A convenient and precise mass spectrometric method for measurement of the deamidation rates of glutaminyl and asparaginyl residues in peptides and proteins has been developed; the rates of deamidation of 306 asparaginyl sequences in model peptides at pH 7.4, 37.0°C, 0.15 M Tris⅐HCl buffer have been determined; a library of 913 amide-containing peptides for use by other investigators in similar studies has been established; and, by means of simultaneous deamidation rate measurements of rabbit muscle aldolase and appropriate model peptides in the same solutions, the use of this method for quantitative measurement of the relative effects of primary, secondary, tertiary, and quaternary protein structure on deamidation rates has been demonstrated. The measured rates are discussed with respect to the hypothesis that glutaminyl and asparaginyl residues serve, through deamidation, as molecular timers of biological events.deamidation ͉ biological clocks ͉ peptides ͉ mass spectrometry T he hypothesis that glutaminyl and asparaginyl residues in peptides and proteins serve, through deamidative transformation into glutamyl and aspartyl residues, as molecular timers of biological events such as protein turnover, development, and aging (1-4) was originally based on the suggestion and then experimental proof that the deamidation of cytochrome c occurs in vivo (5-6) and on reasoning that deamidation is seriously disruptive to biological systems unless it is being used for compensating beneficial biological purposes.Subsequently, it was shown that the first-order deamidation half times in pH 7.4, 37°C ionic strength 0.15-0.20 phosphate buffer of glutaminyl and asparaginyl residues vary over a range of at least 1 day to 9 years as a function of primary sequence and likely over an even wider range as a function of secondary, tertiary, and quaternary structure (3, 7-11). The dependence of deamidation on pH, temperature, ionic strength, and other solution properties was also demonstrated (3,(12)(13). It was additionally shown that the overall in vivo compositions and specific sequence distributions of amide residues in peptides and proteins are supportive of the amide molecular clock hypothesis (1)(2)(3) 14).The first two specific amide clocks to be identified were those that control the in vivo turnover rates of cytochrome c (6, 9) and rabbit muscle aldolase (15-16). Measurements of in vivo turnover rates, in vivo steady-state concentrations, in vivo and in vitro deamidation rates of these proteins, and in vitro deamidation rates of appropriate model peptides demonstrated both sequence dependence and three-dimensional structure dependence of the deamidation of these two proteins. Sequencecontrolled deamidation of the C-terminal sequence . . . Thr-AsnGlu in cytochrome c apparently leads to a changed threedimensional structure that accelerates a second deamidation of cytochrome c. The resulting deamidated forms of cytochrome c are rapidly catabolized in vivo with rates equivalent to the turnover rate. In the case of rabbit muscle aldo...

Nonenzymatic deamidation rates for 52 glutaminyl and 52 asparaginyl pentapeptides in pH 7.4, 37.0 degrees C. 0.15 m Tris-HCl buffer have been determined by direct injection mass spectrometry. These and the previously reported 306 asparginyl rates have been combined in a self-consistent model for peptide deamidation. This model depends quantitatively upon peptide structure and involves succinimide, glutarimide and hydrolysis mechanisms. The experimental values and suitable interpolated values have been combined to provide deamidation rate values in pH 7.4, 37.0 degrees C. 0.15 m Tris-HCl buffer for the entire set of 648 single-amide permutations of ordinary amino acid residues in GlyXxxAsnYyyGly and GlyXxxGlnYyyGly. Thus, knowledge about sequence-dependent deamidation in peptides is extended to include very long deamidation half-times in the range of 2-50 years.

Deamidation of asparaginyl and glutaminyl residues causes timedependent changes in charge and conformation of peptides and proteins. Quantitative and experimentally verified predictive calculations of the deamidation rates of 1,371 asparaginyl residues in a representative collection of 126 human proteins have been performed. These rates suggest that deamidation is a biologically relevant phenomenon in a remarkably large percentage of human proteins.in vivo deamidation ͉ asparaginyl residues D eamidation of asparaginyl (Asn) and glutaminyl (Gln) residues to produce aspartyl (Asp) and glutamyl (Glu) residues causes structurally and biologically important alterations in peptide and protein structures. At neutral pH, deamidation introduces a negative charge at the reaction site and can also lead to structural isomerization. Early work established that deamidation occurs in vitro and in vivo, and that the rates of deamidation depend on primary sequence, three-dimensional (3D) structure, pH, temperature, ionic strength, buffer ions, and other solution properties (1-11). It has been hypothesized (3,5,7,12,13) that Asn and Gln may serve, through deamidation, as molecular clocks which time biological processes such as protein turnover, homeostatic control, and organismic development and aging, as well as mediators of postsynthetic production of new proteins of unique biological value.Deamidation has been observed and characterized in a wide variety of proteins. It has been shown to regulate some timedependent biological processes (8, 9) and to correlate with others, such as development and aging. There are many reports of deamidation under physiological conditions in proteins of biological significance. For examples, see refs. 14-18.Extensive evidence suggests that deamidation of Asn at neutral pH usually proceeds through a cyclic imide reaction mechanism (19)(20)(21). Sometimes the Asp produced by deamidation is isomerized to isoAsp. The in vivo reversal of this isomerization has been widely reported, but reversal of deamidation itself and of the introduced negative charge has not been observed.Deamidation rates depend on the amino acid residues near Asn and Gln in the peptide chain with sequence-determined deamidation half-times at neutral pHs and 37°C in the range of 1-500 days for Asn and 100-Ͼ5,000 days for Gln (7, 13).Sequence-determined Asn and Gln deamidation rates are modulated by peptide and protein 3D structures. Deamidation of peptides is observed at both Asn and Gln, largely in accordance with sequence-controlled rates. Deamidation of proteins, which is usually slowed by 3D structure, occurs primarily at Asn except in very long-lived proteins where Gln deamidation is also observed. In a few instances, 3D structure has been reported to increase deamidation rate.The deamidation rates of individual Asn residues in a protein can be reliably predicted as a result of two recent advances. First, the sequence-controlled Asn deamidation rates of most of the 400 possible near-neighbor combinations in pentapeptide m...

