the surface silanol groups of the column in the presence of a strong electric field and pH buffered mobile phase. All four isomers (D/L-aspartyl and D/L-isoaspartyl) from aspartyl isomerization in a tripeptide were successfully separated on a silica capillary column using sodium phosphate buffer solution at pH 3 [36]. Contrary to elution order found in reverse phase HPLC analysis, the isoaspartyl peptides were more strongly retained on the capillary since their pKa is predicted to be lower than the aspartyl side chain. Also, the products of D130 isomerization from aged and digested rHGH were separated by CE using similar separation conditions [126].

4.6 Nuclear magnetic resonance (NMR) 2D NMR has been used to differentiate the isoaspartyl from aspartyl residues in peptides [127,128]. Scalar coupling of the 1H spin systems between the backbone nitrogen and the Ca, Cb, and amide nitrogen of the neighboring amino acid

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residue in NOESY 2D 1H NMR spectra allows the approximate distances between these groups to be determined for large molecular structures such as proteins [129]. For normal residue linkages, the Ca–N or N–N couplings are typically strong while Cb–N tend to be weaker. However, isoasaprtyl linkages have a methylene group inserted into the backbone that changes the magnitude of these couplings. For example, 2D 1H NMR was used to detect an isoaspartyl residue (isoD56) within a 30-residue tryptic peptide from calbindin D9k that deamidated during purification [127]. The distance between the nitrogen of the G57 and Cb of ˚ while the distance of isoD56 (backbone methylene) was found to be less than 3.5 A ˚ , respectively. This trend Ca and N (of isoD) from N (of G57) were 4.7 and 6 A describes spatially what is attributed to an isoaspartyl residue in the peptide backbone. Additionally, the data showed that the backbone region containing an isoaspartyl residue was in a more extended helical shape compared to the aspartyl version. Another study used 2D 1H NMR to differentiate to the isoforms of a 15-residue peptide using the presence of the Cb – N found in the isoaspartyl spectrum that was not found in the aspartyl spectrum [128]. The advantage of using 2D 1H NMR is the additional structural information acquired that provides insight on how the presence of an isoaspartyl residue affects the higher-order strucutre of the region. However, the disadvantage is that each experiment requires a large amount of sample, which may make the method inapplicable to biological experiments; the samples in the NMR studies described were in the range of 103 M, which is close to the precipitation concentration of most proteins.

4.7 Edman degradation Edman degradation, a chemical method used to sequence N-terminal a-linked peptides, was found to be blocked by an isoaspartyl residue when Smyth et al. attempted to sequence residues 11–18 of pancreatic ribonuclease [130]. For normal a-linked peptides, the addition of phenylisothiocyanate to the amino group facilitates nucleophilic attack of the carbonyl group by the thiol group ultimately resulting in cleavage of the residue and generation of a peptide with n-1 residues and a new amino terminus. When an isoaspartyl residue is the next residue, the carbonyl group is beyond the reach for the attacking thiol due to the inserted methylene group, and the process is interrupted. The failure of Edman degradation at isoaspartyl residues has been used to detect these residues in proteolytic peptides. Furthermore, products of deamidation separated by HPLC determined to have the same masses and sequences by MS/MS or amino acid hydrolysis can be subjected to Edman degradation for differentiation. This method has been useful for experiments involving proteolytic peptides from deamidated proteins [131,132] including ribonuclease A [112,130], calmodulin [99], and human stem cell factor [41] and isomerized Ab peptide from AD brains [133].

4.8 The PIMT enzyme The enzyme PIMT, which selectively methylates isoaspartyl residues, can be used for analyzing asparaginyl deamidation and aspartyl isomerization by detecting the