A method for the quantitative estimation of instability with respect to deamidation of the asparaginyl (Asn) residues in proteins is described. The procedure involves the observation of several simple aspects of the three-dimensional environment of each Asn residue in the protein and a calculation that includes these observations, the primary amino acid residue sequence, and the previously reported complete set of sequence-dependent rates of deamidation for Asn pentapeptides. This method is demonstrated and evaluated for 23 proteins in which 31 unstable and 167 stable Asn residues have been reported and for 7 unstable and 63 stable Asn residues that have been reported in 61 human hemoglobin variants. The relative importance of primary structure and threedimensional structure in Asn deamidation is estimated.biological clocks ͉ proteins T he spontaneous deamidation of glutaminyl and asparaginyl residues causes experimentally and biologically important changes in peptide and protein structures. In asparaginyl deamidation, the primary reaction products are aspartyl and isoaspartyl. Early work on peptide and protein deamidation (1-10) established that deamidation occurs in vitro and in vivo and depends on primary sequence, three-dimensional (3D) structure, pH, temperature, ionic strength, buffer ions, and other solution properties.It was hypothesized (3, 5, 7) and then experimentally demonstrated (2, 8, 9, 11) that deamidation can serve as a biologically relevant molecular clock that regulates the timing of in vivo processes. Substantial evidence supports the hypothesis that Asn deamidation at neutral pH proceeds through a cyclic imide reaction mechanism (12)(13)(14).A procedure is needed whereby the stability of individual amides in peptides and proteins can be reliably estimated. Although it was evident to investigators 30 years ago (2-7) that protein deamidation rates depend on primary, secondary, tertiary, and quaternary protein structure, and numerous examples have been found, it was not possible to devise a useful deamidation prediction procedure until a complete library of deamidation rates as a function of primary sequence was available.A suitable library of sequence-determined Asn rates has now been published (15), and the relevance of this library has been established (16). These rates can now be combined with 3D data to provide a useful deamidation prediction procedure. Each amide residue has an intrinsic sequence-determined deamidation rate, which depends on charge distribution, steric factors, and other aspects of peptide chemistry. This primary rate is modulated by 3D structure, which usually slows the rate. In a few instances, it increases the deamidation rate.We have devised a simple procedure that is useful for predicting the relative deamidation rates of most protein Asn residues. We have tested this procedure on a complete set of all proteins for which, during a review of the literature, we found experiments specifically identifying one or more labile Asn residues in a protein and also a suitable 3...

A completely automatic computerized technique for the quantitative estimation of the deamidation rates of any protein for which the three-dimensional structure is known has been developed. Calculations of the specific deamidation rates of 170,014 asparaginyl residues in 13,335 proteins have been carried out. The calculated values have good quantitative reliability when compared with experimental measurements. These rates demonstrate that deamidation may be a biologically relevant phenomenon in a remarkably large percentage of proteins.asparaginyl residue deamidation ͉ coefficient of deamidation ͉ deamidation index C hanges in peptide and protein structure through the spontaneous nonenzymatic deamidation of glutaminyl and asparaginyl residues have been observed in many in vitro and in vivo experiments. Rates of deamidation of individual amide residues depend upon primary sequence, three-dimensional (3D) structure, and solution properties such as pH, temperature, ionic strength, and buffer ions (1-8).Deamidation at neutral pH introduces a negative charge at the deamidation site and sometimes also leads to ␤ isomerization. These alterations in structure affect the properties of peptides and proteins in chemically and biologically important ways. It has been suggested that in vivo deamidation of proteins serves as a molecular timer of biological events and as a mechanism for postsynthetic production of unique proteins of biological significance (2,4,6,7,9,10). In the case of in vivo protein turnover, the use of deamidation as a molecular timer has been experimentally demonstrated (11-13).Progress in the understanding of deamidation and its potential biological importance has been impeded by the lack of reliable and useful experimental and theoretical information about the deamidation of most proteins. Experimental studies of the deamidation of individual proteins are laborious and time consuming. Until recently, there were no other means by which to estimate the deamidation rates of specific amides.The deamidation rates of individual Asn residues in a protein can now be reliably predicted as a result of two recent advances. First, the sequence-controlled Asn deamidation rates of most of the 400 possible near-neighbor combinations in pentapeptide models have been measured (10); the deamidation rates of a representative group of Gln pentapeptides have been determined (N.E.R. & A. B. Robinson, unpublished work); and the relevance of these rate libraries has been established (14). Second, these rates and the 3D structures of proteins with well characterized deamidations have been combined to produce a computation method that correctly predicts the deamidation rates of most Asn residues for which the 3D structure is known (15). This method has been shown to be Ϸ95% reliable in predicting relative deamidation rates of Asn residues within a single protein, and it is also useful for the prediction of absolute deamidation rates. It has been used to estimate the deamidation rates of 1,371 asparaginyl residues in 126 human pro...