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resulting isoaspartyl residues. Analytical methods employing PIMT use radioactively labeled S-adenosyl-L-methione (AdoMet, 14C or 3H) as the methyl donor that is selectively incorporated onto the isoaspartyl caboxylate group. A popular technique for measuring the isoaspartyl content in peptides by radiolabeling with PIMT is by the vapor diffusion method. In this method, [14C] or [3H] methanol released by quenching the methylation reaction with a mild base is measured by liquid scintillation [118,134]. The methylation step is carried out for 30 min to 1 h and then immediately quenched with a mild base generating radiolabeled methanol. The opened reaction vial is then placed in a sealed tube containing a scintillation cocktail. The [14C] or [3H] methanol vapor diffuses from the reaction solution (or spotted filter paper) into the scintillation cocktail which is then counted to determine the quantity of isoaspartyl residues versus radiolabeled methanol standards. For proteins, the vapor diffusion method is used but precipitation of the protein using trichloroacetic acid is done after methylation and before treatment with base to remove unreacted [14C] or [3H] AdoMet [135,136]. Isoaspartyl residues in cell (E. coli [137], erythrocytes [90], and ooctyes [136]) and tissue homogenates (drosophila [43], Caenorhabditis elegans [138], and mice [45,46]), proteins [101,125,139–141] and peptides [93,99,118,121,122,133,142,143] have been characterized using the vapor diffusion method. Isoaspartyl detection using PIMT is often used in combination with separation techniques, such as HPLC and gel electrophoresis, to localize the isoaspartyl residues in a mixture of proteins or peptides. The vapor diffusion method can be used with HPLC to provide two concurrent chromatograms, one based on UV absorbance and the other derived from radioactive counts of collected fractions. The peptides or proteins can be labeled after being fractionated by HPLC or before separation. Peptides from aged proteins [139–141], such as calmodulin [99], were analyzed using HPLC and PIMT methylation as well as a mixture of histones isolated from nuclei of mouse brain tissue [101,144]. Also, a mixture of proteins can be reacted with PIMT in the presence of radiolabeled AdoMet, separated by gel electrophoresis and then subjected to fluorography [101,136,139,143]. The fluorogram of the gel reveals which proteins, based on molecular weight, contain isoaspartyl residues. The relative intensity of the spots can also provide some information of the relative amount of isoaspartyl formation between the proteins. Finally, the Isoquant kit developed by Promega also uses the PIMT enzyme and HPLC to detect and measure the abundance of isoaspartyl residues by measuring the change in UV absorbance of AdoMet before and after methylation [103]. Localization is not possible but the convenience of the kit helps to quickly and easily provide some information on isoaspartyl content.

5. MASS SPECTROMETRY BASED METHODS FOR STUDYING DEAMIDATION 5.1 Measuring deamidation by isotopic deconvolution and mass defect methods The isotopic deconvolution method for measuring deamidation uses the +0.984 Da shift in the mass spectrum associated with the conversion of –NH2 to –OH. The

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corresponding peaks in the spectrum are a combination of two overlapping species resulting in an atypical isotopic pattern. Assuming the peak intensities are additive, the pattern can be deconvolved quantitatively to its two separate forms by fitting the theoretical isotopic patterns of each contributing species to the experimental pattern. Although a considerable amount of signal averaging is advised to obtain a pattern that accurately represents the sample composition, this method is still much faster and uses less sample than HPLC. Robinson and Robinson efficiently performed a comprehensive analysis of the deamidation rates of asparginyl and glutamyl residues in over 700 peptides that required thousands of analyses using the isotopic deconvolution method on data obtained from a quadrupole mass spectrometer [145,146]. The extent of deamidation in proteins has also been determined by analyzing the proteolytic peptide mixture on a MALDI-TOF mass spectrometer. The advantage of MALDI is that ionization generates only singly charged species therefore producing a mass spectrum of minimal complexity, an important benefit considering the number of proteolytic peptides that can be obtained from a protein digest. For example, the extent of deamidation of human phenylalanine hydroxylase [93,147], calbindin [81], and protective antigen [76] was determined using the isotopic deconvolution method on data from a MALDI-TOF mass spectrometer. Isotopic deconvolution has also been used on high-resolution data from electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) analysis of deamidated ribonuclease A [148] and crystallins from human eye lenses (A. B. Robinson, Private communication). Fragments obtained from top–down analysis of these proteins allowed the localization and extent of deamidation to be measured at several sites therefore eliminating the need for digestion. The mass defect method for measuring deamidation relies on the ability to resolve the 19 mDa mass difference between the A+1 isotope of a non-deamidated peptide and the monoisotopic peak of the deamidated form [23]. Once resolved, the relative intensity of the two peaks corresponds to the extent of deamidation assuming the ionization and detection efficiencies of the two species is the same. FTMS can attain the high resolution necessary to resolve the two forms. For example, a resolution of 280,000 was achieved on an FTMS in order to separate the three deamidated forms of 16-residue synthetic peptide [23]. The mass defect method can also be used on fragments generated by top–down fragmentation (A. B. Robinson, Private communication; [24]). For example, mixtures of wild-type and mutant (Q162E) were mixed at known ratios and subjected to top–down fragmentation by IRMPD [24]. Both forms for fragments containing the Glu/Gln162 site were resolved from one another and the mixture composition revealed by mass defect analysis was found to be close to the expected value.

5.2 The diagnostic bn1+H2O for isoaspartyl residues in CAD MS spectra In 1992, Papayannolpoulos [149] and Carr [150] both reported a bn1+H2O (n is the position of isoaspartyl or aspartyl) fragment ion in the collisionally activated dissociation (CAD) spectrum of peptides with an isoaspartyl residue that was not

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found in the spectrum of the aspartyl form of the same peptide. The fragment ion was also used to differentiate the isoaspartyl from aspartyl forms of a deamidated proteolytic peptide, separated by HPLC, from hirudin, an anticoagulant peptide [151]. The mechanism involves migration of the –OH from the isoaspartyl side chain to the n-1 carbonyl group via an oxazolidone intermediate that rearranges to generate the bn1+H2O ion and an aldinine fragment. Since the isoaspartyl side chain resembles the C-terminus, generation of bn1+H2O for isoaspartyl residues is suggested to resemble the fragmentation channel shown to occur in the low-energy MS/MS spectra of peptides wherein a C-terminal hydroxyl rearrangement generates bm1+H2O fragment ions (m is the length of the peptide) [152,153]. Schindler et al. showed evidence of the hydroxyl transfer mechanism for isoaspartyl residues by performing MS/MS on an O18 labeled peptide [151]. The labeled peptide was synthesized by incubating the succinimide derivative of the peptide in O18 water, so that upon hydrolysis, one of the equivalent oxygens of the carboxyl group of the isoaspartyl side chain was labeled with O18. The low energy MS/MS of the peptide showed the bn+H2O ion being split in a 1:1 ratio of O16:O18 thus proving that there is a migration of –OH from the side chain to the diagnostic fragment. A study in 2000 by Gonzalez et al. showed that the bn1+H2O fragment ion and its complement, the yln-46 fragment ion, can be used to differentiate isoaspartyl from aspartyl residues in sets of synthetic peptides, including D7 and D23 of b-amyloid peptides analogues, and detect isoaspartyl residues in deamidated tryptic peptides from recombinant proteins [28]. The data also demonstrated that the bn+H2O intensity is much larger if a basic amino acid or the N-terminus is on the N-terminal side of the isoaspartyl residue. Intermolecular interaction between the side chain of the basic and isoaspartyl residues is believed to facilitate rearrangement to generate the diagnostic ions. A severe limitation to using the bn+H2O ion to detect isoaspartyl residues may occur when analyzing tryptic peptides, since they should only have C-terminal basic residues unless there is incomplete digestion or there is a histidine present within the tryptic peptide. The complement yln-46 fragment ion can be used to detect isoaspartyl residues in the case of tryptic peptides, but this fragment is usually much less abundant than the bn+H2O fragment ion (the largest yln-46/yln intensity ratio reported was 0.039). Nonetheless, the isoaspartyl residues in two tryptic peptides were characterized by the presence of the yln-46 ions.

5.3 Aspartyl versus isoAspartyl fragment ion ratios in CAD spectra Lloyd and coworkers were first, in 1988, to use fragment ion abundance ratios to differentiate aspartyl from isoaspartyl residues in the MS/MS spectra of peptides using a double focusing magnetic sector mass spectrometer [26]. The MS/MS of the peptides RKDVY and DIRKF-NH2 showed loss of CO from bn+1 to form an+1 while the same loss was much smaller for the isoaspartyl versions (bn+1/an+1(Asp) W bn+1/an+1(isoAsp)). The authors suggest that loss of CO to form the an+1, a stable iminium ion, for the aspartyl form is a much more favored pathway than loss of CO form the isoaspartyl bn+1 to from a primary carbocation. Additionally,

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the same trend was found for a peptide containing a glutamyl residue and its isoglutamyl homologue. A study by Lehman et al. in 2000 found that the intensity of fragment ions resulting from amide backbone bond cleavage (b and y ions) on either side of aspartyl/isoaspartyl showed a reproducible trend that can be used to distinguish isoaspartyl from aspartyl residues [154]. Based on the MS/MS spectra of 15 sets of isomeric peptides, the b/y intensity ratio of complementary b and y ions from cleavage on either side of the aspartyl/isoaspartyl residue were consistently larger for the aspartyl form than the isoaspartyl form. Fragmentation intensity ratios for aspartyl were typically less than 15 times larger than the same fragmentation for the isoaspartyl counterparts although some values were much larger. The trend is believed to be a result of the competition between forming the oxazolone containing the N-terminus (b ion) and direct cleavage to form a terminal amine containing the C-terminus (y ion) [155]. In the ESI process, the amide nitrogen can be protonated thus weakening the C–N bond facilitating nucleophilic attack of the amide bond carbonyl on the C-terminal adjacent amino acid to form the oxazolone. When the residue is isoaspartic acid, formation of the oxazolone is hindered. On the N-teminal side of the isoaspartyl residue, oxazolone formation can be hindered by a similar interaction between the isoaspartyl side chain and the backbone carbonyl since the carboxyl group is closer in proximity to the backbone more so than the aspartyl form. On the C-terminal side, a six-membered ring must be formed that contains the isoaspartyl residue and is kinetically less favored than the five-membered oxazolone structure. Therefore, b ion formation is hindered on both sides of the isoaspartyl residue and direct cleavage is favored resulting in a decreased b/y ion ratio compared to the aspartyl form.

5.4 Immonium ions of isoaspartyl residues The structure proposed by Lloyd for the a1 ion found in the MS/MS spectrum of the peptide DIRKF-NH2, missing from the spectrum for the isoaspartyl version, is essentially the immonium ion for an aspartyl residue (m/z ¼ 88) [26]. Immonium ions are small internal fragments containing one amino acid side chain that result from the cleavage of multiple backbone bonds and are useful for determining the amino acid composition of a peptide. Several studies since then have shown that the intensity of the aspartyl immonium ion found in the MS/MS spectrum of an isoaspartyl peptide is much smaller (or nonexistent) than that found in the aspartyl spectrum [28,154]. Lehmann showed that, for 15 sets of peptides, the aspartyl immonium ion intensity (normalized to another immonium ion in the spectrum) was on the average 5.5 times higher for the aspartyl form over the isoaspartyl form and suggest that such a trend could be used to differentiate the two forms [154]. Gozalez et al. used the aspartyl and isoaspartyl immonium ion intensities to differentiate isomers that could not be differentiated using the bn1+H2O and yln-46 fragment ions [28]. In addition to the aspartyl immonium ion, a fragment ion at m/z ¼ 70 was found in the isoaspartyl spectrum that was not found in the aspartyl spectrum. The ion was suggested to be the immonium ion for an

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isoaspartyl residue that results from a rearrangement of a primary carbocation, a structure suggested to be unstable and therefore not found in the isoaspartyl spectrum. Loss of water from the side chain of the carbocation yields a charged acylium structure of mass m/z ¼ 70. This ion, however, cannot be used as an absolute indicator for the presence of an isoaspartyl since it is the same mass for the proline immonium ion, which is typically a strong signal in the mass spectra of proline-containing peptides.

5.5 Liquid chromatography/mass spectrometry (LCMS) LCMS methods use the RP-HPLC technique because the acid modifiers help the retention and peak shape of peptides on the LC column while assisting protonation and being volatile for MS analysis (see Section 4.3 for details). The localization and extent of deamidation in a protein can be determined from one LCMS run of the proteolytic peptides. For example, nine deamidation sites (aspargine and glutamine) were characterized in gS-crystallin from cataracts as determined from an LCMS analysis of the trypsin digest [68]. Deamidation was first determined by the isotopic deconvolution method and localization of these sites were determined by the MS/MS data, which was necessary because multiple asparagine and glutamine residues were present in many of the peptides. In another study, the carboxyl groups of tryptic peptides were methyl esterified in order to simplify the detection and measurement of two deamidation sites [70]. Detecting the 1 Da shift in an ion trap can be difficult but the +14 Da from the introduced methyl ester at the carboxyl residue (deamidation site) make recognizing deamidation much easier. Also, the methyl ester changes the hydrophobicity of the peptide, shifting it away from the non-deamidated form and therefore simplifying the LC chromatogram for quantitative measurements. The relative quantification of deamidation products can also be determined by LCMS analysis, provided the LC separation is sufficient to separate the isomers. As mentioned above, the isoaspartyl form of a deamidated peptide typically elutes before the aspartyl form and this trend has been used to assign the identities of peptides found in an LC chromatogram [30], but supportive data is often needed to unambiguously make such assignments. Analyses using synthetic peptide standards [60,91] and a mutant form of a protein [40] have been successful in confidently identifying deamidation products separated by HPLC thereby providing reliable quantitative measurements. For example, LCMS/MS was used to measure the extent of in vivo deamidation of a monoclonal antibody using a mutant form as a standard [40]. The antibody in question was isolated and digested in parallel with the mutant form that had an aspartyl residue substituted for the deamidating asparaginyl residue. The retention time of the peptide with the aspartyl substitution from the mutant protein had the same mass-to-charge ratio, MS/MS profile, and retention time as the aspartyl peptide (deamidation product) from the in vivo sample. The information allowed the extent of deamidation and relative quantification of the products to be determined from the LCMS/MS experiment (the identity of the isoaspartyl peptide was assumed based on its retention time with respect to the aspartyl form). Other experiments

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used the retention time and MS/MS profiles of corresponding synthetic peptides (aspartyl, isoaspartyl, and asparaginyl) to measure the deamidation of peptides or the natural abundance of isoaspartyl residues in a protein [60,91].

5.6 Electron capture dissociation Differentiation of aspartyl from isoaspartyl residues in synthetic peptides can be accomplished using electron capture dissociation (ECD) based on fragments generated from cleavage of the Ca–Cb bond [31]. ECD is performed in the ICR cell of an FTMS instrument by irradiating trapped, multiply protonated peptides and proteins with electrons. The fragments are then excited and detected with the high resolution and accuracy attributed to FTMS instrumentation. The c and z fragments typically generated by ECD are a result N–Ca cleavage, but when the residue is an isoaspartyl residue, cleavage of Ca–Cb backbone bond generates the cln+57 and zn-57 diagnostic fragment ions (n is the position of the isoaspartyl/ aspartyl residue and l is the length of the peptide and numbers are nominal masses in Daltons). Figure 6 shows two models, synthetic peptides which demonstrate the z4-57 diagnostic peak. The top spectrum in Figure 6 has no z4-57 peak, an abundant z4-CO2 peak, and an abundant M-60 peak, clearly indicating that Asp4 is, indeed, the a-aspartic acid isomer. The bottom spectrum instead shows an abundant z4-57 peak (it is the second most intense fragment ion peak in the spectrum) and much lower z4-CO2 and M-60 peaks, clearly indicating that Asp4, in this case, is the isoaspartic acid isomer. The cleavage mechanism that generates these diagnostic peaks is similar to a McLafferty rearrangement [156] that results in a stable, even electron enol (zn-57)

Figure 6 Electron capture dissociation spectra of two synthetic peptides, one with Asp (D) and one with isoAsp (isoD). The two peaks Z4CO2 and Z457 show the primary diagnostic peaks with clarity. Because this peptide is similar to a tryptic peptide, with C-terminal arginine, few c ions are observed. (Reprinted with permission from ref. [159].)

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and an odd electron, glycyl-like residue with a Ca radical (cln+57). Rauk has shown that an Ca radical is captodatively stabilized on a glycine residue due to the flanking amine and carbonyl groups [157]. The spectra of the aspartyl peptides showed a peak in the side chain loss region not found in the isoaspartyl peptide spectra representing neutral loss of the aspartyl side chain from the reduced molecular ion ((M+2 H)+d-60). The aspartyl side chain loss from Ca–Cb cleavage indicates the presence of an aspartyl residue, but is less informative when other aspartyl residues are present in the peptide. The cln+57 and zn-57 diagnostic ions were used to detect isoaspartyl residues in a deamidated tryptic peptide, a tryptic peptide from deamidated protein, and to differentiate isoaspartyl and aspartyl tryptic peptides from a protein separated by HPLC [33]. Figure 7 shows a similar separation of the two isoforms of a tryptic peptide from calmodulin, with the spectra below showing the regions for the z10-57, the c6+58, and the M-60 diagnostic peaks. Clearly, the left peak contains isoaspartic acid, and the right one contains aspartic acid. The cln+57 and zn-57 ions were also used to differentiate the isoforms of aspartic acid in synthetic peptides using electron transfer dissociation (ETD) in an ion trap mass spectrometer [32]. Similar to ECD, ETD cleaves N–Ca bonds in peptides and proteins but via interactions with gas-phase electron donors in an ion trap. The advantage of an ion trap is that it is inexpensive instrumentation compared to FTICRMS, but with inferior resolution. Using nitrobenzene anions as

Figure 7 Revised phase HPLC separation of the isomeric forms of the deamidated tryptic peptide (91)VFDKDGNGYISAAELR(106) from bovine calmodulin that were then differentiated by ECD. Mass spectra are zoomed in regions of the ECD fragment spectra where the diagnostic fragment ions used to differentiate the two forms should be located, shaded areas.

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an electron donor, the cln+57 and zn-57 fragment ions were generated, localizing the position of three isoaspartyl residues in the peptide. Also, b and y series backbone fragments were generated, not typically found in ECD spectra, which can be helpful for extra sequence information. Therefore, ETD provides the diagnostic ions for detecting and localizing isoaspartyl residues as well as providing additional fragment ions not found in ECD spectra, but has difficulty resolving higher charge state ions compared to ECD performed in a FTMS.

6. QUANTITATION OF DEAMIDATION AND ITS PRODUCTS 6.1 HPLC combination methods As mentioned above, quantitating the extent of deamidation and the relative abundance of deamidation products can be done with HPLC when used in combination with techniques that can discriminate the multiple forms. Edman degradation, mass spectrometric techniques, and PIMT assays can be performed on collected fractions to differentiate the peptides (aspartyl/isoaspartyl peptides) as long as the peptides are adequately separated.

6.2 CAD methods without HPLC The capability to quantitate the relative amounts of aspartyl and isoaspartyl residues using CAD fragments (bn+H2O, b/y intensity ratio and immonium ions) is possible [154,158]. The experiments illustrating this ability involve performing MS/MS analysis on mixtures of synthetic peptides that vary in isoaspartyl/ aspartyl composition, intending to represent the possible outcomes of asparaginyl deamidation or aspartyl isomerization. Lehman showed that both the b/y intensity ratio and immonium ions could be used to calculate the relative abundance of the two forms [154]. A plot of the b/y intensity ratio from cleavage on the N-terminal side of the peptide VQ(D/isoD)GLR versus aspartyl content showed an asymptotic relationship that could be used as a calibration curve. Also, MS/MS analysis of mixtures of myrGDAAAK and its isoaspartyl counterpart showed a linear relationship between the aspartyl immonium ion (normalized to the lysine immonium ion) and aspartyl composition. However, since these fragments ions are not diagnostic and calculation of the relative intensity the fragments are necessary, at least two points on the calibration curves are needed, and peptide standards are required to generate calibration curves. Alternatively, the bn+H2O fragment ion is diagnostic [149–151]. The relative intensity of bn+H2O was shown to increase linearly with isoaspartyl content when compared to other backbone cleavages that were assumed to be unchanging regardless of sample composition [158]. Despite the advantage, the isoaspartyl peptide standard is necessary to establish the calibration plot.

6.3 ECD method without HPLC Similar to the CAD method using the bn+H2O isoaspartyl fragment ion, the zn-57 diagnostic ion found in the ECD spectra of isoaspartyl/aspartyl peptide mixtures

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was shown to increase linearly with isoaspartyl content for three sets of isomeric peptides that varied in amino acid sequence [159]. The linear relationship between the relative abundance of zn-57 to all backbone fragment ions and isoaspartyl composition was used as opposed to the method using the bn+H2O CAD fragment ion, which used the relative abundance of the diagnostic ion to another fragment ion in the mass spectrum [158]. The relative abundance of most ECD fragment ions from backbone cleavages were found to change with the substitution of one form of aspartic acid for the other, most likely due to gas-phase hydrogen bonding involving polar side chains. Therefore, relative quantitation using the zn-57 fragment ion normalized to another backbone fragment ion may be possible, but only if it does not vary with isoaspartyl composition. Otherwise, normalization to total backbone fragment ion abundance was shown to provide a consistent linear relationship. The ECD method still requires an isoaspartyl version of the peptide to construct the linear plot, however using the deamidated proteolytic peptide from the protein that contains the deamidation site should provide a 3:1 (isoaspartyl: aspartyl) standard for calibration [159]. The proteolytic peptide mixture obtained from protein digestion can be incubated under harsh conditions to provide the 75% isoaspartyl peptide standard to be used for calculating the composition of the mixture from in vivo or in vitro deamidation experiments (Figure 8).

Figure 8 Diagram of a methodology for systematic relative quantitation of Asp/isoAsp ratios in proteins. The methods assume that a small, random coil peptide from an enzymatic digest, under harsh deamidation conditions, will deamidate to the 75% isoAsp typically observed. Provided that caveat holds, the harsh deamidation conditions provide a second calibration point, which allows relative Asp/isoAsp ratios to be measured. This caveat has been verified in several cases, but for any particular protein of interest, it would have to be checked. (Reprinted with permission from ref. [159].)

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7. ISOTOPIC LABELING METHODS The most common ambiguity in mass spectrometric analysis of deamidation and the formation of isoaspartyl residues arises from the simple, nonenzymatic nature of the deamidation reaction. Because the reaction occurs automatically and pseudo-unimolecularly (it requires H2O) at basic pH, it will occur during sample-handling procedures. The most common observation to this effect is that deamidation occurs during enzymatic digestion when trypsin is used, overnight, at pH 8–9, which is a fairly standard condition. Therefore, how do we know which deamidation and isoaspartyl sites are formed under physiological conditions and which are formed as an artifact of our sample processing methods prior to analysis? One method is use of 18O isotopic labeling. 18 O labeling of peptides can be accomplished by digestion of proteins in the presence of H218O. When digestion is performed in H218O, at least one 18O is incorporated into the C-terminus of the newly formed peptide since one molecule of H2O is used to hydrolyze the peptide bond. 18O labeling is easily amenable to analysis by MS since labeled peptides should show a 2 Da shift in their mass spectrum. Interestingly, Schnolzer et al. used MS to show that trypsin can incorporate more than one 18O during peptide bond hydrolysis as indicated by two overlapping isotopic distribution in the mass spectrum (2 and 4 Da shift) [160]. The authors believed that cleaved peptides can interact with the protease again to incorporate another 18O and that the extent of these post-cleavage interactions is peptide dependent. Therefore, tryptic digestion in H218O results in peptide mixtures containing varying amounts of 18 O incorporation, which was later corroborated by other studies [161,162]. However, using consistent digestion procedures can eliminate discrepancies between experiments regarding the amount of 18O that is incorporated and such rigorous protocols have helped to make 18O labeling a viable method for proteomics. Identification of proteins’ C-termini [160,163] and glycosylation sites [164] have been accomplished by 18O labeling and several strategies, reminiscent of ICAT methodology, have been proposed for evaluating relative protein expression using 18O labeling [162,165–167]. Additionally, Grossenbacher et al. labeled two DG sites of recombinant hirudin with 18O by opening their succinimide intermediates (from aspartyl dehydration) with the addition of H218O, therefore suggesting the possibility of detecting aspartyl isomerization and asparaginyl deamidation via an 18O labeling experiment [168]. Finally, both Grossenbacher et al. [168] and Lui et al. [109] found that deamidation occurring in H218O incorporates 18O into the aspartyl or isoaspartyl side chain suggesting the possibility of detecting aspartyl isomerization and asparaginyl deamidation via an 18O labeling experiment. Deamidation of labile asparaginyl residues often occurs during digestion (as opposed to aspartyl isomerization which is typically 40 times slower than deamidation) since elevated temperature and pH for extended periods of time are often required which promotes deamidation. The typical +1 Da shift upon deamidation is often too subtle a change to determine

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the extent of conversion especially when complex isotopic clusters from larger, multiply charged peptides are analyzed. However, the 3 Da shift produced by deamidation in H218O (–NH2 to –18OH) can help determine the extent of unfavorable deamidation that may result from prolonged digestion especially when the deamidation of these larger, multiply charged peptides are investigated. Three proteins (calmodulin, reduced and alkylated ribonuclease A, and lysozyme) were digested in 95% H218O (0.1 M ammonium bicarbonate, pH 8.3) with trypsin (25:1 ratio, respectively) for extended periods of times from which aliquots were taken periodically and analyzed by nanospray FTMS (Figure 9). First, the tryptic peptide from ribonuclease A (residues 67–85) showed only one 18O incorporation upon digestion and completely deamidated after 1 day of incubation as illustrated with an approximate 3 Da shift in the mass spectrum (Figure 9a). After 2 days, the peptide with two deamidations became the more prominent species and this second deamidation site is most likely N71. Although the predicted deamidation half-life for this site is B55 days at pH 7.4, the digest was performed in an environment about 8 times more basic (pH 8.3), which may dramatically accelerate the rate of deamidation within the time frame studied. The tryptic peptide from calmodulin (residues 91–106) initially showed a 1:2 mixture of one and two 18O incorporations, respectively, and continued to shift to a 1:3 mixture after 4 h indicating that this peptide is an excellent substrate for trypsin (Figure 9b). After 24 h, the relative ratio of one to two 18O incorporations remained at 1:3 but the entire isotopic pattern shifted +3 Da as a result of complete deamidation. Similar to the calmodulin peptide, the tryptic peptide from lysozyme (residues 46–61) initially showed a mixture of one and two 18O incorporations (about 1:1, respectively) which shifted slightly in favor of the peptide with two 18O incorporations after 8 h (Figure 9c). Again, this minor shift could be due to the peptide continuing to interact with trypsin. Both forms (peptides with one and two 18O) showed their deamidated counterparts after 1 day of incubation and these deamidated forms were the most prominent after 2 days of incubation. This is expected since the predicted deamidation half-life of the –NS– sequence over –NG– is approximately 10-fold. All three experiments show that the +3 Da mass shifts in the mass spectrum accompanied with deamidation occurring in H2O18 may be a useful technique for detecting and measuring deamidation, especially for routine proteomic experiments in which prolonged digestion periods are often required. The +3 Da mass shift helps to simplify the process of determining the relative abundance of the deamidated and native peptides so that these values can be directly inferred from the spectrum without deconvolution calculations. Although multiple O18 incorporations from digestion may complicate the spectrum, once the relative abundance of the two forms are established (one and/or two 18O), the +3 Da shifts should still provide a better method for detecting and measuring deamidation versus the traditional 1 Da shift found with deamidation occurring in H216O.

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Figure 9 Tryptic digestion of peptides in isotopically labeled water incorporates 18O twice at the C-terminus, and once at each deamidation site.

8. SUMMARY Deamidation of asparagine and glutamine residues to their acidic counterparts is a common PTM on proteins. It occurs non-enzymatically in solution, and with the exception of –NP– and –QP– sequences, it occurs at all Asn and Gln residues

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in all proteins eventually. However, the rates vary dramatically with the primary and higher-order structure of proteins. Deamidation has substantial repercussions in protein stability, often leading to the unfolding and recycling of proteins by the cell. However, the misfolding of deamidated proteins is also associated with many diseases from Amyloid diseases to cataracts to cancer. Deamidation results in a charge change and a +0.984 Da mass shift, both of which are readily detectable by many methods, but it results in a mixture of isomers that are relatively difficult to differentiate. A new mass spectrometric method was recently introduced (and discussed above) which uses ECD fragmentation patterns to distinguish aspartyl and isoaspartyl residues formed from deamidation of asparagine. This method is expected to work similarly for glutamine deamidation. The ECD method not only distinguishes the isomers, it also suggests several methods to quantify them. One method which has been demonstrated in the cytochrome C case, utilizes the characteristic 1:3 branching ratio on deamidation to provide a calibration point which allows relative quantitation. Finally, while deamidation occurs naturally, it also occurs as a samplehandling artifact, most notably during overnight tryptic digestion at basic pH. This artifact can be controlled and monitored by 18O isotopic labeling methods.

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